Download Chapter 1 - Baboola Sumo

The Use of Engineered Microbes as
Medical Agents
By Geoff Graham
Chapter 1 – Introduction and Summary of
the Entire Book
Toward a less volatile existence for all of us
List of Chapters and Appendices
Chapter 1 – Summary of this e-book (this chapter)
Chapter 2 – Concentration of Drugs Near Their Target Tissues
Chapter 3 – Uses for Genetically Engineered Microbes That Remain Outside of Human Cells
Chapter 4 – The Use of Cytotoxic Peptides by Extracellular Xenobiorgs: A Special Case
Chapter 5 – The Use of Extracellular Xenobiorgs Against Cancer
Chapter 6 – The Use of Extracellular Xenobiorgs Against Alzheimer’s Disease
Chapter 7 – The Medical Uses of Intracellular Xenobiorgs
Chapter 8 – The Human Body as a Target for Localized Intervention
Chapter 9 – Access to the Body Via the Lymphatic Drainage System
Chapter 10 – Use of Engineered Nematode Microfilariae as Medical Agents
Chapter 11 – Homing to Chosen Tissues by Way of the Blood
Chapter 12 – Repnumi Regeneration and Senescence
Chapter 13 – Stem Cells and Repnumi Rejuvenation
Chapter 14 – Sources of Genetically Normal Donor Nuclei
Chapter 15 – Assessing and Reporting the Differentiated States of Human Cells
Chapter 16 – Keeping Track of Which Cells Have Been Subjected to Repnumi Rejuvenation
Chapter 17 – Removal of Original Nuclei and Mitochondria from Target Cells of Repnumi
Chapter 18 – Insertion into Cells of Donated Nuclei and Mitochondria in Repnumi
Chapter 19 – Protection of Xenobiorgs from the Immune System
Chapter 20 – Repnumi Rejuvenation of the Central Nervous System
Chapter 21 – Facilitated Telepathy: The Wildest Possibility
Chapter 22 – Remaining Safety Issues
Chapter 23 – Step-By-Step Implementation of Xenobiotherapy and
Repnumi
Chapter 24 – Clearing of Sclerotized Tissue, Proliferation of Rejuvenated Cells, and
Redifferentiation of Differentiated Cells
Chapter 25 – The Lifting of an Ancient Curse: The Social Consequences of Repnumi
Rejuvenation
Chapter 26 – Uses of Xenobiorgs Outside of Medicine
Chapter 27 – A Guide to Resources Useful for Xenobiotherapy and Repnumi Research
Appendix 1 – Quick Internet Access Format
Appendix 2 – New Terminology
Appendix 3 – How readers can help
Appendix 4 – List of Medical Projects Discussed in this Chapter and this E-Book
Appendix 5 – Key words
The Purposes of this E-book and this Chapter
This e-book explores the use of genetically engineered microbes in medicine. It
is mostly speculative, i.e., it synthesizes published information to describe what
doctors and scientists might do with microbes to combat disease and prolong life.
In a few cases, it discusses what doctors and scientists are already doing with
medical microbes.
Repnumi rejuvenation. The ultimate goal of this effort is rejuvenation of
human tissues via a method that is seldom discussed: the rehabilitation of
individual cells, in situ. This would probably require at least 3 things: the
replacement of senesced nuclei by fresh nuclei in the same differentiated state, the
replacement of senesced mitochondria by fresh mitochondria, and the removal of
accumulated intracellular garbage.
Rejuvenation by replacement of nuclei and mitochondria lends itself to the
perfect acronym Renumi (“renew-me”). However, “Renumi” is a proprietary name
for a type of acupuncture { }; hence, to avoid confusion, I use the term Repnumi.
Repnumi will be very difficult. Full rejuvenation of a human being by
Repnumi would be an enormous task. There are some 4 X 1013 cells in a typical
human body {
}, a hundred or more times as many cells as there are stars in
our galaxy { }. Most of these cells are deep inside the body, where there is no
simple way to access them. Although replacement of existing nuclei by
inappropriate successor nuclei would be disastrous, we probably have not
catalogued all of our differentiated nuclear types. Moreover, it is not yet clear
whether mitochondria would also need to be replaced by cell type-specific
successors.
The incremental approach to progress. On the other hand, we need not
accomplish this task all at once. I suspect that an early target of Repnumi
rejuvenation will be the skin of the human face. Facial skin is easily accessible,
http://www.renumi.com/
Bianconi_E Ann Hum Biol. 2013 Nov-Dec;40(6):463-71 An estimation of the number of cells in the human body. M: abst, nin
http://en.wikipedia.org/wiki/ Milky_Way, M: in diameter which contains, nin
relatively forgiving of mistakes (compared with, e.g., heart or brain tissue), and is
very important to the self-image and social success of many of us.
Technological progress is much faster when modest improvements yield
significant rewards. One reason that consumer electronic technology has advanced
rapidly, while nuclear fusion technology has not, is that small increases in
performance of personal computers, smart phones, etc., are very lucrative and spur
further investment. In contrast, there is much less enthusiasm to spend billions of
dollars and years of work merely to bring nuclear fusion technology from 1/3 to
1/2 the way to ignition—because a reactor that cannot ignite its nuclear fuel has
little economic value.
Partly for this reason, this e-book focusses mostly on simpler medical uses for
genetically engineered microbes. The easily achievable uses are critical for
progress, because once they are accomplished, interest in the technology will
increase, and many uses that we cannot now imagine will be tried.
Medical microbes and Repnumi rejuvenation. I expect progress in the
medical use of genetically engineered microbes to lead to progress toward
Repnumi rejuvenation for two reasons. First, genetically engineered microbes will
probably be needed to control Repnumi. Second, moving the biological reagents
needed for both processes to their sites of action and protecting them from the
immune system will require the same knowledge.
Xenobiorgs. I have tried to avoid new jargon in this e-book, but I have
introduced six new terms. In particular, I refer to the medical use of genetically
engineered microbes as xenobiotherapy, and to the microbes themselves as
xenobiorgs. A leukocyte that contains an intracellular xenobiorg is called a
leukocyte/xenobiorg. A list of the new terms used in this e-book is given near the
end of this chapter.
References. Although this chapter is 165 pages in length, it is only a
description of the book’s contents—it is not the book itself. For this reason, I have
given references for only a few assertions that are not repeated in the remaining
chapters. The remaining chapters of the book will be heavily referenced. Any
reader who wants a reference for a statement made in this chapter can email me at
[email protected].
An experiment in better use of information. This book is also an experiment
in the more efficient use of scientific information. Scientists produce mountains of
facts about nature, but make poor use of them. The information that researchers
need is scattered and disorganized—and researchers are usually far too busy to
find, read, organize and synthesize all of the information that they could benefit
from. Yet, there are many retired or unemployed molecular biologists who could
do this intellectual work for active researchers. I hope that this book will inspire
many more attempts like it.
Concentration of Drugs Near Their Target Tissues (A Brief
Summary of Chapter 2)
Localized release of drugs. Repnumi rejuvenation and other advances
envisioned in this book will require methods to move living cells to precise
locations in the body. These methods will be devised sooner if there are near-term
rewards that justify the effort and expense. One such reward may be the ability to
concentrate drugs near their target tissues.
Homing of xenobiorgs to a target tissue. Some human cells can travel through
the body to specific organs. Moreover, disease microbes often concentrate in
specific organs, which suggests they they also may restrict their own location in
the body. Traveling through the body to a specific organ or tissue is termed
homing, and homing of course requires molecular machinery. This machinery
could probably be combined with targeted injection to restrict xenobiorgs to
specific locations within the body. Because of this, it might be possible to
concentrate medical drugs near their intended targets in ways that are not yet
practical.
Chapter 2 explores the possible benefits of this. For example, if a medicine acts
on the heart, what—if anything—would be gained by restricting it to the heart?
Restriction of drugs to target tissues would be beneficial. A look at seven
representative drug types: monoclonal antibodies, lithium, and
analogs/inducers/depleters of five natural hormones, suggests that in all cases
harmful side effects might be reduced by drug concentration at the target.
An analysis of 50 drugs that have been withdrawn from the market reveals
characteristic problems that might be alleviated by spatial restriction. These
include liver toxicity, heart rhythm disruption, and “sending the right signal to the
wrong tissues.” Chapter 2 concludes that about 35 (70%) of the withdrawn drugs
could be improved by restriction to their intended targets—although this does not
mean that all of them would be returned to service.
Drug synthesis, prodrug activation, and scavenging. Xenobiorgs, stationed
in situ near a target tissue, might deliver drugs to that tissue in two ways. First,
they might make and secrete biologically active compounds. Second, they might
convert an inactive blood-borne prodrug to active form.
In addition, xenobiorgs might provide a medical benefit by removing unwanted
molecules from an organ. Removal of the biochemical signals by which cancers
masquerade as wounded tissue is one example. Other examples might include
removal of excess blood glucose, removal of stress hormones, removal of bacterial
toxins, and toxins such as mercury, etc.
A possible new way to increase local drug concentrations. Xenobiorgs might
employ a novel strategy to concentrate bioactive macromolecules near their target
tissues. The strategy is to release two weakly interacting macromolecules that must
cooperate in order to be effective. The probability of such cooperation would be
proportional to the arithmetic product of the two macromolecular concentrations.
As the distance from the target increased, and the concentration of each component
decreased, the probability of cooperative interaction would decrease at a much
greater rate. Examples of cooperating molecules that could serve as the basis for
this strategy are discussed in Chapter 2.
A change in the nature of drugs. Chapter 2 concludes by suggesting that
synthesis and release of drugs by xenobiorgs near their target tissues would change
the chemical nature of many drugs. Many drugs have a chemical structure that is
foreign to biology. This chemistry results from needs to (a) preserve the drug
within a human host long enough for it to reach its target, (b) allow eventual
destruction of or removal from the human host, (c) allow transport through the
blood, (d) allow movement from the blood to a target tissue, and (e) avoid
provoking an immune response.
On one hand, it might be very difficult to engineer xenobiorgs that could
synthesize many of these exotic chemicals. On the other hand, it should not be
necessary, since a bioactive compound made near its target would have no need to
travel through the blood, and could be far more labile than a blood-borne drug.
Indeed, such lability would probably be desirable.
Uses for Genetically Engineered Microbes That Remain
Outside of Human Cells (A Brief Summary of Chapter 3)
Extracellular and intracellular xenobiorgs. Xenobiorgs will probably be
harmless derivatives of human pathogens. Some such pathogens remain outside of
living cells for most of their lives, while others reside within host cells. Thus, both
extracellular and intracellular xenobiorgs could be created.
Chapter 3 focusses on extracellular xenobiorgs. It discusses tasks other than
localized drug delivery that xenobiorgs might be engineered to perform.
Current use of beneficial microbes. Microbes are seldom used as medical
agents. However, bacteriophages are used in some countries to control bacterial
infections, “probiotics” is gaining popularity in western countries, and microbes
are used against dangerous bacterial biofilms in industrial settings. Moreover, there
is interest in using microbes to kill or confuse the bacteria that cause tooth decay.
From “natural” to engineered. Although the microbes used for the above
purposes are usually “natural”, i.e. not engineered with foreign DNA, their genetic
compositions are usually known. Efforts are usually made to learn why they confer
some benefit. The logical next step from this is that they be genetically engineered.
Hence, current medical and industrial use of microbes may provide knowledge
useful in engineering xenobiorgs.
Contrast agents. Xenobiorgs engineered to carry inorganic material might
serve as contrast agents for medical imaging. Because they could be motile, they
might fill networks of tubes. This could be especially valuable in delineating
lymphatic networks, to prevent cancer metastasis (see Chapter 8).
Killing of pathogens. Xenobiorgs could be engineered to attack pathogens.
There are many possible target pathogens, including tooth decay bacteria,
gastrointestinal parasites such as Helicobacter pylori, or the filarial nematodes that
cause elephantiasis (see Chapter 9).
Killing or neutralizing pathogens that do not invade the body, e.g. the bacteria
that cause tooth decay, seems much easier than attacking pathogens that do invade
the body. Accordingly, it will probably be attempted first.
Killing or inactivation of specific human cells. Xenobiorgs could be
engineered to kill or inactivate specific human cells. Cancer cells are the most
obvious possibility, but there are others. These include ghrelin-producing cells that
line the intestine (ghrelin is a hunger-inducing hormone) and adipose cells
(adipocytes).
Local immunological tolerance. Xenobiorgs that could remain alive and near
their target organs might also establish local immunological tolerance. This could
benefit organ transplantation, and autoimmune syndromes such as type 1 diabetes
and Crohn’s disease.
Identification of cells. Xenobiorgs that could sense the internal state of cells
might mark certain cells and/or induce them to proliferate. For example, stem cells
from human organs might be marked with a dye and then isolated by fluorescenceactivated cell sorting.
Other tasks for external xenobiorgs. If they could remain near their target
tissues for weeks or months, xenobiorgs might prevent the graying of hair, prevent
pattern baldness, and prevent benign prostatic hyperplasia. They might stimulate
muscle growth in the feeble (and in bodybuilders), without the side effects of
anabolic drugs. They might discourage monocyte invasion of atherosclerotic
plaque, and might prevent neointima formation in cardiac stents.
The Use of Cytotoxic Peptides by Extracellular Xenobiorgs:
A Special Case (A Brief Summary of Chapter 4)
Pathogen-infected cells and cancer cells are likely targets of xenobiorg attack.
An important method by which cells of all types attack and kill other cells and
viruses is the use of toxic polypeptides. Cytotoxic polypeptides bind their targets
and either lyse them or otherwise disable them. Cytotoxic polypeptides are often
referred to as antimicrobial peptides or AMPs.
Cytotoxic polypeptide distribution. Cytotoxic polypeptides are present nearly
everywhere in the realm of life. They are produced by bacteria, fungi, plants,
invertebrates, and vertebrates. They have been described in the venom of bees,
wasps, spiders, scorpions, and snakes. They are present in shrimp, in fish, and in
the skin of frogs.
Most species that have been examined produce multiple cytotoxic polypeptides.
The roughly 5,000 extant frog species produce an estimated 100,000 different
cytotoxic polypeptides.
In multicellular organisms, expression of cytotoxic polypeptides is often tissuespecific. Cytotoxic polypeptides called histatins are confined to human saliva, for
example. In chickens, some cytotoxic polypeptides are confined to the bone
marrow and the respiratory tract, while others are confined to the liver and the
urogenital tract.
Cytotoxic polypeptide importance. Cytotoxic polypeptides are intensively
investigated for several reasons. First, they are an extremely important part of
innate immunity against pathogens. Secondly, many human endogenous cytotoxic
peptides (see below) may spontaneously kill those tumors that display the Warburg
effect. Third, exogenous cytotoxic polypeptides may become powerful new
weapons against both infectious disease and cancer.
An entire chapter of this e-book is devoted to cytotoxic polypeptides because
they are very well-suited to xenobiotherapy. Xenobiorgs could easily be
engineered to make and release them, and they are most effective when released
near their targets.
Sources of cytotoxic polypeptides. A large number of cytotoxic polypeptides
have already been discovered from natural sources. Efforts to identify even more
through genomic analysis (see below) are underway. Moreover, unnatural sources
such as phage display libraries and rationally designed peptides are also being
exploited.
High-throughput methods have been developed to screen and characterize the
thousands of new polypeptides that the above efforts produce.
♦ Classes of cytotoxic polypeptide
Cytotoxic peptides of interest in this e-book. In this e-book I use the term
“cytotoxic polypeptide” to denote polypeptides that kill individual cells rather than
whole organisms. A polypeptide that lysed cancer cells would be included, even if
the polypeptide was post-translationally modified. However, a polypeptide that
killed by preventing proper nerve transmission, by interfering with the heartbeat, or
by poisoning the liver would not be included. The purpose is to focus on
polypeptides that might plausibly be made by engineered microbes and which
could perform medically useful tasks.
Many types of cytotoxic polypeptide. Cytotoxic polypeptides fall into several
major groups. Bacteriocins―a complex and variegated group―generally kill or
inhibit the growth of bacteria that are closely related to the strain that synthesized
the bacteriocin { }.
Most cytotoxic polypeptides have a broader killing spectrum than do
bacteriocins, and most act by disrupting membranes in their targets. A great many
of these form amphipathic α-helices in solution, but others form β-sheets―and
there seem to be other classes as well. At least a few are also lectins, polypeptides
that bind sugar moieties. Sources such as spider venom sometimes contain multiple
classes of cytotoxic polypeptide.
Some cytotoxic polypeptides are modified post-translationally. These
modifications can include amidation of the carboxy terminus, oxidation, lipidation,
glycosylation, and conversion of L-form amino acids to D-form (chirality
conversion).
Secondary medical effects. Polypeptides secreted by living organisms may
also affect transmission of nerve impulses, blood pressure, or other physiological
characteristics of humans. In some cases, this may be desired; in other cases, it
may be an unwanted attribute of an otherwise useful cytotoxic polypeptide.
http://en.wikipedia.o rg/wiki/Bacteriocin, M: Bacteriocins are prot einaceous, nin
♦ Cytotoxic polypeptide binding to membranes
Much effort has been spent deducing the mechanisms by which cytotoxic
polypeptides bind to and lyse target membranes. Understanding of this would yield
at least seven important benefits.
Rational design. First, it would allow the rational design of new cytotoxic
polypeptides and obviate the need to search for them among familiar and exotic
organisms.
Targeting to specific cell types. Second, it would guide sequence changes that
could target cytotoxic polypeptides to specific cell types, such as cancer cells,
fungi, and gram-positive bacteria. This would inflict maximum damage on target
cells and minimally damage healthy host cells.
Choice of dosages. Third, it would guide the choice of cytotoxic polypeptide
dosages. This is a critical consideration, given that cytotoxic polypeptides bind
target membranes cooperatively.
Use with other peptides or drugs. Fourth, it could guide the use of multiple
different cytotoxic polypeptides against the same target, or the use of cytotoxic
polypeptides along with conventional antibiotics.
Coping with the immediate environment. Fifth, cytotoxic polypeptide action
is influenced by the composition of the target membrane, by the presence of
dissolved proteins in extracellular fluid, and by the ionic strength of that fluid.
Knowledge of these conditions and their consequences could guide the choice of
therapeutic cytotoxic polypeptide and perhaps guide intervention by drugs or other
means to alter the conditions.
Protective screening. Sixth, some soluble polypeptides resemble cytotoxic
polypeptides, but are not cytotoxic. If their binding to membranes were understood
these might protect healthy host cells from therapeutic cytotoxic polypeptides
directed against pathogen-infected cells or cancer cells.
Resistance and cross-resistance. Seventh, and very importantly, it would help
researchers predict when microbial resistance to one cytotoxic polypeptide
automatically causes resistance to a second. The critical need for this knowledge is
discussed below.
Factors that influence binding to a membrane. As discussed in Chapter 4,
the binding behavior of cytotoxic polypeptides is influenced by at least five of their
characteristics. These are their electrostatic charge, hydrophobicity, amphipathic
conformation, curvature, and length. However, different sequences that are the
same in these five ways do not necessarily behave the same.
Electrostatic charge. When binding is based on electrostatic charge, the
cytotoxic polypeptide has a positive charge and the target membrane has a negative
charge. Increasing the ionic strength of the medium weakens this kind of binding.
Hydrophobic interactions. On the other hand, peptide-membrane binding
based on hydrophobicity is not weakened by high ionic strength. An increase in
peptide hydrophobicity can compensate for reduced electrostatic binding in
solutions of high ionic strength.
Modification of peptide ends. Amidation of the carboxy terminus frequently
increases the effectiveness of cytotoxic polypeptides, probably by easing their
entry into lipid membranes. End-tagging of short peptides with stretches of
hydrophobic amino acid often increases their potency (and their resistance to
proteases), probably for similar reasons.
Peptide curvature and kinking. Peptide curvature is important, and a proline
kink near the middle of the peptide can be necessary for binding of a peptide to
bacteria.
The influence of peptide length. Cytotoxic polypeptide length is also
important for binding and lysis. Long polypeptides tend to aggregate on the surface
of target membranes, instead of inserting and forming a pore. Insertion of cytotoxic
polypeptides into membranes is most efficient when the cytotoxic polypeptides are
just long enough to span the membrane and when the hydrophobicity profile of the
inserted cytotoxic polypeptides matches that of the membrane lipids. However,
lytic cytotoxic polypeptides can also be very short, perhaps as short as 4 amino
acid residues.
Multiple mechanisms of cell lysis. Cytotoxic polypeptides lyse their target
cells by different mechanisms. Some known or hypothesized mechanism include
the barrel stave mechanism, the carpet mechanism, and the toroidal pore
mechanism. Pore formation seems to be most effective against bacteria and fungi,
while causing the least damage to human cells. However, cytotoxic polypeptides
can permeabilize bacterial membranes without forming pores.
The influence of target membrane composition. Membrane composition
strongly affects whether there is lysis, and the mechanism by which lysis occurs.
Phospholipids in a membrane promote cytotoxic polypeptide aggregation rather
than pore formation. Moreover, a given cytotoxic polypeptide can lyse different
membranes by different mechanisms.
Partitioning of macromolecules within a target membrane. Most, if not all,
biological membranes include multiple types of macromolecules. Cytotoxic
polypeptides can separate these substances from each other to form dissimilar
domains within the membrane. Such a domain might be enriched in cholesterol, for
example, or cardiolipin. Anionic and zwitterionic lipids can be separated this way.
Formation of these domains can disrupt microbial function, and can also produce
phase boundary defects which allow cell contents to leak.
Lethality without lysis. Cytotoxic polypeptides can probably kill cells without
lysing them. Cytotoxic polypeptides lower the zeta potential of colloids, causing
them to aggregate or flocculate.
Cooperative lysis. Cytotoxic polypeptides often interact to produce their
effects. They may rest harmlessly on the surface of a membrane until they reach a
critical concentration, and then reorient themselves to form a pore. (This fact
argues strongly that localized release of cytotoxic peptides, e.g. by xenobiorgs, is
better than systemic administration).
Multiple cytotoxic polypeptides, either separate or physically joined, in some
cases act synergistically. Their effect together exceeds the sum of their individual
effects. Joined copies of the same cytotoxic polypeptide are sometimes also very
effective.
Although in many cases, cytotoxic polypeptides must be physically free to
aggregate and form pores if they are to be effective, some cytotoxic polypeptides
are effective when bound to the surface of a carrier.
Movement across an outer membrane. Some cytotoxic polypeptides actually
cross the outer membrane and attack targets (such as mitochondrial membranes)
within cells (see below). In some cases, the cytotoxic polypeptides are carried
across the membrane by proteins in the target cell. However, since membranecrossing protein domains are known, there may be other cases where the cytotoxic
polypeptides cross the outer membrane without help.
Effects on cell activity. Another oddity of cytotoxic polypeptide behavior is
that sublethal concentrations of cytotoxic polypeptides may inhibit
macromolecular synthesis in E. coli, or alter protein expression in E. coli.
Presumably this effect occurs in other bacteria as well.
♦ Therapeutic Specificity
Killing of target cells but not host cells. It is desirable for cytotoxic
polypeptides to kill target cells more effectively than non-target host cells. The
greater this difference is, the more useful the cytotoxic polypeptide. Comparative
killing is sometimes expressed as a ratio, with some measure of the effectiveness
against target cells divided by some measure of the damage to non-target cells. As
one example, the inverse of the minimum inhibitory concentration against target
microbes is divided by the concentration needed to lyse 50% of a population of
erythrocytes.
Other harm by cytotoxic polypeptides. Other measures of harm done by
cytotoxic polypeptides are often considered. For example cytotoxic polypeptides
that cause much degranulation of mast cells, and consequent inflammation, are less
useful than peptides that cause less mast cell degranulation.
As mentioned above, much tinkering with peptide sequences and posttranslational modifications has gone toward increasing the lethality of cytotoxic
polypeptides toward target cells while reducing their harm.
Examples of effective cytotoxic polypeptide use. Cytotoxic polypeptides have
been effective against oral and gastrointestinal pathogens, against the enveloped
viruses herpes simplex and HIV, against parasitic fungi and parasitic protozoa,
against Gram-positive bacteria, against Gram-negative bacteria, and against sperm.
They can kill the malaria parasite Plasmodium falciparum, the venereal disease
bacterium Neisseria gonorrhea, the parasitic fungus Candida, the gastric parasite
Helicobacter pylori, and impetigo. They may be effective in treating diabetic foot
ulcers.
Some cytotoxic polypeptides selectively lyse cancer cells, while mostly sparing
red blood cells and fibroblasts.
Several reports indicate that cytotoxic polypeptides can protect mice from
bacterial infection when given systemically, as if they were traditional antibiotics.
Moreover, a cytotoxic polypeptide given systemically was reported to protect
immune-deficient mice from cancer.
Targeting of cytotoxic polypeptides to specific bacteria. However,
investigators also recognize the need for targeting of cytotoxic polypeptides. In one
case, an 8-amino acid segment from a larger protein guided an antimicrobial
peptide to a bacterial target and eliminated that bacterial species from a
multispecies biofilm.
Interactions and side effects. Cytotoxic polypeptides can show synergy with
each other, and also with conventional antibiotics such as β-lactams. Antimicrobial
peptides can sensitize resistant strains to an antibiotic.
Some antimicrobial cytotoxic polypeptides can bind bacterial
lipopolysaccharides and prevent their harmful effects { }. However,
lipopolysaccharide can also prevent the beneficial effects of cytotoxic
polypeptides.
http://en.wikipedia.org/wiki/ Lipopolysaccharide, nin
One problem with the therapeutic use of cytotoxic polypeptides is that some of
them activate the quorum-sensing machinery of bacterial pathogens. Others cause
major releases of microbial cell contents, which is medically undesirable.
♦ pH-Dependent Cytotoxic Polypeptides
Many cytotoxic polypeptides lyse target membranes only at low pH. This
means that they are active only in parts of the gastrointestinal tract or only in
tissues that are respiring anaerobically. Anaerobic respiration (the Warburg effect)
is typical of cancers.
Many of the human body’s own proteins include segments that form lytic
polypeptides at low pH. These polypeptides are presumed to be liberated as the
proteins degrade. The polypeptides probably defend the body against cancer, but
may also explain why even short periods of anoxia are so destructive to some
tissues, particularly the brain. Many lytic polypeptides are expressed in the brain.
Chapter 4 discusses acid-dependent cytotoxic polypeptides and cancers. The
question of why some cancers escape destruction by acid-dependent cytotoxic
polypeptides is also discussed.
Acid-dependence can be a valuable property in a therapeutic cytotoxic
polypeptide, because it can restrict the cytotoxic polypeptide’s activity to solid
tumors. The sequence requirements for acid-dependence are largely known, and
procedures have been described engineering peptides that are lytic only at pH 5.5
and below.
♦ Non-Destructive Entry of Cytotoxic Polypeptides
Some cytotoxic polypeptides can move non-destructively through cell
membranes and attack internal structures, such as mitochondria or internal
parasites. Tarantula venom, for example, contains a cytotoxic polypeptide that
attacks Plasmodium falciparum (malaria) within erythrocytes.
As mentioned above, this nondestructive entry is sometimes due to transport of
the peptide through the membrane by a host carrier. However, there may be other
instances where the polypeptide moves through the outer membrane passively, as
does the Tat protein of HIV-1.
♦ Cytotoxic Polypeptide Sensitivity to Conditions
Many cytotoxic polypeptides are sensitive to conditions typical of the human
body. Many lytic polypeptides, for example, are inactivated by blood serum. NaCl
also inactivates many lytic polypeptides, as does heparin. Ca2+ cations promote
peptide aggregation on cell surfaces, at the expense of pore formation.
Zn2+ ions, on the other hand, increase binding to and lysis of target bacteria by
histidine-rich cytotoxic polypeptides. The presence of two diffusible components
required for activity might sharpen the area where activity occurs, because activity
would be proportional to the product of the two concentrations.
The fact that a given cytotoxic polypeptide is inactivated by blood serum need
not preclude its use. However, it argues for localized release of the cytotoxic
polypeptide, near or in contact with the target. Xenobiorgs that had exited the
blood and moved into the extracellular matrix of a solid tissue might provide the
desired localized release.
Cytotoxic polypeptides isolated from marine organisms are often insensitive to
dissolved NaCl. Moreover, sequence engineering of the cytotoxic polypeptides can
often remove their sensitivities to environmental conditions.
♦ Protection of Producer Cells
In nature, cells that produce cytotoxic polypeptides are usually immune to the
cytotoxic polypeptides they produce. It is desirable, although probably not
essential, that xenobiorgs have a similar immunity.
The self-protection that natural producers of cytotoxic polypeptides have is
beginning to be unraveled, especially in the case of bacteriocins.
Interestingly, some cytotoxic polypeptides are reversibly inactivated by binding
to DNA. This might form the basis of an unconventional method of storing
cytotoxic polypeptides until they are needed.
♦ The Danger that Pathogens May Become Resistant to Human
Innate Immunity
Cytotoxic polypeptides are a very important component of human innate
immunity. If pathogenic bacteria become resistant to cytotoxic polypeptides
through cytotoxic polypeptide overuse or misuse, as has happened with
conventional antibiotics, we may lose much of our natural resistance to pathogenic
microbes.
Microbial resistance to cytotoxic polypeptides is still mostly a mystery. Some
experiments indicate that it emerges very quickly. However, the oral pathogen
Streptococcus mutans has not become resistant to human salivary cytotoxic
polypeptides despite long exposure to them.
It is critically important that we solve this problem before cytotoxic
polypeptides are used medically on a large scale. Otherwise, we may do far more
harm than good.
♦ Mechanisms of Pathogen Resistance to Cytotoxic Polypeptides
Proteases, pumps, and down-regulation. Microbes have multiple ways of
resisting cytotoxic polypeptides. They may secrete proteases to degrade the
cytotoxic polypeptides. Some bacteria have pumps to export cytotoxic
polypeptides that enter their cytoplasm. If they can enter the producer cell, bacteria
can down-regulate the genes that produce cytotoxic polypeptides.
Alterations to the exteriors of target pathogens. A major class of resistance
mechanisms consists of alterations to the cell exterior that passively reduce
cytotoxic polypeptide binding or lysis. Alterations to bacterial polysaccharide
capsules can inhibit cytotoxic polypeptide binding. Altering membrane fluidity can
reduce lysis. Reducing the negative charge on bacterial membranes also reduces
electrostatic binding by cytotoxic polypeptides, which are generally positively
charged.
Bacteria sometimes evolve to bind amino acids to their membrane components.
For example, they may bind lysyl moieties to anionic lipids to reduce their
negative charge. They may also evolve to bind alanyl moieties to teichoic acid in
their membranes. Both modifications reduce cytotoxic polypeptide binding.
The lipid composition of microbial cell membranes strongly influences their
response to cytotoxic polypeptides. Hence, not surprisingly, resistance sometimes
involves a change in membrane lipid compositions.
The cost to a pathogen of adaptation. Some bacterial adaptations to cytotoxic
polypeptides clearly come with a large cost in Darwinian fitness. One form of
resistance, for example, involves loss of bacterial cold shock proteins.
However, some adaptations seem to entail very modest costs. For example,
some bacteria have sensor systems to detect and respond to antimicrobial peptides.
Binding of cytotoxic polypeptides to Staphylococcus aureus induces a response
that involves transcription of multiple operons or regulons. C-terminal amidation
of the cytotoxic polypeptide is needed for this.
Resistance to individual peptides and to peptide classes. Although microbes
can easily become resistant to a given cytotoxic polypeptide, this in itself is not a
cause for great concern. There are a great many natural and pharmaceutical
cytotoxic polypeptides, and when resistance develops to one, a new cytotoxic
polypeptide could be substituted. The problem is that resistance to one cytotoxic
polypeptide may include resistance to many similar cytotoxic
polypeptides―resistance to whole classes of cytotoxic polypeptide may evolve.
The extent to which microbial resistance to one cytotoxic polypeptide confers
resistance to other cytotoxic polypeptides will have a large bearing on the
questions of how useful cytotoxic polypeptides will be in combatting microbes and
how dangerous resistant microbes will be to human innate immunity.
One clue is provided by patterns of cytotoxic polypeptide evolution. The skins
of frogs and toads generally produce multiple cytotoxic polypeptides from multiple
genes. The cytotoxic polypeptide-encoding parts of these genes are clearly under
strong diversifying selection. Clearly, multiple cytotoxic polypeptide types are
advantageous, and frequent minor changes in sequence are also advantageous.
These two facts argue that resistance to cytotoxic polypeptides is usually narrow,
and can be overcome by a sequence change.
However, this interpretation is clouded by the existence of multiple resistance
mechanisms. Do multiple small sequence changes in cytotoxic polypeptides
overcome protease degradation of the peptides, or passive changes to pathogen
envelopes? If small sequences changes are mostly useful in overcoming protease
digestion, might broader resistance based on envelope changes eventually develop?
Bacteriocins as a special case. Even if most cytotoxic polypeptides cannot be
used as drugs because of bacterial resistance and the possibility of subverting
human innate immunity, bacteriocins may offer hope. Bacteriocins have a very
narrow specificity, and may not induce general resistance or cross-resistance with
human innate immunity.
♦ Tumor Resistance to Cytotoxic Polypeptides
Cytotoxic polypeptides are promising agents to use against cancers. Although it
is too soon to tell, cancers may eventually develop resistance to cytotoxic
polypeptides as they do to other drugs. Heparan sulfate protects tumors from
cytotoxic polypeptides, probably by repelling them.
Increased cholesterol in cell membranes is sometimes protective. However, at
least one cytotoxic polypeptide was made more effective by the presence of
cholesterol in the target membrane.
This subject is discussed in greater length in Chapter 5.
♦ Cybernetic Discovery and Cataloguing of Cytotoxic
Polypeptides
Cytotoxic polypeptides are usually short polypeptides, are often produced
facultatively, and are often specific to certain tissues. As a result, they can easily
escape discovery.
One useful method to identify new cytotoxic polypeptides is to search for them
in genomic sequences. In one such study, 317 new cytotoxic polypeptide-like
genes were identified in the Arabidopsis genome.
The fact that cytotoxic polypeptides are usually short limits the confidence with
which they can be identified. Hence, searches for cytotoxic polypeptides within
genomic sequences are usually biased in favor of polypeptides resembling known
cytotoxic polypeptides.
It would be interesting to search the genomes of carrion eaters such as hyenas,
which can supposedly eat any animal matter no matter how putrid.
Several searchable online databases of cytotoxic polypeptides also exist. Some
are dedicated to a particular type of cytotoxic polypeptide, such as bacteriocins.
♦ Cytotoxic Polypeptides Regulate Adaptive Immunity
Some cytotoxic polypeptides, especially those that are host-derived, influence
adaptive immunity. They alter the properties of, or interact with receptors in,
mammalian membranes. By so doing, they influence cytokine release, antigen
presentation, chemotaxis, angiogenesis, wound healing and extracellular matrix
synthesis.
♦ Polypeptides That Bind Without Cytotoxicity
Phage display libraries are sometimes searched for new cytotoxic polypeptides.
These same searches often produce peptides that bind their targets without
damaging them.
Such non-toxic binding polypeptides might label cells non-destructively. They
might also protect healthy cells from cytotoxic polypeptides directed against an
infection or a cancer.
The Use of Extracellular Xenobiorgs Against Cancer (A
Preview of Chapter 5)
A clarification in terminology: two classes of xenobiorg. As mentioned
above, genetically engineered microbes used as medical agents (“xenobiorgs”)
could operate either outside of living cells (e.g. in saliva, in the intestinal lumen, in
the blood, in lymph, in bone marrow, in the extracellular space within many
organs) or within individual cells such as leukocytes, muscle fibers, pancreatic
cells, and neurons. I have termed these “extracellular” and “intracellular”
xenobiorgs, respectively.
Focus on extracellular xenobiorgs. This chapter considers the possible use of
xenobiorgs against cancer. It focuses on extracellular xenobiorgs, but mentions
intracellular xenobiorgs, because many considerations apply to both.
This chapter is in its earliest stages. When a complete first version is ready, it
will probably cover the subjects discussed below.
♦ Subjects left for later chapters
Some subjects related to the use of extracellular xenobiorgs against cancer are
instead left to later chapters.
Movement of xenobiorgs to the proper location. To be effective, xenobiorgs
must travel to the part of the body where they are intended to function. A
discussion of how this might be accomplished is left Chapter 11.
Evasion of the immune system. Xenobiorgs will have to evade the immune
system by some means that damages neither the immune system nor other tissues.
This very complex subject is also left to Chapter 19.
Evasion of intracellular detection. Individual human cells have methods to
detect microbial invaders. If a human cell does indeed detect an invading
xenobiorg, the cell may chemically petition for its own destruction by the immune
system. And even if this does not happen, the cell’s behavior will change
drastically in a way that will greatly reduce its medical usefulness.
Full exploitation of xenobiotherapy will require evasion of intracellular
detection. This need applies mainly to intracellular xenobiorgs, but might also
apply to extracellular xenobiorgs if—by design or by accident—material from the
xenobiorg is transferred into human cells. This issue is addressed in Chapter 7.
♦ Cancer masquerades as wounded tissue
The body has ways of destroying out-of-control cells, but it must also spare and
coddle wounded tissue. Cancer cells “convince” the rest of the body that they are
wounded tissue rather than out-of-control marauders. Cancer cells produce
multiple molecular signals that ward off attack by the immune system and that
facilitate their own nourishment and spread.
Cancer cells often secrete chemical signals that recruit “accomplice cells” to the
tumor. Accomplice cells are non-cancerous cells near the cancers that help the
cancers escape destruction, proliferate or spread. Some accomplice macrophages
prevent other macrophages from destroying the tumor, and also promote invasion
of the tumor by blood vessels (discussed at greater length below). Other
accomplice cells include tumor-associated fibroblasts.
♦ Cancer, the Warburg effect, and low pH
Cancer cells typically display the “Warburg effect.” They produce energy by a
high rate of glycolysis, followed by lactic acid fermentation in the cytosol, rather
than by aerobic respiration. Rapidly growing cancers have glycolysis rates up to
200 times higher than those of their normal tissues of origin—even if oxygen is
plentiful. In such tumors, lactate concentrations rise and the tumors become
acidified. (However, the acidification results from breakdown and nonregeneration of ATP, rather than from the lactate.)
In principle, either the excess lactate or the low pH of cancers could be used by
xenobiorgs to identify and destroy cancers. As discussed below { }, the low pH
has considerable potential for this.
Search for the following string, butwith the foreslash removed: One/feature of solid cancers …
♦ Cancer and hypoxia
Tumors, especially solid tumors, are highly hypoxic. This hypoxia increases
invasion of the tumor by blood vessels (angiogenesis), reduces adhesion of the
cancer cells to each other and to the extracellular matrix, and increases
invasiveness.
The proteins that promote these characteristics are probably secreted by tumors
within exosomes, small bodies with a diameter of between 30 and 100 nanometers.
In one set of experiments, conditioned medium from hypoxic tumor cells increased
angiogenesis by about 3-fold.
In those same experiments, reoxygenation only slightly restored the normoxic
phenotype. Reoxygenation actually increased invasiveness.
The reduced cohesion of cancer cells caused by hypoxia was was accompanied
by changes in several proteins involved in cohesion such as E-cadherin, α-catenin,
vinculin, and Snail.
Hypoxia increases the production of matrix metalloproteinase, which helps
tumor cells burrow through the extracellular matrix. Hypoxia decreases production
of Tissue Inhibitor of Matrix Metalloproteinase (TIMP), a process that further
activates matrix metalloproteinase.
TIMPs also stabilize complexes between E-cadherin and β-catenin. E-cadherin
is often lost at the invasive front of a tumor, and this loss represents an important
metastatic step.
Hypoxia causes at least some cancer cells to secrete more of the angiogenic
factors angiogenin, Vascular Endothelial Growth Factor, interleukin-1α,
interleukin-3, chemokine CXCL1, and Platelet-Derived Growth Factor-BB.
The hypoxia of cancer cells is a characteristic that might be exploited to destroy
them, and at least one cancer drug binds cancer cells based on this characteristic.
Xenobiorgs can also use the hypoxia of cancers to destroy the cancers while
preserving healthy tissue. Cases where this has already been done are discussed
below {Search for the following string, but with the foreslash removed: Cases/of
selective killing of tumors…}.
♦ Cancer and angiogenesis/vasculogenesis
Angiogenesis is the formation of new blood vessels from existing blood vessels.
Vasculogenesis is the formation of blood vessels, where none exist, from migrating
endothelial precursor cells. Both can provide tumors with blood vessels, and at
least one of the two—and perhaps both—is necessary for tumors to survive and
spread.
There is a clear correlation between angiogenesis and tumor progression—and
hence mortality.
Xenobiorgs might inhibit tumor growth and spread by inhibiting angiogenesis
and vasculogenesis. In addition, they might exploit either process to find, invade,
and attack tumors (see below).
♦ Cancer and the basement membrane
Cancers often interact abnormally with basement membranes, and invasion of
the basement membrane characterizes malignant, as opposed to benign, tumors.
The abnormal interactions between tumors and basement membranes might be
targeted by xenobiorgs. At least one potential target for therapy, tumor matrix
metalloproteinase, is discussed below.
♦ A potential screening method
It is now possible to mark cell lines with differently colored spontaneously
fluorescing proteins (green fluorescent protein and its derivatives from the jellyfish
Aequorea victoria, and DsRed from the mushroom coral Discosoma). Mixing and
plating normal and cancerous cells of different colors and then treating them with
candidate xenobiorgs (perhaps from an undefined, heterogeneous population)
could allow isolation of xenobiorgs that only attacked cancer cells.
♦ Homing to cancer
For xenobiorgs to be useful against cancer, some way of getting them to the
cancer must be found. This could be difficult if not all locations of a disseminated
cancer are known, or if the cancer is still undiscovered. However, several methods
are plausible.
Lymphocyte infiltration. In some cases, lymphocytes can recognize and
infiltrate tumors, even when the lymphocytes cannot kill the tumors. If this
mechanism of recognition were known, it might be exploited by extracellular
xenobiorgs.
Endothelial cell infiltration. In addition, tumors need a blood supply. Those
tumors that thrive are able to attract blood vessels to them. This may mean that
endothelial cells or endothelial cell precursors will migrate to them. If so, such
cells could be engineered to express endostatin or some other tumor inhibitor.
Alternatively, they might be loaded with bacteria or viruses that preferentially
replicate in tumors. Moreover, if the molecular machinery that guides homing of
endothelial cells to tumors could be established, it might be copied in extracellular
xenobiorgs.
Ratios of signaling molecules. As discussed below, tumors often make
abnormally much or abnormally little of signaling molecules that govern their
interaction with surrounding tissue. Typically the changes are not great—perhaps
only a factor of 3. However, the ratio between an overexpressed signaling
molecule and an underexpressed one might be as much as 10. If, for example, the 3
most overexpressed signaling molecules and the 3 most underexpressed signaling
molecules of a given cancer type were assayed by a xenobiorg, the xenobiorg
might reliably locate the tumor—and perhaps even discover an undiscovered
tumor.
A model for efficient hunting. A model for local tumor hunting by xenobiorgs
already exists in the human body. This is the milling around of B cells in germinal
centers of the immune system. This milling around allows the B cells to contact as
many antigen-presenting cells as possible. It could be a model for hunting of
cancer cells by xenobiorgs in some organ or space such as bone marrow.
Tracking of cells. Both xenobiorgs and cancer cells can be labeled with
fluorescent proteins and tracked. At least in experimental animals, this should
allow visualization and quantification of the success of xenobiorgs in finding
tumor cells.
Mimicry of tumor migration. Many cancer types migrate from their tissue of
origin preferentially to other organs. Breast cancer and multiple myeloma, for
example, are made much worse by the fact that they metastasize to bone marrow.
As another example, acute lymphoblastic leukemia invades liver, spleen, lymph
nodes and brain.
The metastatic behavior of specific cancers is largely explainable by known
facts about the signaling molecules that they express. For example, CXCR4 is
present on acute lymphoblastic leukemia (ALL) cells. These cells home to regions
that express stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4.
Xenobiorgs might locate metastasizing cancers by expressing the same homing
molecules on their surfaces.
The signaling molecules that control programmed cell movements during
development and recovery from injury are also known and could be exploited to
send xenobiorgs to desired tissues.
Medical marking of tumors. If the site of a tumor is known, it might be
possible to medically mark that site using either a surgically implanted chemical
beacon, genetically engineered host cells, or implanted xenobiorgs. The medical
mark would attract either immune system cells or xenobiorgs to destroy the tumor.
Many chemical beacons used by the human body, including SDF-1, are well
characterized.
♦ Tools to destroy or disable cancer cells
Killing of tumors and neutralization of tumor resistance to therapy. The
usual goal of cancer therapy is to kill or inactivate cancer cells while doing
minimal damage to healthy surrounding tissue. This can involve either of two
approaches. The first is to exploit some cancer-specific characteristic to selectively
injure the cancer cells. The second is to neutralize specialized resistances to
therapy that cancers often develop, so that the cancers will at least be no more
resistant to therapy than are normal cells.
The first approach suffers from two limitations. First, the cancer may have no
special characteristics that make it selectively vulnerable. Second, even if it has
such characteristics, it may lose them under selective pressure.
Activation of a prodrug. Xenobiorgs that infiltrated a tumor might transform a
harmless prodrug into a toxin too dangerous to administer systemically. If such a
toxin were too unstable or had too much affinity for solid tissues to diffuse far
from the site where it was created, it might be an effective anti-cancer medicine.
Testing cell interiors for cancerous transformation. Some cancers can be
distinguished by antigens that they carry on their surface. However, in many cases,
it will be necessary to sample the internal conditions within cells to determine
whether they are cancerous. This might be done in several ways.
Some bacteria can move between the cells of a solid tissue. These might sample
all of the cells of a solid tumor, even those not accessible via blood or lymph, as
well as healthy cells in the vicinity.
It might also be possible for xenobiorg to insert diagnostic proteins, mRNA
molecules, or transcribable genes into target cells.
Reverse-orientation receptors. In addition, it might be possible to temporarily
insert a protein receptor, such as an immunoglobulin receptor, into a target cell in
reverse orientation. Thus whereas a normal immunoglobulin receptor samples
proteins outside the cell and reports to the cell interior, these would sample
proteins inside the cell and report to a xenobiorg outside the cell.
Cancer-specific genetic markers. Internal conditions within cancer cells that
contribute either to the malignant phenotype or to therapy resistance might include
a high level of the protein “survivin”, high levels of carbonic anhydrase 9 (a
marker for tumor hypoxia), high levels of the human telomerase reverse
transcriptase hTERT, high levels of the transcription complex NF-κB, high levels
of transcription factors such as AP-1, high levels of the oncogene HER2, high
levels of the glycosyltransferase MGAT5 (which promotes metastasis), and high
levels of glutathione (in radiation-resistant cancers).
It might not be necessary to test for overexpressed proteins themselves. In many
cases, the same promoter that drives the overexpressed gene could be inserted into
cancer cells and used to drive expression of some anti-cancer protein or signal
protein.
Killing metastatic cancers using tissue-specific characteristics. Brain and
breast tissue differ in protein, mRNA, polysaccharide, and lipid composition.
Breast cancer often metastasizes to brain. If the metastatic breast cancer cells retain
characteristics of breast cells, they might be identified and killed within brain
based on those characteristics. Xenobiorgs might contribute to this.
The rationale behind neutralization of a cancerous phenotype. There is little
to be gained by sparing cancer cells from destruction. Hence, if cancer cells could
be identified in situ with certainty and killed effectively, with little collateral
damage, there would be no point in trying to reverse either their malignant
phenotype or their resistance to drugs and radiation. Instead, it would be better
simply to kill them.
However, there may be cases where the distinction between cancer cells and
healthy tissues is inefficient, or where the toxins used to kill cancer cells inevitably
spread to and damage surrounding healthy cells. In such cases, it might make sense
to subject all of the cells in an area to some treatment which left the normal cells
unchanged but reversed some abnormal and undesired characteristic of cancer
cells.
Several techniques might be used for this. One is RNA interference.
A second neutralization technique is to insert a protein which will downregulate some malignant property of cancer cells while leaving normal cells
unaffected. As an example, the nuclear transcription factor NF-κB promotes
angiogenesis and invasiveness in cancers, while inhibiting apoptosis (cell suicide)
in cancers. However, the human growth hormone gene inhibits NF-κB. It might be
inserted into all cells in an area, restoring normal NF-κB in the cancer cells.
A third neutralization technique might be to destroy tumorigenic or therapyresistance genes in both normal cells and cancers, in the hope that the normal cells
will not be adversely affected. Site-specific endonucleases can now be designed
that will cut chosen unique DNA sequences within a given genome. These
techniques may be extended to create molecules that will perform less drastic
alterations such as methylation, insertion of a short DNA sequence, or deletion of a
short DNA sequence—all having the effect of silencing a single gene.
One way to get therapeutic proteins into cells is to fuse the proteins to a protein
transduction/nuclear localization domain (e.g. the amino acid sequence
YGRKKRRQRRR) so that they can penetrate the cell membrane and enter the cell
nucleus. Alternatively, they might be injected by xenobiorgs, or a gene that makes
them might be injected.
Other signaling molecules have been suggested for treatment of cancer, based
on results with experimental tumors. These include the chemokines CCL16,
CCL17, CCL19, CCL21, CCL22, CCL27, and CXCL4.
♦ Destroying cancer by disruption of angiogenesis and
vasculogenesis
As discussed above, cancers require a generous blood supply that must be
delivered by newly formed blood vessels. Formation of new blood vessels can be
by angiogenesis (formation of new blood vessels by the sprouting of endothelium
from preexisting vessels) or vasculogenesis (formation of new blood vessels from
marrow-derived endothelial stem cells).
TRAIL. The protein TRAIL (tumor necrosis factor-related apoptosis-inducing
ligand) selectively induces apoptosis in a variety of transformed cells while sparing
normal cells. It seems to act, at least in part, by damaging tumor vasculature.
Endothelin. Under some circumstances the human peptide endothelin can
promote local blood vessel constriction and inhibit tumor growth. (However,
endothelin has mixed effects.)
Other angiostatic factors. Wikipedia { } lists another 29 natural human
http://en.wikipedia .org/wiki/Angiogenesis_inhibitor
factors that are angiostatic (i.e, they inhibit blood vessel formation by either
angiogenesis or vasculogenesis).
Extracellular xenobiorgs might release angiostatic factors near tumors and thus
disrupt or reduce the tumor blood supply. These inhibitors of blood vessel
formation are likely to be dangerous if used systemically—particularly in patients
at risk for ischemia.
Interference with angiogenic machinery. In addition, xenobiorgs might
interfere with angiogenic proteins in the tumor vicinity. Angiogenic factors include
Basic Fibroblast Growth Factor, Vascular Endothelial Growth Factor, CXCL8, and
the integrins αVβ3 and αVβ5.
One method of interference is to release single-chain artificial antibodies to a
given protein. A second method to interfere with signaling molecules to release
natural or artificial decoy proteins, i.e. mimics of signaling partners, but which are
inactive.
A complication: multiple pathways to stimulate blood vessel formation.
Although restricting the blood supply of tumors was once regarded as the magic
“silver bullet” against cancer, the technique has not yet fulfilled these expectations.
The main reason is that multiple factors can independently promote angiogenesis
or vasculogenesis and that if one is blocked, the tumor will eventually evolve to
produce others. For example, when Vascular Endothelial Growth Factor is
prevented from acting, Fibroblast Growth Factor, Platelet-Derived Growth FactorBB, Granulocyte-Macrophage Colony-Stimulating Factor, and/or angiopoietins
may emerge to continue stimulation of new blood vessels. This complication does
not necessarily invalidate the strategy, but it indicates that thoroughness will be
required.
♦ Use of cytotoxic polypeptides to disable cancer cells
Cytotoxic polypeptides are discussed mainly in Chapter 4, however their use
against cancers is mentioned in this chapter (Chapter 5).
Some cytotoxic polypeptides that kill microbes also kill cancer cells, but do not
harm normal tissue. In many cases, the reasons for this selectivity are not known.
Also, some reports do not distinguish between resistance caused by the cancerous
state and resistance that might be characteristic of the tissue that a cancer
originated from.
Sensitization by acid. One feature of solid cancers that may well contribute to
their increased sensitivity to some cytotoxic polypeptides is their acid nature.
Many of the cytotoxic polypeptides produced naturally in the human body—often
as breakdown products of larger proteins with other functions—become cytotoxic
only at low pH. This arrangement may itself be a defense against cancer (albeit an
expensive one, because it is probably increases the destructiveness of hypoxia that
has other causes). In any case, polypeptides that are cytotoxic only under acidic
conditions can reliably be engineered and might be useful against tumors if
delivered locally by xenobiorgs.
Binding to surface phosphatidylserine. The specificity that some cytotoxic
polypeptides have for cancer is not due to cancer’s acidity. Of these, at least some
kill cancer cells by binding to surface phosphatidylserine and depolarizing the cell
membrane. It has been suggested that cancer cells might evolve resistance to this
form of polypeptide cytotoxicity.
Lysis of mitochondrial membranes. Although most cytotoxic polypeptides
kill cancer cells by rupturing the outer membrane, some enter the cells and instead
rupture the mitochondrial membrane. This usually triggers apoptosis (cell suicide),
but is likely to be deadly in any event.
Killing by amylin. Because the pancreatic peptide amylin (aka IAPP) can be
very deadly to β cells of the pancreas, it has been suggested as a therapy for
pancreatic cancer.
Resistance due to heparin sulfate. Heparan sulfate on the surfaces of tumor
cells can inhibit the anti-cancer activity of some cytotoxic antimicrobial peptides,
probably by repelling them via negative electrostatic charges.
Cytotoxic polypeptides that are selectively toxic to cancer cells seem promising
for cancer therapy, particularly if they are delivered locally by xenobiorgs. Many
candidates exist, and multiple peptides could be used at once, to prevent evolved
resistance to any single one from undermining the therapy.
Resistant cancers, if they evolved, would pose no danger to the public health,
because cancer is not contagious.
One complication is that the xenobiorg itself should probably be invulnerable to
the cytotoxic polypeptide that it delivers.
♦ Selective killing of tumors by bacteria: known cases
Cases of selective killing of tumors by bacteria have already been reported.
Killing of cancers by Clostridium. Bacteria of the genus Clostridium are
obligate anaerobes that produce endospores. Clostridium spores selectively
germinate in solid tumors and are very oncolytic. However, their oncolysis is
almost always interrupted sharply at the outer rim of the viable tumor tissue where
the blood supply is sufficient.
Clostridium spores can be engineered to express anti-cancer proteins. An
obvious possibility is to engineer them to express anti-angiogenic proteins.
Killing of cancers by engineered Salmonella. The bacterium that has been
used most successfully against cancer in experimental animals is Salmonella
typhimurium. The strains are genetically altered to reduce their virulence. Strains
that have been made auxotrophic for (i.e. nutritionally dependent on) the amino
acids leucine and arginine grow well in cancer cells but hardly grow at all in
normal cells. Strains engineered to express interleukin-2 decrease angiogenesis
into the target tumors and increase tumor necrosis.
It has been argued that both the accumulation of the Salmonella typhimurium
within the tumor and the subsequent damage to the tumor vasculature depend on
the action of Tumor Necrosis Factor.
Salmonella leucine-arginine auxotrophs have been used to kill human prostate
cancer and human breast cancer orthotopically transplanted into mice. The
orthotopically transplanted tumors are very prone to metastasis. Nevertheless,
about 40% of the cancer-implanted mice remain free of cancer for the rest of their
normal-length lives.
♦ Tools to neutralize secreted or cell-surface cancer products
Although the best use of xenobiorgs against cancers would be to kill the
cancers, xenobiorgs might also tame cancers somewhat by interfering with the
products that cancers secrete.
Destruction of hydrogen peroxide. Xenobiorgs might remove H2O2 from the
vicinity of melanoma cells. H2O2 is produced by both melanoma cells and tissues
that they invade. The effect is mixed, since H2O2 kills many melanoma cells, but
the overall result seems to be an increase in metastasis. The enzyme catalase
destroys H2O2, and can reduce liver metastasis of melanoma cells in mice.
Xenobiorgs could easily be engineered to secrete catalase.
Single-chain antibody mimics. Most of the self-promoting chemicals that
cancers secrete are proteins. Xenobiorgs might neutralize these proteins by
cleaving them, by modifying them, or by sequestering them—but to do any of
these, the xenobiorgs must first recognize them. Xenobiorgs are likely to be
derived from bacteria, and hence to be bound by the limitations of bacteria.
Bacteria cannot be made to secrete structurally accurate antibodies, but can be
engineered to secrete at least some single-chain antibody mimics that bind chosen
proteins. The extent to which this is possible will determine much about the ability
of xenobiorgs to shut down intracellular and extracellular host machinery.
Decoy ligands and receptors. Xenobiorgs have one other opportunity to
neutralize signaling proteins that cancers secrete: the use of inactive (“decoy”)
ligands or receptors. It is often possible to create an inactive derivative of a ligand
or receptor that still binds its cognate protein. By competition, these reduce binding
of the authentic ligand or receptor to its cognate and thus act as inhibitors. Many
natural inhibitory proteins of this type have been described.
Secretion of antagonists. Finally, xenobiorgs might counteract, rather than
neutralize, cancer signaling molecules by secreting proteins with antagonistic
effects. As one example, the chemokine ligand CCL21 is angiostatic.
Targets of secreted antibody mimics. Monoclonal antibodies directed against
VCAM-1 can reduce the invasiveness of melanoma cells. Other plausible targets
include ICAM-1, mannose receptors, and VLA-4, metalloproteinases, interleukin1β, and many chemokine receptors.
In cases where the target protein is bound to cancer cells, it might be useful to
link a single-chain antibody mimic or a decoy protein to some toxin that will kill
those cells.
Inhibition of matrix metalloproteinases. Matrix metalloproteinases are zincdependent endopeptidases that can degrade most components of the basement
membrane and extracellular matrix. Malignant cells overproduce these
metalloproteinases and thus can move through the extracellular matrix. Membrane
type 1 metalloproteinase (MT1-MMP), is particularly important to tumor
metastasis.
Matrix metalloproteinases, especially MT1-MMP, are promising targets for
inhibition by xenobiorg-synthesized proteins.
Counteracting metastasis to bone. Metastasis to bone is an especially
dangerous complication of breast cancer. Once in the bone, the cancer cells
stimulate bone breakdown and sometimes also the synthesis of disorganized,
useless bone. Metastatic breast cancer cells do not destroy bone directly; instead
they stimulate osteoclasts to dissolve the bone. Dissolution of the bone matrix
releases growth factors that may further stimulate the invasive breast cancer cells
in a runaway reaction.
Invasive breast cancer cells produce at least 38 distinct growth factors or
growth factor receptors that directly or indirectly promote bone resorption. Any of
these might be neutralized by extracellular xenobiorgs present in the marrow. The
xenobiorgs might bind and destroy the active growth factors or receptors, or they
might secrete decoy growth factors or growth factor receptors.
Alternatively, extracellular xenobiorgs might be engineered to secrete factors
that oppose bone resorption.
Heparan sulfate as an anti-cancer target. Heparan sulfate is a linear
polysaccharide found in all animal tissues. Heparan sulfate in the extracellular
matrix may assemble chemokines for presentation to migrating cells (including
metastasizing cancer cells) and may protect chemokines from degradation. Binding
to heparan sulfate can be necessary for chemokine activity. Xenobiorgs might
modify heparan sulfate and associated molecules in ways that could inhibit cancer
metastasis.
The Use of Extracellular Xenobiorgs Against Alzheimer’s
Disease (A Preview of Chapter 6)
It may be possible to use extracellular xenobiorgs as agents against Alzheimer’s
disease. Some special means to move the xenobiorgs through the blood-brain
barrier and into the brain would presumably be necessary and, of course, some
means to evade the immune system would be necessary.
African trypanosomes of the subspecies Trypanosoma brucei gambiense can
cross the blood-brain barrier, and evade the human immune system by successive
antigen replacement. The bloodstream form can now be propagated axenically. T.
brucei gambiense can be genetically transfected, although it apparently cannot yet
be stably genetically engineered. If the damaging effects of T. brucei gambiense
could be lessened by genetic engineering, and if it could be engineered to express
chosen genes, it might become the basis of a brain-targeting xenobiorg.
The most promising activity for an anti-Alzheimer xenobiorg to perform would
be to remove the soluble β-amyloid monomers or oligomers that damage brain
tissue. However, since ions of zinc, copper, and iron may help precipitate βamyloid plaque, a xenobiorg might ameliorate Alzheimer’s disease by removing
these metal ions from the brain.
African trypanosomes and Alzheimer’s disease are both discussed at greater
length in Chapter 7.
The Medical Uses of Intracellular Xenobiorgs (A Summary
of Chapter 7)
Intracellular xenobiorgs are more important. Intracellular xenobiorgs are
more important to the basic purposes of this e-book than are extracellular
xenobiorgs. It is the intracellular xenobiorgs that could guide Repnumi
rejuvenation, the in situ replacement of aged nuclei and mitochondria by
appropriate youthful counterparts. It is the intracellular xenobiorgs that could stop
cancer before it starts, and shield leukocytes from HIV. Generally, it seems more
effective medically to influence the behavior of native human cells—as
intracellular xenobiorgs would do—than to add some new and alien cell type.
More difficult and perhaps more dangerous. Unfortunately, intracellular
xenobiorgs will be harder to use safely within the human body than extracellular
xenobiorgs will be. Human cells have elaborate and effective machinery to detect
microbial invaders, and will lose their medical usefulness if they in fact do detect
invaders. This machinery can probably be defeated—the most promising technique
would involve “dummy” receptors made by the invading xenobiorg—but it may or
may not be safe to do so.
The chance that an intracellular xenobiorg would spread to non-target cells in
the human host can probably be reduced to near-zero, and the chance that the
intracellular xenobiorg might evolve into a pathogen that spreads between people
can surely be eliminated. But what if an intracellular xenobiorg genetically donates
its evasion machinery to a genuine infectious pathogen? The result might be a
more dangerous infectious disease; however, it is still unclear whether this could
actually make a disease pathogen either more successful or more dangerous.
Chapter 7 discusses the many possible uses of intracellular xenobiorgs as well
as the technical difficulties that must be overcome.
♦ Medical tasks that intracellular xenobiorgs might perform
♦♦ Intracellular xenobiorgs could enhance the ability of leukocytes to
fight infections
Leukocytes (white blood cells) that fight bacterial (and other microbial)
infections often fail because the bacteria resist their killing methods, or kill the
leukocytes, or both. Nevertheless, the leukocytes are in general well-equipped to
kill microbes. Giving the leukocytes a small number of new abilities might greatly
increase their success.
Conversion of prodrug to antibiotic near an infection. One new ability that
could prove valuable is the ability to convert an inactive prodrug to an active
antibiotic, but only near an infection. Antibiotics that are too dangerous to
administer systemically might be converted either chemically or by desequestration to active form near their bacterial targets. Through the years, many
antimicrobial substances have been discovered, but never developed into drugs.
Use of leukocytes carrying intracellular xenobiorgs might give many of these old
drug candidates a second chance.
Prevent bacteria from neutralizing cytotoxic polypeptides. Another valuable
new ability might be the ability to prevent bacteria from neutralizing cytotoxic
polypeptides. (Cytotoxic polypeptides are discussed at length in Chapter 4.)
Leukocytes respond to a bacterial infection by producing antibacterial cytotoxic
polypeptides, which both kill bacteria and attract more leukocytes. However, many
bacteria degrade these cytotoxic polypeptides. There are multiple natural protease
inhibitors that intracellular xenobiorgs within leukocytes might produce to prevent
this degradation.
In addition, the intracellular xenobiorgs might produce new cytotoxic
polypeptides that resist degradation.
Production of harsher vacuole toxins. Leukocytes often kill bacteria by first
ingesting them into vacuoles and then flooding those vacuoles with toxins.
Intracellular xenobiorgs might produce new and more virulent toxins and
combinations of toxins to kill ingested bacteria, while at the same time secreting
agents that protect their host cell from those toxins.
Neutralization of false signals from bacteria. The human immune system
needs self-generated chemical restraints to prevent it from damaging its host’s
tissue. Pathogenic bacteria mimic these chemical restraints, and thus partly disable
the immune system. However, intracellular xenobiorgs might produce single-chain
antibodies that bind and neutralize those false signals and thus allow a full immune
response.
Removal of lymphocytes infected by HTLV-1. Human T-Lymphotrophic
Virus I (HTLV-I) causes adult T-cell leukemia and tropical spastic paraparesis,
both serious diseases. HTLV-I infects about 1% of the CD4+ T-lymphocytes of a
diseased person. It would be beneficial to destroy the infected lymphocytes.
HTLV-1 alters the surfaces of infected CD4+ T-lymphocytes to evade cells
(cytotoxic T-lymphocytes and “natural killer” cells) that would normally kill them.
However, natural killer cells might be engineered with intracellular xenobiorgs that
could restore their ability to recognize and destroy HTLV-I-infected CD4+ Tlymphocytes.
The same strategy might be extended to combat HIV-1 infections.
The concept is discussed in Chapter 7.
Engineered preservation or destruction of antibacterial DNA traps. At least
3 leukocyte types (neutrophils, mast cells, and eosinophils) form their own nuclear
or mitochondrial DNA into traps that combat invading microbes. The traps oppose
pathogens both by confining them to a hostile local environment and by preventing
their spread through the body. However, many pathogens take countermeasures
against traps, including using deoxyribonucleases to destroy the traps.
Disruption of traps destroys their antimicrobial activity. This might be due to
simple diffusion of the antimicrobial toxins in the traps, or might be a defense to
protect the rest of the body from those toxins if the trap ruptures.
Intracellular xenobiorgs might give trap-forming leukocytes the ability to
inhibit bacterial deoxyribonucleases, and thus preserve the traps. Many secreted
inhibitors can be imagined, including monomeric human actin, which inhibits
many deoxyribonucleases.
The same strategy could be used against the many virulence factors that
pathogenic bacteria secrete within traps.
Ironically, there is also one case (involving infections of the middle ear by
Haemophilus influenza) where existence of the traps appears to benefit the bacteria
rather than the host. In this case, leukocytes engineered to secrete
deoxyribonuclease at the site of infection might benefit the host.
Enhancement of defective neutrophils. Both cystic fibrosis patients and
newborn infants (especially premature infants) have defective neutrophil function,
which seriously weakens their immune systems.
Cystic fibrosis patients suffer from a seeming immunological paradox: their
lungs contain huge amounts of antimicrobial peptide and an abundance of
neutrophils. Both of these would be expected to kill invading bacteria, but neither
does. Indeed, they seem to disable each other.
In some parts of the world, as many as 25% of newborn infants suffer bacterial
sepsis. Neutrophils of neonates cannot perform many of the functions that
neutrophils in older people perform.
Intracellular xenobiorgs might improve neutrophil function in both cystic
fibrosis patients and in infants. Chapter 7 discusses measures that might be tried.
♦♦ Intracellular xenobiorgs could give white blood cells the ability to
detect and correct antigenic and biochemical changes deep within
the body
Another potential use of circulating white blood cells carrying intracellular
xenobiorgs is to probe abnormal conditions deep within the body. These
abnormalities could be either antigenic (i.e., consisting of proteins or
polysaccharides that can arouse an immune response) or biochemical. The
appearance of new antigens, changes in cell membrane lipid composition, growth
of regions of anaerobic metabolism, and development of ionic abnormalities may
all indicate conditions deserving medical scrutiny.
Many white blood cells percolate through solid organs, seeking foreign antigens
or other indicators of disease. Hence, white blood cells (leukocytes) carrying
intracellular xenobiorg passengers could interrogate most cells of a chosen target
tissue, if they had some molecular means to do so.
Early detection of pancreatic cancer. Pancreatic cancer is the fourth most
common cause of cancer death in both the United States and worldwide. By the
time it is diagnosed, it is nearly always not survivable. However, pancreatic cancer
is actually a slow-growing cancer that could be cured if caught only 6 months
before symptoms appear.
The antigen MUC5 is absent from all cells of the normal pancreas, but appears
on 79% of advanced pancreatic cancers. Pancreatic cancers with MUC5 are more
aggressive than those without MUC5. Cytotoxic T-lymphocytes can be created that
will kill cells expressing MUC5, and thus help destroy the cancer.
It is not certain that early-stage pancreatic cancers express MUC5; but if they
do, leukocyte/xenobiorg composites expressing an engineered receptor for MUC5
might filter through the pancreas and detect cases of early-stage pancreatic cancer.
They might then release a chemical signal into the blood that would prompt further
medical investigation (methods by which leukocyte/xenobiorgs might report
abnormal antigens are discussed below).
Early detection of cervical cancer. Cervical cancer is the third deadliest
cancer in women worldwide. There are about 500,000 new cases and 280,000
deaths from it each year.
The enzyme carbonic anhydrase IX is overexpressed in several cancer types,
including cervical cancer, but its expression in normal tissues is low. Its expression
correlates with advanced stage and poor prognosis in both cervical and other
cancers. It may be necessary for cancer survival and spread.
Unfortunately, cervical cancers do not shed carbonic anhydrase IX into the
blood. Hence, monitoring carbonic anhydrase IX in blood serum will not reveal
cervical cancer.
Leukocyte/xenobiorgs engineered to express an engineered receptor to carbonic
anhydrase IX might provide a method to detect cervical cancer. This possibility is
discussed in Chapter 7.
A slightly different method to detect cervical cancer is described 3 paragraphs
below.
Detection of antigen combinations. Unfortunately, antigens expressed by
cancers are often present on healthy cells in other locations within the body. Such
healthy cells may shed low levels of these antigens into the blood, masking any
signal from an early stage cancer. Moreover, most antigens partly degraded by the
time they leach into the blood, complicating efforts to detect them. Hence, blood
tests for cancer have fundamental limitations.
Circulating leukocyte/xenobiorgs might provide doctors with an ability that
blood tests do not. They could detect combinations of antigens that are not found
together in normal tissue, and whose presence together in a tissue suggests cancer
or some other pathology. In the following discussion, the term antigen signature
refers to a combination of antigens specific to a given tissue type or physiological
state (such as cancer).
A simple but useful antigen signature might consist of a tumor-carried antigen
combined with a tissue-specific antigen. Detection of the two antigens together
could tell doctors that a tumor was present and in what tissue. (Many antigens are
limited to tumors, but not to any one type of tumor.)
However, as discussed below, tumor signatures often involve more than two
antigens.
Unlike cancer antigens that eventually leach into the blood, cancer antigens still
attached to the cancer that produced them are likely not to be partly degraded. This
is likely to make them easier to detect.
Abnormal signatures of cervical cancers. Some human cervical cancers carry
two proteins that are not present together in normal tissues. Protein p16 is tumor
suppressor protein that inhibits cell proliferation, while protein Ki-67 accompanies
cell proliferation and may be necessary for cell proliferation. The two proteins
never occur together in normal tissue, but occur together in many cervical cancers
caused by human papilloma virus. Detection of these proteins together, perhaps
along with carbonic anhydrase IX (mentioned above), during a routine screening,
might alert doctors that further investigation is warranted. (Effective, inexpensive
screening for cervical cancer is especially important in poor nations, where
medical resources must be allocated very carefully.)
Abnormal signatures of glioblastomas. Glioblastomas are the deadliest form
of brain cancer. Glioblastomas spread in part by inducing angiogenesis, the
proliferation of existing blood vessels near the glioblastoma. However, the antigen
signatures of blood vessels induced to proliferate by glioblastomas differ from the
signatures of blood vessels induced to proliferate by normal processes. The
abnormal angiogenesis signature that is part of glioblastoma-induced angiogenesis
might be used in early detection of glioblastomas.
Antigen signatures of other cancers. Multi-antigen signatures for breast
cancer, malignant pleural mesothelioma, and renal cell carcinoma have been
reported. Antigen signatures can distinguish between the “clear cell” and
“papillary” variants of renal cell carcinoma. This is discussed in Chapter 7.
Detection of antigen signatures. To be useful, circulating leukocyte/
xenobiorgs would have to detect ominous antigens or combinations of antigens,
and report them somehow to doctors. Detection is discussed here, while reporting
is discussed below.
Natural sampling and engineered receptors. Some human cells can nibble
antigens from target cells, a kind of natural antigen sampling. Leukocyte/
xenobiorgs that could do this could carry target antigens into the blood, where they
could be harvested and analyzed. This possibility is discussed in Chap
Sampling of antigens using engineered receptors. It is also possible to
construct hybrid receptors whose extracellular domains, transmembrane domains,
and intracellular domains derive from different sources. These might be encoded
by intracellular xenobiorgs, inserted into the host leukocyte membrane and used to
detect cancer antigens on human cells.
In this scheme, the extracellular domains that recognized human cancer
antigens would probably be derived from mouse B cells selected for their ability to
react with the human cancer antigens. The transmembrane domain would transmit
the resulting signal to the intracellular domain inside the host leukocyte. This
would activate some enzyme that would make a secretable chemical signal.
Sampling of cells’ internal state using reverse-orientation receptors. In
addition to detecting surface antigens, leukocyte/xenobiorgs might be able to detect
antigens within target cells, as long as those antigens were near the cell membrane
of the target cell. They would do this be inserting a reverse-orientation receptor
into the target cell.
As described above, cell surface receptors normally consist of three parts: an
external domain joined to a transmembrane domain joined to an internal domain.
Normally, the external domain recognizes a ligand and changes conformation as a
result. The transmembrane domain also changes conformation, and by doing so,
transmits the binding signal through the membrane. The internal domain also
changes conformation, and by doing so reports to the cell interior that binding of
the ligand has occurred.
It might be possible to construct an artificial receptor in reverse orientation, so
that an internal domain detected a ligand within the cell and reported this binding
to the outer surface of the cell. The internal domain would very likely be derived
from a single-chain antibody constructed to bind some molecule whose presence
near the cell membrane yielded information about the cell state. Examples might
be viral antigens, proteins present near the cell surface only in cancers, and
proteins present only in specific cell types.
This reverse receptor would be inserted into a target cell membrane by a
leukocyte/xenobiorg. The leukocyte/xenobiorg would then monitor the external
domain for conformation change for some period, perhaps 5 minutes.
It would be impractical and destructive for a xenobiorg or leukocyte/xenobiorg
to insert reverse-orientation receptors into cells chosen at random from the body;
hence, the xenobiorg would have to have some “reason” to test a given cell. This
reason might be the presence of a surface antigen on that cell, the presence of the
cell in some region of the body such as a lymph node, the secretion by the cell of
some chemical, or something else.
Flexibility of antigen detection methods. The simplest scheme for using
leukocyte/xenobiorgs to detect abnormal antigen signatures would involve
simultaneous detection of multiple target antigens by a single leukocyte/xenobiorg.
The xenobiorg would have surface receptors able to detect all proteins of the
abnormal antigen signature; if all of the receptors bound targets simultaneously,
the leukocyte/xenobiorg would report binding (reporting methods are discussed
below).
However, this scheme might work poorly for two reasons. First, it might
involve putting more receptor types on the leukocyte/xenobiorg surface than the
surface could hold. Second, antigenic heterogeneity in the target cell type might
decrease the number of target cell regions where all antigens in the signature were
present—which would decrease the ability of the leukocyte/xenobiorg to detect the
target cell type.
To circumvent these difficulties, arrangements could be devised to report
antigens detected by the leukocyte/xenobiorg over a time interval of minutes to
hours. If the leukocyte/xenobiorg bound all of the antigens in the signature, it
would report this, even if binding took place at different times as the
leukocyte/xenobiorg moved through the tissue being examined.
In addition, leukocyte/xenobiorgs moving through a tissue might communicate
with each other via some diffusible chemical or even by fluorescence (at very short
range). Hence, antigens detected by different individual leukocyte/xenobiorgs
could be reported as a signature.
Reporting of abnormal conditions. To be useful, leukocyte/xenobiorgs that
detected abnormal antigen signatures would have to report them in a way that
doctors could detect. One possibility is that leukocyte/xenobiorgs carrying
substances detectable by medical imaging techniques might arrest and accumulate
at tissue sites having abnormal antigen signatures. Otherwise, leukocyte/
xenobiorgs would probably have to continue their journey through the solid tissue
of interest, enter the lymphatic system, and then enter the blood.
Leukocyte/xenobiorgs in the blood might report an abnormal signature by
releasing some chemical or macromolecule into the blood. Alternatively, they
might rearrange their own DNA in a way that could by detected by polymerase
chain reaction analysis of DNA from a blood sample. Either way, they would
probably have to replicate quite a few times in order to generate a detectable
signal.
Intracellular xenobiorgs against cancer. Much of the contents of Chapter 5
(which discusses the use of extracellular xenobiorgs to fight cancer) applies also to
intracellular xenobiorgs. Leukocyte/xenobiorgs might release DNA traps
(mentioned above) to prevent cancer from metastasizing. Furthermore, simple
changes in gene expression of leukocytes, which intracellular xenobiorgs might
direct, can greatly increase the effectiveness of leukocytes against cancer.
Alzheimer’s disease. Alzheimer’s disease is the most common form of
dementia. It is incurable and fatal. In 2006, there were some 26.6 million
Alzheimer’s sufferers worldwide and this number is expected to grow to 100
million by 2050.
The β-amyloid peptide, which is thought to kill neurons and cause the disease,
is created from Amyloid Precursor Protein. The β-amyloid peptide exists in several
variants, of which the variant consisting of 42 amino acids is the most dangerous.
Leukocyte/xenobiorgs might detect and report the β-amyloid peptide.
Leukocyte/xenobiorgs might also preemptively degrade the Amyloid Precursor
Protein so that the most dangerous β-amyloid peptide cannot be formed from it.
Alternatively, they might take up and sequester the dangerous peptide(s). As yet
another alternative, they might produce chelators of iron, copper, and zinc ions—
which would probably ameliorate the disease.
Chapter 6 discusses Alzheimer’s disease, how how xenobiorgs might combat it,
in greater detail.
Diseases with high polyamine levels. Biologically significant polyamines
include spermidine, spermine, putrescine, and cadaverine. Spermidine and
spermine are essential for human life. Putrescine and cadaverine are toxic in large
doses, but putrescine may be essential in small doses.
Polyamines can be detected by cell surface receptors.
Polyamines are elevated in cancers, and help cancers to spread. They are also
elevated in infections by the ulcer-causing bacterium Helicobacter pylori, and
contribute to tissue damage and progression to gastric cancer. Polyamines
exacerbate nerve damage in Parkinson’s disease and ischemic stroke.
Leukocyte/xenobiorg combinations might detect polyamines in various tissues
and warn of damage. In addition, they might ameliorate the harmful effects of
polyamines in cancer, H. pylori infection, Parkinson’s disease, and stroke by
sequestering or degrading polyamines or blocking polyamine synthesis.
Strokes also release arachidonic acid, which might be useful in guiding
leukocyte/xenobiorgs to the stroke area.
♦♦ Intracellular xenobiorgs could give blood cells the ability to remove
harmful chemicals from the blood.
Removal of harmful chemicals from the blood. Intracellular xenobiorgs
might be used to modify leukocytes or other blood cell types so that they remove
dangerous substances from the blood. There are many substances whose partial or
total removal from the blood would benefit a person’s health: these include glucose
in diabetics, low density lipoprotein in many people, trimethylamine-N-oxide in
people who consume large amounts of L-carnitine (found in red meat and often
taken as a supplement), and circulating viruses in people with viremia.
Removal of homocysteine. Homocysteine is a good candidate for removal by
intracellular xenobiorgs circulating within blood cells. Homocysteine is
biologically corrosive, and elevated blood concentrations of it probably cause
arteriosclerosis, heart disease, stroke, Alzheimer’s disease, and bone fractures in
the elderly. Many people, including heavy drinkers, have high levels of blood
homocysteine. Chapter 7 gives a simple calculation which suggests that adding a
modest number of leukocyte/xenobiorgs to a person’s blood could lower
homocysteine levels substantially.
♦♦ Intracellular xenobiorgs could preserve functions of hair and skin
cells
Preservation of hair color. Graying of hair is the most obvious sign of human
aging, and is usually unwelcome. Although youthful hair color can be restored by
dyeing, dyeing can be expensive and must be repeated frequently as hair grows, to
prevent gray roots from showing. Dyeing may also be a health hazard. A safe,
permanent method to prevent graying would be widely used.
Graying is caused by loss of melanocyte stem cells within hair follicles. The
stem cells either commit suicide (“apoptosis”) or differentiate and are not replaced.
Hair follicles and the melanocyte stem cells within them are accessible from the
skin’s surface. Xenobiorgs might enter the stem cells and prevent their loss, by
blocking both apoptosis and terminal differentiation.
Since mutated melanocyte stem cells produce malignant melanomas, it might be
dangerous to disable death mechanisms of melanocyte stem cells unless some very
secure method were devised to kill or inactivate any cells that became cancerous.
Because hair coloration is a cosmetic procedure, not necessary to human health,
society might decide that the risks of intracellular xenobiorg use to preserve hair
color outweigh the benefits. Chapter 7 lists surrogate systems that might be used to
estimate the dangers.
Stem cells of other types are lost by apoptosis and differentiation. Intracellular
xenobiorgs might preserve these as well.
Reversal of baldness. Baldness increases with aging, especially in men.
Although it is mainly a cosmetic problem, hair loss can also expose the head to
damage from the sun and to increased heat loss.
Forced expression of the protein β-catenin within ordinary epidermal cells can
probably induce them to form new hair follicles. Intracellular xenobiorgs might
induce ordinary scalp epidermal cells to form hair follicles, and also protect those
follicles from the influences that caused the baldness in the first place.
Stem cells within hair follicles can also differentiate into neurons, glia, smooth
muscle cells, adipocytes, and other cell types, and thus might be used to treat
degeneration of many tissue types. Use of intracellular xenobiorgs to induce hair
follicles may be studied for this reason also.
Tanning. Although tanning of the skin is often desired, especially by lightskinned people, tanning that is induced by ultraviolet light is damaging and
dangerous. Tanning occurs when specialized skin cells called melanocytes produce
melanin. However, production of melanin by other skin cells might also produce a
naturalistic and sun-protective tan. Intracellular xenobiorgs installed within skin
might produce melanin constitutively or in response to some harmless trigger.
Skin is relatively forgiving of mistakes. It may seem excessive to resort to
genetically engineered microbes to treat cosmetic problems such as graying,
baldness and paleness of the skin. However, skin is easily accessed and (compared
to brain or heart tissue, for example) forgiving of mistakes. If the uses described
here are safe and tolerated by society, doctors will gain valuable experience in the
use of intracellular xenobiorgs that could preserve more vital tissues such as brain
and heart (see below).
♦♦ Leukocyte/xenobiorgs might locate, chemically treat, avoid, stabilize,
or remove soft plaque
Diagnosis and treatment of atherosclerotic lesions. Atherosclerosis is the
leading cause of morbidity and death worldwide. Four of every 10 deaths result
from atherosclerosis. In the USA alone, atherosclerosis costs society $350 billion
annually. Clearly, society would benefit from ways to diagnose and treat
atherosclerosis.
Atherosclerosis involves biochemical changes to arterial walls that are at least
partly understood. When those changes are prevented, atherosclerosis progresses
much less. Atherosclerosis involves increases in arginases (particularly arginase
II), superoxide, other reactive oxygen species, caveolin I, asymmetric dimethyl-Larginine, L-NG-nitroarginine methyl ester, ornithine and urea, and decreases in Larginine, and nitric oxide.
Leukocyte/xenobiorgs might chemically announce, locate and mark
atherosclerotic patches either from the lumen of the artery or from outside the
artery. Access to atherosclerotic patches from outside the artery might be possible
for two reasons: first, arteries are often paralleled by lymphatic vessels, and
second, many leukocytes can move through solid tissue toward a target.
Leukocyte/xenobiorgs that lodged in or near atherosclerotic plaque might
remove chemical species that promote atherosclerosis and secrete chemical species
(arginine or nitric oxide) that retard it.
Generalizations about atherosclerotic plaque. Atherosclerotic plaque is an
accumulation of material on artery walls. It consists of macrophages, and debris
from macrophages, as well as cholesterol, fatty acids, calcium and varying
amounts of fibrous connective tissue. It is unhealthy, but is present in most
humans, including even young children. The accumulation of material is always
between the endothelial (inner) lining of the artery and the smooth muscle wall of
the arterial tube.
Macrophage involvement. Macrophages that have ingested oxidized low
density lipoprotein form the earliest stages of atherosclerotic plaque. Advanced
atherosclerotic plaque contains many “foam cells”, macrophages that have
accumulated large amounts of lipids. Atherosclerotic plaque is very hard to
monitor because coronary arteries are narrow and continually moving, and because
arteries often widen in response to the plaque buildup—although this does not
prevent sudden rupture of the plaque and infarction. If an artery widens too much,
it may burst—and cause sudden death.
Uses of intracellular xenobiorgs. Intracellular xenobiorgs, present within
macrophages, might combat atherosclerosis in several ways.
First, as passengers within macrophages, they might label dangerous regions of
atherosclerotic plaque. Chapter 7 suggests several ways in which they might do
this.
Second, xenobiorgs within macrophages might alter those macrophages so that
they no longer joined and contributed to plaque. Chapter 7 suggests several
possible methods.
Third, macrophage/xenobiorgs might invade regions of soft plaque and stabilize
it. They might seal the plaque to prevent entry of additional macrophages, and
might alter the plaque so that blood clots could not form over it.
Fourth, macrophage/xenobiorgs could enter the plaque and slowly digest or
remove it. Since this might destabilize the plaque, some method would have to be
found to prevent destabilization.
♦♦ Intracellular xenobiorgs could defend cells from pathogens
General Principles. Xenobiorgs residing within human cells might protect
those cells from invasion by pathogens. Ordinarily, this would be a disfavored
method of combatting disease, because other method such as vaccines, antibiotics,
and good hygiene would give a better return on effort. However, there may be
some exceptions.
Good candidate diseases include (a) those which infect a limited and welldefined set of tissues or, (b) those which infect a bottleneck tissue before they
infect other tissues, or (d) those that must be carried through the body by blood
cells. An obvious candidate is the human immunodeficiency virus HIV, which is
spread through the body by a specific set of lymphocytes.
Intracellular xenobiorgs and HIV. Although Human Immunodeficiency
Virus-1 damages many tissues, it is thought to infect only immune system cells
(CD4+ helper T cells, macrophages, and dendritic cells). If a patient’s immune
system is rendered unable to support proliferating HIV, the patient will be cured of
AIDS—however, this is only known to have happened once, through a medical
fluke.
Intracellular xenobiorgs might be implanted into the bone marrow cells of
AIDS patients. The procedure would probably require ablation of the patient’s
immune system, followed by autologous transplantation of hematopoietic stem
cells harboring intracellular xenobiorgs. One important type of cell that supports
HIV replication, CD4+ T-lymphocytes, is killed by HIV by 3 distinct mechanisms.
Intracellular xenobiorgs would have to be designed that could block all 3.
Although autologous bone marrow transplantation might be feasible in rich
countries, it would be an impractical treatment in poor countries, where most AIDS
infections occur. However, if most HIV infections begin by invading a limited area
of tissue, intracellular xenobiorgs might be used to defend those tissue areas. The
xenobiorgs (probably derived from bacteria) might be applied as a lotion or by
injection into the tissue to be protected. The foreskin (and perhaps the rest of the
penile epidermis) of uncircumcised men is one plausible target. Areas of the
cervixes and vaginas of women are another plausible target. In addition, the lymph
channels and nodes that drain the genital areas of men and women are conduits of
HIV spread throughout the body—and intracellular xenobiorgs stationed in them
might block HIV spread. Chapter 7 discusses the molecular biology of this.
Intracellular xenobiorgs and human papillomavirus. Human papillomavirus
is more dangerous to women (in whom it causes cervical cancer) than to men (in
whom it may cause penile cancer). Human papillomavirus is even more dangerous
when present along with HIV.
Although circumcision of men is an excellent option for reducing the spread of
human papillomavirus, intracellular xenobiorgs in penile tissue might provide the
same protections against papillomavirus as against HIV. Intracellular xenobiorgs
might protect other parts of the penis, and protect sexually active men who refuse
circumcision.
There are excellent vaccines against human papillomavirus that can protect
women from infection. However intracellular xenobiorgs might protect against a
wider range of papillomaviruses and might protect women after they have been
infected.
Emergency protection against influenza and other respiratory viruses.
Intracellular xenobiorgs might also protect cells against virus infection on an
emergency basis, if the tissues to be protected are easily accessible. For example,
many type H5N1 influenza virus strains originate in chickens but infect humans.
Fatality rates are over 60%, and direct human-to-human transmission is feared,
raising the possibility of a pandemic that could kill tens of millions of people. In
addition, there are occasional outbreaks of other respiratory viruses such as
coronaviruses, including Severe Acute Respiratory Syndrome (SARS)—for which
there is still neither a vaccine nor a cure.
Intracellular xenobiorgs might replace or supplement antiviral drugs, and
perhaps reduce the number of lung cells infected. Protection might take several
forms. Intracellular xenobiorgs might prevent the virus from entering their host
cells, prevent the virus from replicating in their host cells, or prevent virus
escape―perhaps by killing their host cells.
Presumably, the xenobiorg endosymbionts would be delivered to the lungs by a
spray.
Antiviral compounds made by intracellular xenobiorgs. Intracellular
xenobiorgs could not synthesize the wide variety of exotic compounds that the
pharmaceutical industry can; this is a disadvantage. However, they might convert
some prodrugs to active form near sites of potential infection.
In addition intracellular xenobiorgs could make and use single-chain antibodies,
aptamers, retrocyclins, other antimicrobial peptides, lectins, proteases, cell surface
receptors, specialized lipids, and oxidizing agents. These are discussed in Chapter
7.
♦♦ Intracellular xenobiorgs might prevent cells from becoming
cancerous
Chapter 5 and other places in this e-book discuss using xenobiorgs to kill
existing cancers. However, it would be far better to prevent cancers from
developing in the first place.
Intracellular xenobiorgs might protect people against cancer if they could be
stationed within cells that are likely to become cancerous. They could detect
expression of genes associated with cancerous transformation and, if necessary,
inactivate or destroy their host cell.
Breast and prostate cancer. The human female breast and the human male
prostate are glands that produce feared cancers. Within each organ, a subset of
cells produce most of the cancers. These cells might be occupied by anti-cancer
intracellular xenobiorgs. Chapter 7 discusses these in detail. People known to be at
elevated risk, particularly in the case of breast cancer, would likely be the first
patients.
Other cancers. Many other cancers, including ovarian cancer, lung cancer, and
pancreatic cancer might conceivably be prevented by pre-seeding of tissues with
intracellular xenobiorgs. Again, people at high risk for these cancers would likely
be the first patients.
Pigmented nevi and malignant melanoma. In addition, because skin is easily
accessible and relatively forgiving of mistakes—and because people care greatly
about their appearance—an early use of intracellular xenobiorgs may be to remove
pigmented nevi (moles). Furthermore, malignant melanoma may be one of the first
cancers to be prevented by intracellular xenobiorgs.
Teratomas derived from pluripotent stem cells. Pluripotent stem cells may
become very useful in reconstructing damaged or aged tissues. However,
pluripotent stem cells carry with them the risk of teratoma formation. Intracellular
xenobiorgs within transplanted pluripotent stem cells might reduce this risk by
killing cells that became transformed.
♦♦ Intracellular xenobiorgs might kill or inactivate other types of
delinquent cell.
“Delinquent” cells defined. Cancer cells are derived from normal cells, but no
longer contribute to the health of their parent organism; instead, they damage that
health. Foam cells (mentioned above) are macrophages that contribute to
atherosclerotic plaque, rather than performing the functions of healthy
macrophages. Cells with mutated mitochondrial genomes oxidize low density
lipoprotein and poison the body with superoxide anions. Cancer cells, foam cells
and superoxide anion generators are examples of cells that have become liabilities
to their parent organism, and which the parent organism would be better off
without. I refer to such cells as “delinquent” cells.
Many types of delinquent cells can be imagined. As one example, cells might
become unusually active sources of virus particles. As a second example, cells
might inappropriately express digestive enzymes in vital organs such as the heart
or brain. As a third example, cells might express inappropriate signaling molecules
that disrupt function of the surrounding tissue.
Diseases caused by protein expansion or contraction. Chapter 7 focuses on
delinquent cells that have undergone genetic changes that do not lead to cancer
(cancer is discussed above), but which harm their human carrier. These include
Huntington’s Disease and 8 other “polyglutamine” neurological diseases caused by
expansion of intragenic triplet tandem arrays. They also include “polyalanine
diseases”, abnormally short tandem arrays within the apolipoprotein(a) gene, and
diseases caused by changes lengths of non-coding tandem arrays,.
Other genetic changes. Other changes of interest, which may or may not be
deleterious, occur in both germ line and somatic cells. These include changes in
expression and transposition of LINE-1 elements, changes in unstable genetic
palindromes, aneuploidy, “copy number variation” of chromosome segments, and
insertion of processed pseudogenes into the genome.
It seems likely that as more is learned about the aging process, more types of
delinquent cells will be discovered.
Therapy with intracellular xenobiorgs. Intracellular xenobiorgs might protect
neurons from expansion of proteins such as huntingtin (the altered protein in
Huntington’s Disease) by suppressing the synthesis of host protein MSH3, a
protein necessary for array expansion. Alternatively, they might selectively
inactivate the abnormal gene altogether and supply a replacement themselves. (Use
of intracellular xenobiorgs to create human proteins will be tricky, because human
proteins are often modified in ways that bacteria could not duplicate.)
Intracellular xenobiorgs installed within germ line cells might kill or inactivate
germ cells with clearly abnormal and detrimental genetic changes.
♦♦ Intracellular xenobiorgs might kill the mosquitos that transmit
malaria.
Malaria is spread to human hosts by female mosquitos of the Anopheles genus.
Both sexes of Anopheles feed on nectar. However, because nectar’s protein content
alone cannot support oogenesis, the female must take one or more blood meals
from an animal (human in this case) source. This blood meal inoculates a human
with malaria.
Hosting of malaria imposes a burden on female mosquitos, as well as on
humans. Intracellular xenobiorgs within human blood cells might be engineered to
increase that burden, perhaps to the point of killing the female mosquito.
If some means were used to increase the protein content of the nectar meals that
Anopheles mosquitos take so that the meals could support oogenesis, the behavior
of female mosquitos might change due to selective pressure. In a large population,
some of them would presumably skip the step of biting a human host; if these
succeeded in reproducing, the tendency to skip biting a human host might increase
within the mosquito population. This would be expected to happen if either (a)
mosquitos in the same population interbreed extensively, or (b) competition
between mosquitos is severe, so that only the most successful propagate. The
tendency to skip the blood meals from humans might accelerate if human blood
cells contained xenobiorgs that killed mosquitos.
Malaria parasites cannot survive or propagate in plants, and it is very unlikely
that they could evolve to do so. A mosquito population in which both sexes took
all of their meals from plants would also be a population purged of malaria. Hence,
if certain conditions were met, mosquito populations might become both nonbiting and malaria-free.
On the other hand, use of high-protein plant nectar to purge mosquito
populations of malaria might well backfire. It might turn out that the extra protein
simply increased the number of mosquitos without curbing the females’ appetite
for human blood.
An experimental attempt should be made to purge a mosquito population of
malaria parasites by increasing the protein content of plant nectar and “poisoning”
the blood of an animal host. The experiment should involve animal rather than
human hosts and should involve some means of increasing the protein content of
plant nectar that is not heritable. It should probably take place on some malarial
Pacific island where there are no humans. If the experiment succeeded, the control
technique might be tried among human populations.
♦♦ Intracellular xenobiorgs might protect against inappropriate
apoptosis.
Apoptosis compared with necrosis. The cells of a person’s body can die in
two distinct ways. The first is necrosis, which resembles normal, uncontrolled
death, i.e. loss of homeostasis and subsequent degeneration. The second is
apoptosis, a tightly controlled form of cell suicide. Some cell deaths seem to be a
mixture of the two.
Purposes and adverse consequences of apoptosis. Apoptosis is an ancient
process, and is vital to normal development, defense against cancer, and defense
against infectious disease. However, apoptosis that occurs at the wrong time
contributes enormously to human morbidity and mortality. Most autoimmune
diseases, almost all neurodegenerative diseases, almost all diseases involving
ischemia (insufficient blood flow to an organ), and many other diseases are made
much worse by the fact that they trigger apoptosis. Almost any disease or injury
that takes longer than 5 minutes to kill a person is exacerbated by apoptosis.
Apoptosis is a complex process and is under very complex regulation.
Intracellular xenobiorgs would have several ways of preventing it. In addition,
intracellular xenobiorgs could serve as research tools to help dissect apoptosis,
which is still incompletely understood. Chapter 7 discusses these issues.
Apoptosis and intracellular xenobiorgs in various diseases. Intracellular
xenobiorgs might ameliorate a number of injurious processes that are made worse
by apoptosis. One of these is ischemia-reperfusion injury which affects the heart,
brain, kidneys, liver, and intestines—and which constantly hinders organ
transplantation. Skin might also be protected from scleroderma, cutaneous lupus,
and toxic necrolysis by intracellular xenobiorgs.
Intracellular xenobiorgs might protect the gastrointestinal tract from apoptosis
that occurs in Crohn’s disease, ulcerative colitis, necrotizing enterocolitis, and
infection with Helicobacter pylori. Intracellular xenobiorgs might protect lung
tissue from apoptosis that occurs in cystic fibrosis—or might replace the defective
Cystic Fibrosis Transmembrane Conductance Regulator proteins that cause cystic
fibrosis. Intracellular xenobiorgs might prevent the apoptosis that occurs in alveoli
in chronic obstructive pulmonary disease, but might also ameliorate chronic
obstructive pulmonary disease by secreting antibacterial peptides, by defending
lung cells from viral invasion, and by restraining the growth of airway tissues—
including (perhaps) by inducing some airway cells to apoptose.
Intracellular xenobiorgs might prevent or ameliorate diabetes by protecting the
β cells of the pancreas from glucose-induced apoptosis. The very complex
molecular etiology of diabetes and possible interventions by intracellular
xenobiorgs are discussed at length in Chapter 7.
Protection of healthy cells during cancer treatment. Intracellular xenobiorgs
might also protect healthy cells against treatments used in cancer chemotherapy.
These treatments including radiation, chemotherapy, and heat—with anticancer
viruses being developed. All of these treatments, including anticancer viruses,
either cause or might cause, apoptosis in affected cells. Although intracellular
xenobiorgs could not protect cells from the DNA damage caused by radiation and
chemotherapy it might ameliorate some harmful sequelae.
In the case of radiation therapy of cancer, the most vulnerable healthy tissues
are probably those near the site of irradiation. In the case of chemotherapy of
cancer, the most vulnerable cells are probably those that divide rapidly—such as
marrow cells, intestinal cells and cells in hair follicles. These, and other vulnerable
areas such as fingernails and toenails, might be protected by intracellular
xenobiorgs, even though whole-body protection is probably impractical.
Neural diseases and injuries, apoptosis, and xenobiorgs. Inappropriate
apoptosis contributes importantly to Alzheimer’s disease. Intracellular xenobiorgs
might ameliorate Alzheimer’s disease by preventing this apoptosis, and also by
interfering with α-1-antichymotrypsin and other proteins that contribute to
amyloid plaque polymerization and neuron inflammation.
Ischemic stroke, amyotrophic lateral sclerosis (Lou Gehrig’s disease),
Huntington’s disease (mentioned above), Parkinson’s disease, and spinal cord
injury, are also worsened by apoptosis. Chapter 7 discusses ways in which
intracellular xenobiorgs might protect tissues in these conditions.
Protection of kidneys by intracellular xenobiorgs. Kidneys can be injured by
obstruction of a ureter. Such obstruction can be caused by a kidney stone, by a
tumor, by pregnancy, and by blood clots. This type of injury is worsened by
apoptosis.
Kidneys can also be injured by contrast agents used in medical imaging and by
cyclosporine, an immunosuppressant drug used to prevent rejection of transplanted
organs. Intracellular xenobiorgs might lessen the damage of both processes.
Medical imaging presents a scheduled and temporary challenge to the kidneys,
which might allow intracellular xenobiorgs to be temporarily inserted into sensitive
tissue.
Limiting the damage due to osteoarthritis and rheumatoid arthritis.
Arthritis is a painful and debilitating disease that exists in two distinct forms:
osteoarthritis and rheumatoid arthritis. Both feature dedifferentiation of and
apoptosis of chondrocytes, the cells that maintain cartilage.
Cartilage is both maintained and destroyed by chondrocytes. A key goal of
osteoarthritis research is to find ways both to preserve chondrocytes and to shift
their activity toward building of cartilage.
Rheumatoid arthritis is a chronic inflammatory disorder that affects both joints
and other organs. It often causes joints to fuse.
In rheumatoid arthritis, damaged joints may become depleted of functioning
chondrocytes. Researchers hope to inject either transplanted chondrocytes or
transplanted chondrocyte precursors from other regions of a patient’s body into
damaged joints in order to rebuild the joints. It may be necessary to expand the
cells in culture first.
So far, attempts to reconstitute functioning joints this way have failed, mainly
due to a lack of knowledge of the fate of transplanted chondrocytes or chondrocyte
precursors. Intracellular xenobiorgs might help track the fate of transplanted
chondrocytes or precursors, and might eventually prevent apoptosis or other
processes from depleting the population of active, useful chondrocytes.
Chapter 7 discusses the molecular biology of preserving and directing
chondrocytes.
Protection of bones from osteoporosis. Osteoporosis is skeletal fragility
caused by insufficient deposition of calcium phosphate and other minerals. It is the
most frequent skeletal disorder and is most common among post-menopausal
women.
The main cause of osteoporosis is accelerated bone resorption by osteoclasts.
However, inappropriate apoptosis of osteoblasts and osteocytes also contributes.
Preventing this apoptosis or replacing the lost osteoblasts and osteocytes might be
useful in treating osteoporosis. Mesenchymal stem cells can differentiate into
osteoblasts, and are present at several location in the body, including adipose (fat)
tissue.
Chapter 7 discusses the potential benefits and likely difficulties of using
intracellular xenobiorgs either to preserve osteoblasts and osteocytes or to promote
their replacement in situ using mesenchymal stem cells.
Protection of liver cells. Apoptosis of liver cells helps protect the liver from
viral infections and liver cancer. However, apoptosis of liver cells contributes
greatly to damage from various liver disorders or stresses. These include alcoholic
fatty liver disease, non-alcoholic fatty liver disease, cholestasis (the obstruction of
bile flow), acetaminophen overdose, hepatitis B and C, and liver transplantation.
Intracellular xenobiorgs could protect liver cells from apoptosis in all of these
conditions.
The human liver has two special properties that might make it a better candidate
to host intracellular xenobiorgs. The first is that it has great powers of regeneration.
This might be exploited to spread intracellular xenobiorgs through a patient’s liver,
since if an intracellular xenobiorg’s host cell divides, the intracellular xenobiorg
presumably can also proliferate and persist in both descendant cells.
The second special feature of liver is that humans generally have much more
liver function than they really need. Patients can lose 60% to 75% of their liver
function before showing symptoms. Thus, use of intracellular xenobiorgs to protect
just a portion of the liver might have great health benefits.
Protection of heart tissue from apoptosis. Unlike liver tissue, heart tissue
cannot regenerate. Hence, protection of heart tissue is vital to human health. As
mentioned above, heart tissue is susceptible to ischemia-reperfusion injury, where
much of the damage is done by reperfusion and involves completion of apoptosis.
However, apoptosis also contributes to dilated cardiomyopathy. Chapter 7
discusses how intracellular xenobiorgs might protect heart cells from apoptosis.
Protection of skeletal muscles. Oculopharyngeal muscular dystrophy is a lateonset triplet expansion disease that causes the nuclear protein PABPN1 to form
abnormal tubules. It is invariably fatal, and exerts much of its effect by inducing
apoptosis in skeletal muscles.
Intracellular xenobiorgs might prevent this apoptosis or—even better—silence
the disordered gene. Most patients have a second, normal copy of the gene.
Moreover, intracellular xenobiorgs could themselves supply the normal protein
PABPN1.
Even if only a minority of muscles, such as those involved in swallowing, could
be treated, patients might benefit. Swallowing of food into the lungs, and
consequent aspirational pneumonia, is a major cause of death in oculopharyngeal
muscular dystrophy patients.
Congenital muscular dystrophy type 1A is a second disease of skeletal muscle
that involves apoptosis. Intracellular xenobiorgs might supply the missing protein
in patients with congenital muscular dystrophy type 1A, might silence a defective
allele if necessary, and might also prevent apoptosis in vulnerable muscles or
motor neurons if necessary.
Ullrich congenital muscular dystrophy and Bethlem myopathy are skeletal
muscle diseases caused by mutations in the genes encoding collagen VI. Myoblasts
from Ullrich patients are predisposed to apoptosis, and drugs that suppress
apoptosis benefit them.
Xenobiorgs within muscle cells might suppress apoptosis. They might also
silence defective collagen VI genes, and replace the defective product with normal
collagen VI.
Prevention of eclampsia and pre-eclampsia. Eclampsia and pre-eclampsia are
serious complications of pregnancy. Pre-eclampsia may progress to eclampsia.
Pre-eclampsia is thought to result from apoptosis of the extravillous
trophoblasts that grow out from the placenta and penetrate into the decidualised
uterus, ensuring the fetus of an adequate blood supply. Intracellular xenobiorgs
might protect these trophoblasts from apoptosis; Chapter 7 discusses the
possibilities.
Treatment of arterial pulmonary hypertension. The use of drugs to curb
harmful apoptosis has a serious drawback. Drugs are likely to inhibit apoptosis
throughout the body, and apoptosis is sometimes necessary for good health. Cancer
is a famous example of a disease made worse by failure of apoptosis to occur
properly. Arterial pulmonary hypertension is another example (see below). Both
diseases might be exacerbated by anti-apoptotic drugs. In contrast, intracellular
xenobiorgs might restrict the inhibition of apoptosis to chosen tissues.
Intracellular xenobiorgs might also promote apoptosis in tissues where it is
required, one example being pulmonary arterial hypertension. Pulmonary arterial
hypertension is a disease of the pulmonary vasculature. It involves an increased
pulmonary vascular resistance which eventually causes right heart failure and
premature death. The median survival time from diagnosis is only 2.8 years.
Pulmonary arterial hypertension begins with injury to the lung vasculature,
usually by any of several viruses, followed by overgrowth of the endothelial layers
and resistance of those layers to apoptosis. However, in early stages of the disease,
there may be too much apoptosis, rather than too little.
Access to the lung vasculature is easy, and intracellular xenobiorgs could be
delivered by nebulized spray to them. Either naked bacteria or bacteria within
leukocytes might be administered. The advantage of the latter might be that many
leukocytes can pass through endothelial cells via a process called transcellular
diapedesis (see the Chapter on Homing). It might be easy for the leukocytes to
disgorge their intracellular xenobiorg passengers into the endothelial cells they
were passing through.
Intracellular xenobiorgs might have an advantage over other therapies in that
they could sense the condition of their host cell and respond accordingly. This
might be important if the vasculature simultaneously included cells too prone to
apoptosis along with others that were too resistant.
♦♦ Intracellular xenobiorgs and the engineering of adipose tissue
Adipose tissue, also called body fat, is unique. It is abundant, largely
dispensable, and much of it is easily accessible. Because it exists in many areas of
the body, it might be possible to engineer regions of it to secrete chemicals that
influence only one organ, such as the heart. Also, most people have too much
adipose tissue, and would be healthier with less of it.
Removal of adipose tissue by intracellular xenobiorgs. Intracellular
xenobiorgs within adipocytes (the cells of adipose tissue) might alter their hosts to
take up fewer fatty acids, to efflux more of them, and to proliferate less frequently
than they do. Intracellular xenobiorgs might dispose of excess fatty acids by
promoting non-shivering thermogenesis in white adipose tissue, by degrading the
fatty acids directly, or by absorbing them and carrying them to the intestinal lumen
where they would be excreted. They might also be programmed to kill induce
apoptosis in their host adipocytes, presumably after depleting their lipid stores.
Chapter 7 discusses the molecular biology of these ideas.
Removal of blood glucose. Adipose tissue is the major site for conversion of
carbohydrate to fat in mammals. Glucose is transported into adipocytes and
converted to fatty acids. These are then incorporated into triacylglycerides as
discussed above, and stored in the lipid droplet.
Given the unhealthiness of high blood sugar, there would seem to be little value
in blocking the uptake of sugar. However, intracellular xenobiorgs might be
programmed to accelerate the removal of glucose from the blood, and thus help
control diabetes. Since this would cause fat to build up, it would likely only be
advantageous if intracellular xenobiorgs also supplied some way to remove the fat.
Removal of unwanted depleted adipocytes. In formerly obese people who
have undergone gastric bypass surgery, lipid-depleted adipocytes often form an
“apron of skin.” This is undesirable, and might be improved if intracellular
xenobiorgs could enter and kill the adipocytes.
Breast augmentation. Breast augmentation sometimes involves transplantation
of adipose tissue. However, fat-graft augmentation of the breast is usually limited
to one brassière cup-size, or less, and complications are common. Intracellular
xenobiorgs might also swell adipocytes in breast tissue, and encourage the
proliferation of adipocytes and associated cells. This could allow non-surgical
breast augmentation with entirely natural materials.
Marking of stem cells. Adipose tissue includes cell types other than
adipocytes. One such cell type, adult stem cells, can be reprogrammed into
pluripotent stem cells, without the need for feeder cells. If intracellular xenobiorgs
were to spread through adipose tissue, they might mark the stem cells, making
them easier to isolate.
Intracellular xenobiorgs might mark all of the different cell types in adipose
tissue, facilitating analyses of adipose tissue structure. The possible use of
intracellular xenobiorgs to mark stem cells is mentioned again further in this
chapter.
Control of macrophages in adipose tissue. Intracellular xenobiorgs might
enter macrophages and control macrophage behavior. Much of the harm done by
obesity involves inflammation, perhaps in part mediated by macrophages.
Intracellular xenobiorgs within macrophages might reduce their destructive effects.
It is also possible that if adipocytes were selectively destroyed by intracellular
xenobiorgs, a dangerous surplus of macrophages might result. Rapid shrinkage of
adipose tissue from gastric bypass surgery might also create a surplus of
macrophages. Xenobiorgs within surplus macrophages might prevent them from
doing harm.
Altering the characteristics of visceral adipose tissue. Visceral adipose
tissue—the adipose tissue that surrounds organs—is more dangerous than is
adipose tissue in other locations. If the characteristics of visceral adipose tissue that
make it exceptionally dangerous were known, intracellular xenobiorgs might alter
those characteristics.
Some of the differences between visceral adipose tissue and adipose tissue in
other locations may result from difference in their endocrine functions. Adipose
tissue is a major endocrine organ. It produces a number of important hormones,
including leptin, tumor necrosis factor, interleukin-6, estradiol, adiponectin,
plasminogen activator inhibitor-1, chemerin, retinol-binding protein 4, visfatin,
apelin, and perhaps resistin.
Chapter 7 discusses these hormonal products of adipose tissue, how they might
be manipulated by intracellular xenobiorgs, and what might be gained.
Peptide YY3-31. One anti-obesity hormone is peptide YY3-31, which
suppresses hunger, shows promise as an anorectic drug, and is reduced in amount
in the blood of obese people. It is also useful in removing aluminum that has
accumulated in the brain.
Enterostatin. Enterostatin is a pentapeptide, and a degradation product of
colipase, an obligatory cofactor for pancreatic lipase. Enterostatin is forms in the
small intestine, but subsequently enters blood and lymph.
Enterostatin reduces food intake, and selectively reduce fat intake. It reduces
body weight and body fat. It makes the stomach feel full. Animals prone to
consume dietary fat and to become obese have less enterostatin than normal.
Intracellular xenobiorgs stationed in adipose tissue might secrete enterostatin
into the blood and help control obesity. On the other hand, enterostatin has side
effects that might have to be countered if such a scheme were used. Chapter 7
discusses this.
Cholecystokinin. Cholecystokinin is a set of peptides derived from a progenitor
protein, preprocholecystokinin. Cholecystokinin stimulates the digestion of fat and
protein and suppresses hunger. However, cholecystokinin has undesirable side
effects that depend on which members of the set are expressed. The possible use of
intracellular xenobiorgs to turn adipose tissue into cholecystokinin secretors is
discussed in Chapter 7.
Ghrelin. Ghrelin is a peptide hormone that is 28 amino acid residues in length.
It is produced by cells in the stomach and pancreas, and induces hunger. To induce
hunger, ghrelin must be modified (octanoylated) after its translation at its third
amino acid residue (serine).
Intracellular xenobiorgs might be used to modify adipocytes to take up and
sequester or destroy ghrelin, which in turn might reduce hunger. Since ghrelin has
many effects throughout the body, such a strategy would be complicated—and
might or might not be feasible. Chapter 7 discusses the idea.
People with Prader-Willi syndrome have abnormally high concentrations of
ghrelin and are insatiably hungry. Overeating induced by this hunger inevitably
kills Prader-Willi patients. Prader-Willi patients are good candidates for ghrelinreduction therapy by xenobiorgs within adipocytes.
Tumor necrosis factor. Tumor necrosis factor, as its name implies, destroys
cancer cells. However, it also promotes inflammation that exacerbates human
pathologies that include Alzheimer’s disease, depression, inflammatory bowel
disease, toxic shock, cancer-induced cachexia (wasting), and many other
autoimmune disorders.
Tumor necrosis factor is produced in part by adipose tissue. Within adipose
tissue, activated macrophages probably produce most of the tumor necrosis factor.
Xenobiorgs installed in adipose tissue might reduce the amount of tumor
necrosis factor produced by obese people. The most promising strategy is probably
to install xenobiorgs within a minority of adipocytes and engineer the
adipocyte/xenobiorgs to take up and destroy tumor necrosis factor or to secrete a
diffusible inhibitor such as a decoy receptor or a single-chain antibody. They might
also secrete a diffusible factor that would reduce tumor necrosis factor synthesis by
neighboring macrophages.
Alternatively, and less plausibly, intracellular xenobiorgs might invade all or
most macrophages within a patient’s adipose tissue. They could then selectively
downregulate tumor necrosis factor production.
Chapter 7 discusses this idea in more detail.
Interleukin 6. Interleukin 6 has complex, and seemingly contradictory, effects
on a person’s body. IL-6 is produced by adipocytes, which may be the reason that
obese people have elevated C-reactive protein. Adipocyte/xenobiorgs could be
programmed to make either more or less IL-6, perhaps in restricted areas of the
body, as a patient’s health seemed to require.
Adiponectin. Adiponectin is a protein that is secreted into the blood only by the
placenta and by adipose tissue. However, adiponectin amounts in the blood are
inversely correlated with the body fat percentage in adult humans. Weight
reduction increases circulating adiponectin levels.
Adiponectin has a number of effects that counteract the unhealthy effects of
obesity, and obese people might benefit from increased adiponectin synthesis.
Intracellular xenobiorgs within adipose tissue might increase adiponectin secretion.
Chapter 7 discusses this possibility.
Other possible manipulations of adipose tissue-related factors. Adipose
tissue might plausibly be manipulated by intracellular xenobiorgs to change
concentrations of the adipose-related proteins aromatase/estradiol, tissue
plasminogen inhibitor, chemerin, retinol-binding protein 4, visfatin, apelin,
Chapter 7 discusses these possibilities.
Adding extraneous hormones to adipose tissue. It might be possible to use
intracellular xenobiorgs to engineer adipose tissue to express hormones that are
ordinarily not expressed in adipose tissue, or to remove from the blood hormones
unrelated to adipose tissue function.
It may also be possible to transplant adipose tissue to locations where it is not
normally present and to engineer that adipose tissue to express chosen hormones,
using intracellular xenobiorgs.
♦♦ Extension of cell life via telomerase and other methods
Telomerase repeats. Telomerase is an enzyme that adds tracts of non-coding
tandemly repeated DNA (“telomerase repeats) to the ends of chromosomes. The
added DNA protects the ends of chromosomes from shortening that would
otherwise be caused by chromosome replication. It also protects chromosome ends
from damage inflicted by exonucleases, and from ligases that might otherwise join
chromosomes at their ends.
Telomerase activity needed for replication. Because replication of a linear
double helix unavoidably removes material from the two ends, unlimited safe
proliferation of cells requires unlimited addition of telomerase repeats to
chromosome ends. Moreover, because continued replication of a chromosome that
had lost its telomerase repeats would endanger the chromosome’s integrity,
regulation has evolved to block chromosome replication when telomerase repeats
become too short. The result is that cells lacking telomerase activity cease
replication.
Although telomerase is highly expressed in embryonic cells and in adult tissues
that must divide regularly, it is expressed at very low levels in most adult tissues.
This means that most adult tissues have a limited capacity for proliferation.
The limited capacity for proliferation that most human adult cells have may
limit their life span, and this in turn might limit the life span of people. It has
occurred to many observers that adding telomerase activity to human cells that lack
it might restore their ability to proliferate, and thus lengthen the lives of people.
Chapter 7 discusses the arguments for this idea and the stronger arguments against
it.
Protection against diseases that shorten telomeres. Although use of
intracellular xenobiorgs to express telomerase within cells is unlikely to protect
against senescence, there are a number of pathologies where telomerase repeats are
abnormally short in some tissues, either because telomerase itself is defective, or
for some other reason. These diseases include aplastic anemia, dyskeratosis
congenita, Werner syndrome, ataxia telangiectasia, ataxia-telangiectasia like
disorder, Bloom syndrome, Fanconi anemia, and Nijmegen breakage syndrome.
Intracellular xenobiorgs that encode telomerase or other factors that protect
telomeres might protect vital tissues in people with these diseases.
Protection against anti-cancer drugs that shorten telomeres. Since
telomerase is reactivated in many cancers, and an alternate pathway of replacing
telomerase repeats appears in other cancers, drugs that inhibit telomere growth are
under consideration as anti-cancer agents. If these drugs are used, intracellular
xenobiorgs might be used to protect particularly vulnerable tissue such as bone
marrow and intestinal cells from the drugs’ effects.
Chapter 7 also discusses the telomere-protective compounds carnosine and
anserine.
Temporary suspension of p53 and retinoblastoma proteins. To restore
proliferative ability to human tissues that had lost it, intracellular xenobiorgs might
have to temporarily suspend activity of two tumor suppressor proteins, p53 and
retinoblastoma protein. Although disabling tumor suppressor proteins is inherently
risky, intracellular xenobiorgs might themselves destroy their host cell if that cell
developed characteristics of cancer.
As stated above, this is unlikely to be a good general strategy for extending the
human life span, but might prolong the life of selected tissues.
♦♦ Assessment of cells’ differentiated state
A potential use of intracellular xenobiorgs would be to assess a cell’s
differentiated state and to report that information to the cell surface. This is
discussed somewhat in Chapter 7 and to a much greater extent in Chapter 16.
Repnumi rejuvenation. If an intracellular xenobiorg could enter a target cell,
reliably read that cells’ differentiated state, and report that information to the cell
exterior—all without perturbing the cell—it could set the stage for Repnumi
rejuvenation, the replacement of a cell’s nucleus and mitochondria by more
youthful counterparts of exactly the same type. Chapter 16 is devoted to discussing
this possibility.
As discussed above, xenobiorgs might use reverse-orientation receptors to
probe the interior states of cells. This would obviate the need for the xenobiorg to
enter the target cell.
Isolation of specific cell types. There are many purposes in research and
medicine for which it would be advantageous to isolate a specific cell type. A
classic example is the need to isolate pluripotent stem cells, but other pure cell
types such as rare leukocytes varieties or rare transient stages would also be useful.
Intracellular xenobiorgs might invade a population of cells, assess the
differentiated state of each cell, and (when appropriate) mark the cell with a
fluorescent protein or surface antigen. The cells could then be purified by
fluorescence-activated cell sorting.
Intracellular xenobiorgs might also be programmed to block any of the gene
expression or spatial changes that accompany tissue differentiation. This could
allow researchers to assess the importance of specific changes to overall tissue
differentiation.
Intracellular xenobiorgs within a heterogeneous tissue might also limit the
action of medicines to desired target cell types only.
In some cases, doctors might want to selectively remove certain cell types from
a population. The antigen-presenting cells that cause graft-versus-host disease in
bone marrow transplants are good candidates for this.
Mapping of solid tissues, especially over time. Intracellular xenobiorgs might
report the differentiated states of cells in living solid tissues, and could report
changes in gene expression over time in response to stimuli of interest. The
reactions of gastrointestinal cells or liver cells to food, or the reaction of pancreatic
β cells to changes in blood glucose levels are possible examples.
Investigations of tissues often consist of bulk measurements made on masses of
cells that resemble each other. Intracellular xenobiorgs could report characteristics
of specific cell types within a tissue.
Researchers generally classify cells types as distinct based on their appearance
or by the antigens that they display. However there may be hidden differences
between cells that have indistinguishable appearances and antigen sets. Potential
examples include cells that have rearranged their chromatin in preparation for
changes in gene expression and cells that have repositioned their organelles as part
of a change in function. Intracellular xenobiorgs could reveal this.
Early detection and tracking of cancer. Precancerous cells and cells in the
early stages of cancer differ morphologically from normal cells. These differences
might be detected by intracellular xenobiorgs.
Cancers are complex cell populations. Within a cancer, some cells may be more
dangerous than others, and cancer cells may cooperate to further a cancer’s growth
and spread. Different cell types within a cancer may interconvert, which
undermines therapies aimed at so-called “cancer stem cells.”
Keeping track of cell types within an experimental or clinical cancer is
notoriously difficult. With their complex abilities, intracellular xenobiorgs might
allow cancers to be followed much more closely.
Detection of hidden injury. Cells that have been damaged, or which are
migrating or proliferating to compensate for damage to neighboring cells, are in
many cases subtly different from undifferentiated cells. Their complement of
expressed genes may be different, of course, but their morphology often changes
slightly also. The position of the nucleus within a cell, the position of individual
regions of chromatin within the nucleus, nuclear rotation, the relative volume of
the nucleus, the appearance of the cytoskeleton, the nature of connections between
neighboring cells, the charge difference across the outer membrane, and other
features can change in response to tissue injury.
The ability to detect hidden injury might allow doctors to detect rheumatoid
arthritis, Alzheimer’s disease, cancer metastasis, viral infection, and other forms of
tissue damage much earlier. Intracellular xenobiorgs might give researchers not
only the ability to detect hidden damage, but the ability to explore the consequent
behavior of neighboring cells. Healthy cells in the vicinity of a diseased cell may
be abnormal in their morphology, movements, and autocrine/paracrine signaling.
And of course, in many cases new cells, especially immune system cells, will
invade an injured tissue. Intracellular xenobiorgs could help sort this out.
Organs affected by infectious disease are likely to be affected unevenly.
Furthermore, some cell types within an infected organ may be preferentially
infected. Intracellular xenobiorgs could reveal this.
In addition, it could be important to know the proportion of cells in a tissue that
are dead, either in a living organism, or after some procedure such as
freezing/thawing. Intracellular xenobiorgs could reveal this also.
Tracking of circadian cellular rhythms. Many human tissue types, especially
cells of the immune system—but also neurons, fibroblasts, hepatocytes, kidney and
adrenal cells—show periodic changes. (These changes are known loosely as
“circadian” rhythms, although the term “circadian” technically specifies only those
rhythms with a 24-hour cycle.) Rhythms can have important consequences, such as
changing the effects of vaccination and perhaps of immunosuppressive drugs,
depending on the time of day that they are administered.
Intracellular xenobiorgs might track circadian changes, and elucidate the
interaction between circadian timing and tissue function. They might elucidate the
effects of drugs, diseases, and other influences on the circadian rhythm of tissues.
Intracellular xenobiorgs might reveal tissues that lost either their normal rhythm or
their inter-cellular synchrony and which might therefore in the process of losing
function.
♦♦ Creation or preservation of stem cells
The usefulness of human stem cells. The usefulness of non-human stem cells
into multiple normal specialized human tissues. This ability is called pluripotency.
Stem cells are of great medical interest because they might replace damaged
human tissues. There are too many candidate tissues to list (the human body is
composed of at least 210 distinct cell types), but they include tissues of the heart,
brain, spinal cord, retina, kidneys, pancreas, liver, bones, skin, teeth, hair follicles
and so on.
Human stem cells might also create human tissues within an artificial setting,
such as implanted in an experimental animal. Such cells could be very useful in
studying the actions of drugs, hormones, and toxins, and could also be useful in
studying human tissues that are important but hard to study, such as preimplantation embryos.
The usefulness of non-human stem cells. Non-human stem cells have been
used to engineer the germ lines of experimental animals, and thereby have greatly
increased the value of those animals as research objects. Non-human stem cells
may someday enable the production of animal products such as meat, milk, fur,
ivory and feathers without the need to raise, butcher, and dispose of whole animals.
This could in turn have important economic, environmental, and public health
benefits.
Difficulties in working with human stem cells. Although mouse stem cells are
easy to work with, and have shown that stem cells have great promise, human stem
cells are much less tractable. They are hard to isolate and even harder to maintain
in a state of pluripotency.
In addition, stem cells sometimes falsely appear to form new and desired
structures when transplanted into a target tissue. Closer examination shows that
they have instead induced pre-existing local tissues to form or preserve those
desired structures. As examples, transplanted stem cells can induce local cells in
heart or kidney to form blood vessels and to resist apoptosis (cell suicide).
Intracellular xenobiorg uses in stem cells. Genetically engineered bacteria
that could enter and remain in stem cells without disturbing them might perform
several useful tasks. First, they could mark the stem cells, making it clear whether
biological structures were derived from those stem cells or from cells affected by
the stem cells. Second, they could preserve isolated stem cells from apoptosis or
differentiation. Third, they might induce differentiated diploid human cells to
become pluripotent stem cells—a process that is already accomplished by other
methods.
Advantages of intracellular xenobiorgs. Intracellular xenobiorgs have several
likely advantages as agents of stem cell preservation. First, they are a way to move
active genes into cells without integration of DNA into host nuclear or
mitochondrial genomes. Second, they could be engineered to shut down and
disintegrate or exit their host cell when no longer needed. Third, they can be
engineered to express multiple genes in any desired combination, order, and level
of expression. Fourth, they can express proteins within host cells without the
complications of RNA interference. Fifth, they could increase the efficiency and
accuracy of genetic engineering of the nuclear genome, if this were desired. Sixth,
they could shorten the process of selection of pluripotent stem cells, could prevent
associated changes in gene expression, and could obviate the need for selection
using drugs and drug resistance. Seventh, intracellular xenobiorgs might not only
preserve stem cells from apoptosis and unwanted differentiation, they might also—
on receiving a signal—start the stem cells down a desired differentiation pathway.
Intracellular xenobiorgs might also be very useful in investigating stem cells.
They would be unique among artificial regulatory agents in their ability to start and
stop multiple actions independently. As such, they could be very useful in
dissecting the regulatory networks that control pluripotent stem cells.
Disadvantages of intracellular xenobiorgs. Several drawbacks to the
proposed use of intracellular xenobiorgs apply not only to their use in stem cells,
but generally. First, their host cells have elaborate machinery to detect their
presence—and if an intracellular xenobiorg is detected, the host cell will respond
in ways that make the host cell almost useless. Second, they cannot secrete RNA
into their host cells in cases where this is desired. Third, it is likely to be difficult to
regulate the copy number of intracellular xenobiorgs within their host cells; very
likely, exactly one xenobiorg per host cell would be desired, but this could be very
hard to achieve.
Undifferentiation signatures. Chapter 7 discusses what is now known about
totipotent and pluripotent stem cells, including “undifferentiation signatures”, the
sets of expressed genes that control their behavior. Several proteins that might be
made by intracellular xenobiorgs and used to control pluripotent stem cell behavior
are discussed. These include alkaline phosphatase, Bcl-2, fibroblast growth factor
2, Nanog, Oct4, p53, Wnt1 and Wnt 3, and telomerase among others. The potential
creation and use of nitric oxide by intracellular xenobiorgs is also discussed.
♦♦ Protection of cells from toxins
Intracellular xenobiorgs Spatial restriction of immunosuppression from
environmental toxins or carcinogens. As mentioned above, they might protect
sensitive cells in marrow, intestinal lining, hair follicles, and other tissues from
cancer chemotherapy. They might protect skin cells from the sun, brain cells from
toxic Alzheimer’s plaque, and heart and brain cells from brief periods of anoxia.
They might protect endothelial cells of blood vessels from alcohol, cigarette
smoke, and free radicals generated by digestion of fats.
Most importantly, intracellular xenobiorgs might protect liver cells from toxic
products of cytochrome P450 activity. Most drugs and other foreign organic
compounds are changed by action of the cytochrome P450 system in the human
liver. These changes work generally to make the compounds more soluble, rather
than to detoxify them, and in some cases make the compounds more toxic.
One group of toxic compounds, the aflatoxins, is produced by Aspergillus
flavus and Aspergillus parasiticus. Aflatoxins, especially aflatoxin B1, are potent
liver toxins and carcinogens. Yet, especially in poor countries, staple foods such as
chilies, corn, cotton seed, millet, peanuts, rice, sorghum, sunflower seeds, tree nuts,
wheat and many spices are often contaminated.
Although it would be better to prevent aflatoxin contamination of food or to
remove the aflatoxin contamination from food, these can be very difficult.
Intracellular xenobiorgs might instead protect liver cells by inactivating aflatoxins
or aflatoxin metabolites that entered the cells.
Intracellular xenobiorgs might also protect liver cells from acetaminophen
toxicity.
♦♦ Removal of intracellular garbage
Cells have machinery to degrade surplus or damaged proteins. One system is
the ubiquitin-proteasome system. A second system includes lysosomes.
However, proteins that have become oxidized can form large amorphous
aggregates within cells. If these proteins become sufficiently oxidized, crosslinks
between them accumulate that resist proteolysis. Larger numbers and sizes of
highly oxidized aggregates accompany aging.
Intracellular xenobiorgs might be engineered to remove protein aggregates from
cells. The most promising strategy might be for a single xenobiorg to divide and
for one progeny xenobiorg to engulf damaged groups of protein, exit the host cell,
enter the blood, travel to the intestine, move through the intestinal wall into the
intestinal lumen, and exit via the feces. Alternatively, intracellular xenobiorgs
might be engineered to digest protein aggregates that human cells cannot.
Chapter 7 will discuss these ideas.
♦♦ Cassettes for gene therapy
Intracellular microbial parasites can secrete proteins into their host cell. In at
least some cases, these proteins function. Thus it is reasonable to hope that
intracellular xenobiorgs, based on intracellular microbial parasites, might be
engineered to supply beneficial proteins to their host cells.
Intracellular xenobiorgs might be engineered to stimulate bone or muscle
growth in people deficient in either. They might restore function to the defective
melanocytes of albinos. They might restore normal superoxide dismutase function
to neurons in patients with some forms of amyotrophic lateral sclerosis (motor
neurone disease, Lou Gehrig’s disease). They might be engineered to emulate the
action of fibrate drugs in liver cells, increasing high density lipoprotein synthesis
and reducing triglyceride synthesis.
Intracellular xenobiorgs might convert adipocytes, or other cells that do not
normally express insulin, into insulin-producing cells. However, the number of
living converted cells would have to be monitored by magnetic resonance imaging
or some other procedure. The engineered cells would have to be able to sense the
amount of circulating insulin and adjust their output accordingly.
Intracellular xenobiorgs might release small amounts of the protein leptin near
the hunger centers of the brain. This might reduce a person’s appetite and weight.
In other cases, where a patient needed to eat, intracellular xenobiorgs might release
orectic factors near hunger centers of the brain.
Intracellular xenobiorgs might release anesthetic or anti-inflammatory factors
near the site where they were needed. They might release soporifics near sleep
centers of the brain. They might release promoters of bone growth near fragile
bones.
Knowledgeable molecular biologists can probably think of many other
possibilities.
For intracellular xenobiorgs to supply proteins to a human cell that would then
be secreted would require several steps. A given protein would have to be made by
the xenobiorg, exported to the host cell cytoplasm, processed in any way
necessary, and then secreted by the host cell.
Intracellular xenobiorgs might exploit the cell machinery that secretes proteins
as individual molecules.
Alternatively, intracellular xenobiorgs might export proteins from the host cell
via exosomes. Exosomes are vesicles of 30-90 nm diameter that are secreted by a
wide variety of mammalian cell types. Exosomes can transfer both proteins and
RNA molecules between cells, including, for example, cardioprotective
substances. Exosomes might be good export vehicles for products produced by
intracellular xenobiorgs.
In addition, intracellular xenobiorgs might themselves travel between cells—
perhaps in some cases moving contents of one cell into another.
One advantage of engineering human cells with intracellular xenobiorgs, rather
than by modifying the human nuclear genome, is that the engineering could be
reversed easily and without harm to the host cell.
In many genetic diseases where a deleterious mutant protein is made,
intracellular xenobiorgs might silence the host gene(s) and supply a normal version
of the protein instead. One example, discussed above, is Huntington’s disease.
Intracellular xenobiorgs might also reduce the expression of genes whose
expression is unhealthily high. For example, they might reduce cholesterol
synthesis in the liver, emulating statin drugs.
An enormous number of proposals for gene therapy have been made, and
intracellular xenobiorgs are good candidates to fulfill many of them.
♦♦ Directors of gene conversion
Intracellular xenobiorgs might be engineered to direct targeted gene conversion
in human cells. Gene conversion is a process by which one DNA sequence
replaces a similar, but not identical, DNA sequence within a larger DNA molecule.
In principle, targeted conversion is the best method of gene therapy. It both
removes unwanted DNA sequences and replaces them with desired sequences. It
corrects gene sequences in situ, leaving intact all of their surrounding regulatory
sequences and not altering the gene’s position in the nucleus. Unlike the case
where genes would be expressed from within intracellular xenobiorgs, gene
conversion allows gene expression to follow its normal spatial path, starting with a
transcribed gene and ending with a fully processed protein (if processing is
necessary). Morever, unlike the case where genes would be expressed from within
intracellular xenobiorgs, gene conversion presents no challenges in maintaining
correct copy number of the introduced gene.
In principle, almost any defective human gene could be corrected by
conversion. Chapter 7 discusses one exciting possibility, the conversion of at least
some of the Human Leukocyte Antigen (HLA) genes that can thwart both organ
and stem cell transplantation. This would allow donor HLA genes to be converted
to sequences already present in the recipient, and reduce the risk of tissue rejection.
The focus is on hematopoietic stem cell (bone marrow) transfer, since this is a
mature technology.
Targeted gene conversion requires site-specific nucleases, and great progress
has been made in the design of these. The most promising strategy to design sitespecific nucleases exploits the zinc finger DNA-recognition motif, but there are
other promising approaches as well.
Use of intracellular xenobiorgs to direct targeted gene conversion would require
engineering of several molecular processes. The DNA sequences intended to
replace host nuclear DNA sequences (the “replacement sequences”) would have to
be added to the xenobiorg and kept stable within the xenobiorg. Once the
xenobiorg had entered and taken up residence within the host cell, the replacement
sequences would have to be extruded, transported to the nucleus, taken to the
chromosomal site where conversion is intended, and then used in gene conversion.
It would not be necessary to transport extruded DNA sequences to the nucleus
if the intracellular xenobiorg already resided in the nucleus. This idea is not too
farfetched—some intracellular bacterial parasites do reside in the host nuclei.
However, it seems more likely that intracellular xenobiorgs would reside within a
cytoplasmic endosome.
The same molecular processes that convert DNA sequences can also insert large
sequences into the target DNA molecules. This might allow insertion of
functioning genes into tissues that do not normally express those genes, or even
insertion of genes that are not present in the human genome.
♦♦ Monitoring of cells
Intracellular xenobiorgs might also monitor and report on the condition of
individual cells. As a simple example, they might secrete a chemical into the blood
if their host cell became cancerous, ischemic, or virus-infected.
Some bacteria accumulate crystals of magnetite and/or greigite that orient them
with respect to the Earth’s magnetic field. Conceivably, this or some other means
might be used to allow bacteria to display information to an external
electromagnetic field. One can imagine a situation many years from now when
medical patients are scanned by a device resembling a magnetic resonance imaging
device, and the device records a kilobyte of information about each of the patient’s
4 X 1013 cells. Such an arrangement might alert physicians to many impending
problems long before they become medically evident.
In the very distant future, intracellular xenobiorgs within a person’s brain cells
might display and receive signals that could enable facilitated telepathy. This wild
and highly speculative possibility is discussed in Chapter 21.
♦ Intracellular microbial parasites that could form the basis of
intracellular xenobiorgs
♦♦ The choice of starting material: choosing the best microbe
Intracellular xenobiorgs would probably be derived from some existing species
of microbe. This microbe would very likely be a human or animal parasite, and
would probably be a bacterium. (However, it is also possible that intracellular
xenobiorgs would instead be derived from human mitochondria; see below.)
Desirable and undesirable characteristics. In choosing microbes as starting
material, researchers should consider several issues:
Is the microbe obligately or facultatively (optionally) intracellular?
Facultatively intracellular microbes seem easier to work with.
How easily is the microbe grown in culture?
How easy is it to genetically engineer the microbe?
How destructive is the microbe to host tissues? Does it damage cells as it enters
them? If it occupies a phagosome or endosome, does it disrupt it? If it damages
cells, could that damage be reduced by genetic engineering of the microbe? Can it
promote an immune reaction that may damage the human host?
Is the microbe motile?
Can the microbe spread from cell to cell within solid tissues? If so, how
damaging it this?
Is the human host likely to have pre-existing immunity (either innate or
adaptive) to the microbe which might interfere with its use as an intracellular
xenobiorg?
How contagious is the microbe? Are there natural barriers, such as the need for
passage through an insect (e.g. in the case of malaria), that would prevent the
xenobiorg derivative from reverting to a harmful and contagious pathogen? If not,
can other reliable containment be devised?
Does the microbe home to any particular organ or tissue, such as lymph nodes
or neutrophils? Depending on circumstances, this could either increase or decrease
its value. Moreover, it might be possible to transfer this ability from one xenobiorg
candidate to another.
Whether or not to use a disabled dangerous pathogen. An additional issue to
be considered is whether it would be better to begin with a virulent pathogen that
has been well-studied, or a less harmful parasite that has received less attention.
Dangerous pathogens would include Listeria monocytogenes (which causes
listeriosis), Mycobacterium leprae (leprosy), and Bordetella pertussis (whooping
cough), while less dangerous members of the same genera would include
Mycobacterium lepraemurium, Listeria innocua, Listeria ivanovii, and Bordetella
bronchisepta.
It seems to me that thoroughness of understanding is more important than
harmlessness of starting material, and that it would therefore be better to begin
with a dangerous human pathogen that has been well studied and then disabled
with certainty. However, the issue is far from settled.
Candidate pathogens. Chapter 7 considers a number of pathogens that can
spend at least part of their lives inside human cells. These include Escherichia coli
K12, members of the Shigella subgenus, members of the Salmonella genus,
Bordetella pertussis, Yersinia pestis, Listeria monocytogenes, Coxiella burnetii,
Legionella pneumophila, Group B Streptococcus, Brucella abortus, Pseudomonas
aeruginosa, Anaplasma phagocytophilum, Chlamydia trachomatis, Chlamydophila
pneumonia, Francisella tularensis, Rickettsia rickettsia and related species,
Mycobacterium tuberculosis and M. leprae, Plasmodium falciparum, Leishmania
donovani, Trypanosoma cruzi, Toxoplasma gondii, and members of the genus
Babesia. Chapter 7 evaluates each of these as a potential basis for intracellular
xenobiorgs.
Xenobiorgs derived from human mitochondria. The biggest drawback to
converting a microbial pathogen into an intracellular xenobiorg is that human cells
have sophisticated and capable methods to detect microbial invaders. If a cell
detects an invader, the cell’s behavior will change in ways that almost completely
destroy the cell’s usefulness.
Chapter 7 suggests ways to defeat human cells’ ability to recognize invaders.
However, if it proves impossible to hide a pathogen-derived xenobiorg from the
host cell, there is another possible alternative: deriving an intracellular xenobiorg
from human mitochondria. Mitochondria are thought to be derived from
proteobacteria, and contain an individual genome which in humans has 37 genes. It
might be possible to expand the mitochondrial genome with additional useful
genes.
Mitochondria are not attacked by their host cells and do not trigger abnormal
behavior in their host cells. On the other hand, they have no system to secrete
proteins into the cytosol and leakage of their contents into the cytosol triggers
apoptosis (cell suicide). Chapter 7 discusses the possible approaches, and the likely
difficulties of converting mitochondria into xenobiorgs.
♦♦ Intracellular xenobiorgs could defend against intracellular
pathogenic single-celled microbes
Intracellular xenobiorgs might be engineered to protect their host cell from
invasion by pathogenic microbes. Hence, Chapter 7 also discusses each of the
above-mentioned microbial species as potential invaders to be defended against.
The usefulness of intracellular xenobiorgs to defend against invading microbes
will probably be determined by a number of factors:
Is the disease caused by the microbial parasite serious enough that it is likely to
be intensively studied in the future, independently of any usefulness the parasite
may have as an intracellular xenobiorg?
How vulnerable are the invading microbes to traditional medical defenses such
as antibiotics and vaccines? Either of these is less drastic than a defense involving
intracellular xenobiorgs, and would ordinarily be preferred.
Are there any human populations, such as premature infants, the
immunocompromised, or the elderly that especially need protection, and might not
be protectable by ordinary medical means?
How many different human cell types are infected by the microbial parasite?
Are there too many to protect?
Are there any “gateway” tissues, such as the lungs, the intestinal lining or
genitalia, that must be infected first, and which could be protected by intracellular
xenobiorgs?
Are the invading microbes spread through the body by any cell types such as
monocytes, macrophages or neutrophils?
Does the parasite itself have the means to block superinfection (infection of
cells that are already infected)? If so, could this be used by intracellular xenobiorgs
to prevent the original infection?
Many infections do not progress rapidly and kill their host, but instead become
chronic. This seems to be to the parasites’ long term advantage. Because it aids the
establishment of a chronic infection, parasites may evolve to allow themselves to
be partly blocked or even largely killed off by the body’s defenses. Might these
defenses be manipulated to completely destroy the infecting microbes and resolve
the infection?
Does the progression of the disease depend on host genotype? Could
components of resistance in one host be transferred by intracellular xenobiorg to
sensitive host?
How long can the disease exist in a quiescent state before manifesting itself?
If the disease is serious, it presumably damages tissues. Is this damage due to
the disease agent itself or to subsequent action of the immune system (as in the
case of Chagas disease and chlamydia)?
How well is the molecular machinery of infection understood? Does that
machinery have any obvious vulnerabilities that intracellular xenobiorgs could
exploit to suppress an infection or reduce the harm that the infection does?
What is the energy source that the invading microbe uses, once in the cell?
Could it be temporarily blocked by a resident intracellular xenobiorg?
Are the disease properties of the invading microbe affected by quorum sensing?
If so, could this be exploited to reduce the microbe’s virulence?
Can the microbe that becomes the basis for an intracellular xenobiorg safely be
made resistant to medical antibiotics, so that it can be used in conjunction with
them?
Does the disease agent spread directly from cell to neighboring cell via
paracytophagy?
Does the disease invade or otherwise damage germ line DNA, such that
subsequent generations’ health may be compromised? Intracellular xenobiorgs
might be engineered to protect germ plasm or destroy the cells if they became
infected.
♦ Engineering the invasion of host cells by xenobiorgs
Transport through the body and insertion into host cells. Intracellular
xenobiorgs must somehow be emplaced within desired host cells. This involves
two steps. First, they must be transported through the body to their intended host
tissues—a problem discussed in Chapters 8, 9, 10, and 11. Second, when they have
reached the vicinity of their future host cells, they must invade those cells. This
second problem is discussed in Chapter 7: Chapter 7 discusses methods by which
microbial parasites invade human cells, and how these methods might be exploited
by genetic engineers. Chapter 7 also touches upon the related problem of moving
xenobiorgs across the blood-brain barrier.
Multiple mechanisms of entry. Mechanisms by which microbes enter cells
follow no evolutionary trend. Instead they seem either to have arisen independently
of each other or to have diverged rapidly.
General methods of microbe entry into cells include coiling of a host cell
pseudopod around the entering bacterium, entry via the “zipper” mechanism, the
violent entry method of Bdellovibrio, and microsporidian polar tubes among
others. The bacterium Salmonella enterica var. Typhimurium has 3 distinct ways of
entering cells.
Endocytosis. Endocytosis is an entry process in which the parasite does not
destroy the continuity of the host cell envelope, and enters surrounded by hostderived membrane. Endocytosis is probably the best entry option for xenobiorgs.
Parasites that enter by endocytosis include malarial parasites, Shigellae,
Rickettsiae, T. cruzi trypomastigotes, and Chlamydiae. Some of these parasites
have a specialized organelle of entry or attachment, while others do not. Some of
them expend energy to enter the host, while others do not. Chapter 7 discusses
these as options for inserting xenobiorgs into human host cells.
Host-specified vs. parasite-specified. Some parasite entry into host cells is
host-specified, while in other cases it is parasite-specified. Entry into so-called
“professional” phagocytes is usually specified by the host cell. Entry into other
cells is almost always parasite-specified. Plans to insert xenobiorgs into specific
cell types will have to account for this.
Motility and cell type-specificity. Two additional factors that designers of
intracellular xenobiorgs should consider are xenobiorg motility and possible cell
type-specificity of parasitization. Motile xenobiorgs may be better able to contact
their future hosts and initiate uptake than non-motile xenobiorgs would be. In
addition, numerous examples from human and animal diseases show that microbes
can selectively parasitize specific cell types. Depending on circumstances
xenobiorg engineers might exploit this or be forced to overcome it.
Molecular machinery by which microbes enter host cells. Much has been
learned about the molecular machinery that governs microbe uptake. Chapter 7
discusses Listeria internalins, Yersinia invasin, type IV pili of Francisella,
Bordetella filamentous hemagglutinins, Streptococcus pilin proteins, bacterial wall
teichoic acid and lipoteichoic acid, integrin receptors, massive localized ruffling,
microfilament function, complement receptor 3, pertussis toxin, laminin-binding
protein, fibrinogen-binding protein, C5a peptidase, ubiquitin ligase, cholesterolrich domains, lipid rafts, antibody enhancement, and other factors. Genetic
engineers might assemble components of this machinery to program the entry of
xenobiorgs into chosen cell types.
Chapter 7 also discusses the possible use of minicells derived from bacteria to
perform initial steps of uptake of xenobiorgs.
Minimization of damage to host cells. Insertion of xenobiorgs into cells
should damage the cells as little as possible. Pathogenic microbes differ greatly in
how much they damage the cells that they invade. Invasion by Coxiella burnetii is
quite benign. Bacillus pertussis, Streptococcus agalactiae, Leishmania donovani,
Chlamydia, and Rickettsiae are—in varying degrees—more harmful.
Uptake of microbes by phagocytic cells can trigger an immune response.
However, this can also be avoided.
♦ Evading detection and destruction by the host cell
The need to evade detection. For intracellular xenobiorgs to be medically
useful, they must not disrupt normal functioning of their host cell. Since human
cells react drastically to intracellular parasites, some means to prevent detection of
intracellular xenobiorgs must be used. This will not be easy, because cells have
elaborate and efficient machinery to detect invaders.
An increase in danger? There is an additional complication. Giving microbes
the ability to evade detection while in cells might increase their danger as disease
agents. The problem is not that xenobiorgs would themselves revert to
pathogenicity; the likelihood of this could probably be reduced to nearly zero. The
danger is instead that they might transfer their evasion machinery to active
pathogens. Before intracellular xenobiorgs are used in most cell types, this possible
hazard will have to be assessed.
Wolbachia. Chapter 7 discusses a natural example of a bacterium that lives
within the cells of many arthropods, including many insects. The bacterium is
genus Wolbachia, which lives in many insect cell types without disrupting them.
Wolbachia can confer benefits on its host, including resistance to several types of
virus, resistance to insecticide, resistance (of a mosquito) to malaria parasites, and
other benefits. Chapter 7 discusses the lessons that Wolbachia might hold for the
design of intracellular xenobiorgs.
Obligatory endosymbionts in Amoeba proteus. Amoeba proteus is a
eukaryotic protozoan. When Amoeba proteus is co-cultured with bacteria that
destructively parasitize it, an interesting evolution occurs in only a few hundred
generations of the amoeba. The bacteria become less destructive to the amoebae,
and the amoeba become dependent on the bacteria—which assume the role of
obligatory endosymbionts.
Obligatory endosymbionts in immortalized human cells. It seems likely that
Amoeba proteus changes genetically and that the bacteria do not. This limits this
example’s usefulness as general model for xenobiorg-host cell accommodation, but
it leaves open the possibility that this system might be a model for accommodation
between immortalized human cell lines and intracellular pathogens. Immortalized,
non-cancerous human cell lines can proliferate indefinitely, and might serve as
useful medical agents, if they could be controlled and if they could be engineered
to evade a patient’s immune system. Co-cultured, genetically engineered bacteria
might facilitate both goals.
A big potential benefit of this idea is that selection might avoid the need for a
great deal of trial and error. Moreover, a general benefit of bacteria that have
become obligatory endosymbionts is that the bacteria can be selectively poisoned,
making it easy to kill the host cell as well.
Host responses to microbial infection. Mammalian cells respond to invasion
by bacteria and other microbes by altering, upward or downward, the expression of
hundreds of genes. Some of the response depends on signaling between infected
cells. Chapter 7 describes this response along with the regulation that coordinates
it.
Mammalian cells also have features that can destroy invading microbes.
Destruction of intracellular xenobiorgs would, of course, end their usefulness;
however, even detection of the xenobiorgs, without their destruction, would negate
most of their value.
Location of xenobiorgs in cells. There are several possible locations for
xenobiorgs. They may be encased in one of several types of intracellular vacuole,
they may be free in the cytoplasm, or they may even be free in the nucleus.
However, the best place for them would probably be within an intracellular
vacuole.
Detection of xenobiorgs within endosomes. A critical part of the system that
detects bacterial invaders is the set of Toll-like receptors, which recognize specific
molecule types associated with pathogens. At least four Toll-like receptors are
present within endosomes (a type of intracellular vacuole). One of these, TLR3,
detects viral double-stranded RNA. Two others, TLR7, detect viral single-stranded
RNA. TLR9 detects unmethylated CpG dinucleotides in DNA; these are
characteristic of bacterial DNA.
In addition to the Toll-like receptors, Retinoic Acid-Inducible Gene-I-Like
Receptors, and Nod-Like Receptors also recognize molecules from pathogens.
The system for detecting molecules characteristic of pathogens is termed the
“innate” immune system. Innate immunity is extremely complex and not yet
entirely understood. It may be, for example, that toll-like receptors that are thought
to detect viral RNAs may detect other pathogen RNAs as well.
Dummy innate immunity receptors. There are several conceivable methods to
defeat the innate immune system. One might be to engineer xenobiorgs to have
fewer of the molecules that trigger innate immunity. However, while this might be
useful in reducing interaction between xenobiorgs and the innate immune system,
it seems implausible that it could be done thoroughly enough to eliminate that
interaction, unless the xenobiorg was derived from human mitochondria.
It might also be possible to silence the genes that encode proteins of the innate
immune system. This might be done in any of several ways. However, the result
would certainly make the host cells functionally deficient, and might provoke a
regulatory cascade that would completely destroy host cell usefulness.
The best option to defeat the innate immune system would seem to be to encase
intracellular xenobiorgs within cytoplasmic vacuoles that are monitored by innate
immune system receptors, but to engineer the xenobiorg to synthesize nonfunctioning (“dummy”) copies of those receptors. The dummy receptors would
occupy the vacuole containing the xenobiorg and would exclude functioning
copies of the receptor created by the host cell. In a molecular sense, the dummy
receptors would “assure” the host cell that the vacuole was free of intracellular
microbes.
Chapter 7 discusses in detail the problem of engineering intracellular
xenobiorgs to defeat detection by the innate immune system.
Nourishing xenobiorgs within cytoplasmic vacuoles. Microbes that invade
human cells must overcome several problems. First, they must obtain adequate
nourishment—which can be difficult since they must compete with the host cell
biosynthetic machinery for nutrients such as amino acids. This problem may be
more severe if the invading microbe is enclosed within a cytoplasmic vacuole.
Protecting xenobiorgs within cytoplasmic vacuoles. A second problem is that
microbes generally enter cells within vacuoles called phagosomes, and that these
phagosomes ordinarily fuse with lysosomes. Lysosomes are acidic (pH 4.8), and
contain nucleases, proteases, glycosidases, lipases, phosphatases, sulfatases,
phospholipases and other acid hydrolases that function optimally in acidic
conditions. Lysosomes also contain antimicrobial peptides.
Invading microbes enclosed in phagosomes cope with lysosome fusion in one
of three ways. First, they may escape from the phagosome into the cytoplasm
before a lysosome fuses with the phagosome. Second, they may prevent lysosome
fusion with the phagosome. Third, they may remain in the phagosome, allow
phagosome-lysosome fusion to occur, and then thrive in the hostile environment.
Listeria, Shigella, Rickettsia, Francisella, and Trypanosoma cruzi, escape from
the phagosome into the cytoplasm. Mycobacterium, Legionella, Chlamydophila
pneumoniae, E. coli K12, Salmonella and Toxoplasma gondii inhibit phagosomelysosome fusion. Coxiella persists in mature phagolysosomes (phagosomes that
have fused with lysosomes).
Prevention of phagosome-lysosome fusion. Residence of intracellular
xenobiorgs within phagosomes that have not fused with lysosomes is probably the
best strategy for protecting them.
The cell wall of Mycobacterium tuberculosis prevents fusion of phagosomes
and lysosomes. It does this by blocking the bridging molecule, early endosomal
autoantigen 1 (EEA1) of the host. However, vesicles filled with nutrients continue
to fuse with the phagosomes, thus nourishing the bacteria. Interestingly, the
bacteria appear to protect only the individual phagosomes in which they reside
from fusion with a lysosome.
Leishmania donovani promastigotes produce a lipophosphoglycan that
prevents lysosome-phagosome fusion. Similarly, Salmonella typhimurium
produces the SpiC protein that inhibits interactions between the Salmonellacontaining vacuole and lysosomes.
Tolerance of conditions within a phagolysosome. Amastigotes (not
promastigotes, as mentioned immediately above) of Leishmania donovani can
withstand conditions within mature phagolysosomes, perhaps owing to protection
by pterins. On the other hand, Leishmania donovani may produce enough ammonia
by hydrolyzing proteins to raise the pH of the phagolysosomes and inactivate the
acid hydrolases. Yersinia pestis and Mycobacterium tuberculosis also prevent
phagolysosomes from acidifying, and thus preserves themselves. Coxiella burnetii
also thrives within mature phagolysosomes.
Many microbes produce enzymes or other substances that might protect them
from the harsh conditions within phagolysosomes. Mycobacterium tuberculosis
can neutralize reactive oxygen intermediates. Pseudomonas aeruginosa must have
strong defenses against oxidation, since it makes a pigment that oxidatively
stresses other cells; α-ketoglutarate derived from histidine may be the protective
substance. The lipopolysaccharide wall of gram negative bacteria protects against
singlet oxygen. Carotenoid pigments protect of Sarcina lutea and Staphylococcus
aureus from singlet oxygen. Streptococcus agalactiae produces a carotenoid
pigment that protects it from hydrogen peroxide, hypochlorite, superoxide, and
singlet oxygen. Aspergillus fumigatus and Burkholderia cepacia produce melanin,
which is also anti-oxidant. Glutathione may protect against some reactive oxygen
intermediates. Salmonella enterica var. Typhimurium uses the superoxide
dismutase SODCI to detoxify superoxide anions. Anaplasma phagocytophilum can
detoxify superoxides, perhaps also using superoxide dismutase.
Preventing damage. Microbial parasites living within mammalian cells often
damage those cells. However, the example of Wolbachia (discussed above) shows
that intracellular parasites need not damage their hosts. In the case of at least 3
parasites, Coxiella, Rickettsia, and Chlamydia, there are conditions where the
parasites can multiply without damaging their host cells. Coxiella burnetii can exist
and replicate within human cells for months. The most important determinant of
whether intracellular microbes damage their hosts is the rate of microbe
replication—with higher replication rates causing damage. For some purposes,
non-replicating variants of a bacterium such as Legionella pneumophila might
provide a good basis for construction of an intracellular xenobiorg.
Preventing antigen presentation. Successful use of intracellular xenobiorgs
will also require that the xenobiorgs do not attract a T cell-mediated immune
response that kills their host cell. This will in turn require one of two things: either
that protein fragments from the intracellular xenobiorg are not presented on the
host cell surface, or that some other means be found to prevent presented
fragments from inducing an immune response. This subject is discussed in
Chapters 7 and 20.
♦ Manipulation of host cells by intracellular xenobiorgs
Can xenobiorgs manipulate host cell gene expression? An important purpose
of intracellular xenobiorgs would be to manipulate gene expression within host
cells. Intracellular xenobiorgs could do this in many ways. They might secrete
DNA-binding proteins, or other transcription factors that would enter the host cell
nucleus and alter gene expression. They might silence gene expression using RNA
interference. They might export proteins that would function in the host or be
secreted from the host. Conceivably, they might also export messenger RNA that
would be translated into functional protein by the host cell.
An important question, however, is whether proteins and other macromolecules
secreted by intracellular xenobiorgs could actually function within host cells.
Might host cells have evolved some process to restrict the movement or the
function of parasite-synthesized macromolecules, especially proteins? As
discussed below, it appears that parasite-encoded proteins can enter the host cell
nucleus and bind DNA, can affect the activity of nuclear transcription factors, and
can alter events in the host cell cytoplasm.
Secretion of DNA-binding proteins. Anaplasma phagocytophilum is a gramnegative bacterium that infects neutrophils and other cells. It resides in early
endosomes.
Anaplasma phagocytophilum secretes the protein AnkA, which passes through
the endosome membrane and moves into the host cell nucleus, where it complexes
with nuclear proteins and binds chromatin. In this case, the binding seems to be
biased toward A+T-rich sequences, but otherwise sequence-independent.
Secretion of multiple proteins by Salmonella. Salmonella enterica var.
Typhimurium is a bacterium that parasitizes both humans and mice. It invades cells
of the animal host, typically macrophages, and takes up residence within a host
cell’s cytoplasm. It resides within a special vacuole called the SalmonellaContaining Vacuole.
Salmonella enterica var. Typhimurium has two separate Type III Secretion
Systems. The first is encoded by a region of the bacterial chromosome called
Pathogenicity Island-1, and injects proteins into the host cell from outside. The
second is encoded by Pathogenicity Island-2, and injects proteins into the host cell
from inside the Salmonella-Containing Vacuole. During infection, some 32
proteins are injected from within the Salmonella-Containing Vacuole into the host
cell cytoplasm. Many are known to act within the host cells.
Non-Salmonella protein fragments have been linked to a Salmonella protein
injected from within the Salmonella-Containing Vacuole, and used to provoke an
immune response. A multiple mutant of this same Salmonella strain replicates very
poorly within host cells, but still delivers engineered protein—and is under
development as a vaccine vector.
♦ Safety and other topics
Chapter 7 discusses several additional topics as well. One is the ability that
some pathogens have to move directly between cells of a solid tissue, and how
intracellular xenobiorgs might exploit this.
Chapter 7 also discusses safety, especially containment of intracellular
xenobiorgs. Preventing intracellular xenobiorgs from evolving into disease agents
or contributing genes to existing disease agents would likely involve several
precautions.
The first precaution is to delete genes that enable the source pathogen (the
microbe from which the intracellular xenobiorg is derived) to live outside the body
and to spread between hosts.
The second precaution is to engineer the xenobiorg so that it depends on
nutrients, and perhaps on exogenously added proteins, that the xenobiorg would
not encounter outside of a laboratory. Xenobiorgs would carry a limited supply of
these with them during their service as medical agents, and die when they were
exhausted.
The third precaution is to make the xenobiorg highly sensitive to one or more
chosen antibiotics. These would have to be chosen carefully, so that the
vulnerability of the xenobiorg to antibiotics did not complicate additional antibiotic
therapy that a patient might need.
The fourth precaution is to disable any molecular machinery that enables the
xenobiorg to transmit its genes to other microbes.
The fifth precaution is to engineer any xenobiorg genes considered potentially
dangerous so that they require specialized cellular machinery for expression—
machinery unlikely to be present in other pathogens.
Chapter 7 also describes specialized reagents and engineering techniques that
could be useful to researchers investigating intracellular parasites.
The Human Body as a Target for Localized Intervention (A
Brief Summary of Chapter 8)
Chapter 8 explores the human body as a target for xenobiotherapy, and explores
the logistical, financial, and institutional limits that xenobiotherapy faces.
The main subject of this e-book is how to transport engineered microbes to
some chosen organ of the body and then use those microbes to perform some
medically beneficial act. The most exciting of these possible beneficial acts is the
replacement of senescesced nuclei and mitochondria with more youthful
counterparts—which I refer to as Repnumi rejuvenation. Repnumi rejuvenation is
also logistically the most difficult medical task discussed in this e-book—and since
Chapter 8 explores the limits of what xenobiotherapy might accomplish, it focuses
on Repnumi rejuvenation.
Human tissues and the paths to reach them. Chapter 8 evaluates the human
body as a target: What kinds of tissues are present, and what are the obstacles to
reaching and treating them? What are the basic constraints on moving engineered
microbes to specific organs, and on replacing the nuclei and mitochondria of cells
there? In particular, what are the constraints given the most pessimistic reasonable
assumptions?
Typical vs atypical human tissues. The human body can be considered as
consisting of typical and atypical tissues. Typical tissues are relatively immobile,
with cells having a single nucleus that contains a normal diploid genome. Their
function does not depend on deviation from the normal set of 46 chromosomes or
changes in DNA sequence. The various cell types that compose human skin are
good examples of this.
Atypical tissues include those with multiple nuclei (muscle and liver) and nonsolid tissues such as white blood cells. They also include cells whose function
depends on a heritable genetic rearrangment. Memory B cells and memory T cells
are examples of this latter.
Chapter 8 discusses the problems of inserting xenobiorgs into tissues and then
replacing the nuclei and mitochondria. It considers both typical and atypical
tissues—with the atypical tissues likely to be more difficult targets.
In order to invade tissues, xenobiorgs must first be transported to those tissues.
Chapter 8 considers three possible methods of transport.
Injection of xenobiorgs into chosen tissues. The first method of moving
xenobiorgs to a specific tissue is simply to inject them, using a syringe. The human
body is surprisingly tolerant of slender puncture wounds, and almost any target
outside the cranium and spinal cord can be reached by injection. Chapter 8
discusses this, along with the problems of accurately targeting injected substances.
Access to chosen tissues via the lymphatic system. The second method of
moving xenobiorgs to a specific tissue is entirely hypothetical—it has never been
tried. This method is to use the lymphatic system that drains a specific volume of
solid tissue to instead access that tissue.
The lymphatic system drains tissues, rather than supplying them, and flow of
lymph away from tissues is enforced by a system of valves. Nevertheless, despite
obstacles, the lymphatic system has potential advantages as a method of accessing
tissues, and should be considered. Moreover, learning to navigate the lymphatic
system using xenobiorgs may provide an enormous advantage: the lymphatic
system is a major avenue of cancer metastasis, and if that metastasis could be
monitored or blocked, many lives would be saved. Chapter 8 briefly discusses
xenobiorg navigation of the lymphatic system. It is treated at greater length in
Chapter 9 and 10 (see below).
Access to chosen tissues via the blood. The third method reach target tissues is
to access them via the blood. This is the body’s own method of sending cells to
chosen destinations, and the method that this e-book concentrates on. However, it
faces a key difficulty: the blood bathes all of the body’s tissues indiscriminately,
and targeted cells traveling with the blood must somehow recognize where they are
and exit the blood into the target tissue. The body’s machinery for doing this is
very elaborate, has not yet been fully elucidated, and still makes little sense.
Chapter 11, rather than Chapter 8, discusses the molecular details involved in
sending engineered cells through the blood to a specific target tissue, arranging for
the cells to exit the blood into the target tissue, and then arranging for them to
move through the extracellular matrix to chosen cells. Chapter 8 instead examines
the logistics of the process.
The logistics of access via the blood. Chapter 8 estimates the number of cells
in the human body and the number of cells in a representative organ (the heart).
Chapter 8 estimates the likely efficiency of Repnumi replacement, along with the
possibility that a single replacement nucleus or mitochondrion might rejuvenate as
many as 1024 target cells. Chapter 8 then estimates the number of cells that could
be safely added to the human blood supply. It uses all of this information to
estimate how long total rejuvenation of the human body might take.
Chapter 8 also estimates the time it would take to completely rejuvenate an
adult human body assuming that only one tissue type at a time could be
rejuvenated. The human body has at least 210 distinct tissue types, but the true
number of cell types that differ materially might be much larger. It may be that
many tissues that appear homogeneous are not.
The process by which human cells leave the blood and enter tissues is called
diapedesis. Diapedesis is itself destructive and might limit the rate at which
Repnumi could be performed.
Financial costs. Chapter 8 also estimates the financial costs and benefits of
Repnumi rejuvenation. The costs would include the cost of treatment and the value
time taken away from work. The financial benefits would include increased
productivity in old age, less dependence on the government for financial
assistance, and less dependence on social services.
Chapter 8 compares possible scenarios for administering Repnumi rejuvenation
with the existing state of nursing home facilities and with kidney dialysis
arrangements.
Chapter 8 concludes that the costs of Repnumi rejuvenation might be too high
for American society bear, even for a procedure as desirable as rejuvenation.
Sources of expense. Repnumi’s greatest expense will probably be the
preparation of donor cells carrying nuclei and mitochondria appropriate for
donation to each of the body’s many hundreds of different tissues. The second
greatest expense may be the careful injection and tracking of Repnumi donor cells.
Another expense will be the testing of biological function recovery, and the need to
insure that function is not recovered too unevenly.
Phasing in of new medical procedures. In practice, of course, if Repnumi
rejuvenation is possible, it will be phased in slowly. Some tissues will commonly
be treated before others are. Good candidates for early treatment are the skin of the
face and of the scalp.
Before any rejuvenation is attempted, many of the simpler procedures suggested
in this book will very probably have been tried—and some will probably be
commonplace.
Access to the Body Via the Lymphatic Drainage System (A
Brief Summary of Chapter 9)
Access to specific regions of the body via the lymphatic drainage system.
Human cells navigate from one part of the body (such as the bone marrow) to
another part (such as an inflamed organ) through the blood. This process is termed
“homing.” Nearly all, if not all, of the enormous scientific literature on homing
concerns homing through the blood—and this is also the main method of moving
engineered microbes to specific organs considered in this e-book.
However, Chapters 9 and 10 consider an alternate way of moving xenobiorgs to
chosen tissues: navigation via the lymphatic drainage system. Chapter 9 considers
the lymphatic drainage system itself: how it is organized, how valves within it
control the flow of fluid (which moves in only one direction), and how xenobiorgs
might move against the flow and into the tissue that is the source of the lymph.
Chapter 10 considers a special type of multicellular organism, the motile
microfilaria of certain parasitic nematodes, that might be exploited to move
passenger xenobiorgs upstream.
Lymphatic drainage collects fluid from tissues. The human circulatory
system consists of two parts: the blood circulatory system and the lymphatic
drainage system. The blood circulatory system consists of arteries, blood
capillaries and veins. Because the blood supply is under pressure and because
blood vessel walls are porous, fluid moves from the blood into solid tissues. Unless
it is removed, this fluid will swell the tissues, causing edema. The lymphatic
system collects this fluid and pumps it (against pressure) back into the blood.
Lymphatic drainage prevents unhealthy accumulations of fluid that would
otherwise occur spontaneously.
The lymphatic drainage system is organized like a tree, with the vessels that
collect lymph corresponding to the smallest twigs and the largest lymphatic vessels
that receive the combined volume of lymph corresponding to the tree trunk. This
tree-like organization, combined with the thoroughness with which the lymphatic
system drains tissues, might be important advantages in concentrating drugs or
other medical agents uniformly within a complex structure such as an infected lung
or a cancer growing in bone and muscle.
The smaller branches of the lymphatic drainage system differ antigenically
from the larger branches. This might be useful in helping xenobiorgs navigate the
system.
Lymphatic capillaries. The first vessels in the lymphatic drainage system are
lymphatic capillaries. Lymphatic capillaries are permeable to fluid. Their main
purpose is to absorb excess fluid from the tissues they lie in.
A defining feature of lymphatic capillaries is that they contain no valves to
control the flow of fluid. They are culs-de-sac that follow a tortuous course,
frequently merge, and sometimes expand into dilated regions called ampullae.
Lymphatic capillaries are the smallest lymphatic vessels, about 20 µm in
diameter. This is large enough to allow all leukocytes to flow through, and
probably large enough to allow cells to slip past each other. However, the
smallness of lymphatic capillaries, combined with the twisted paths that the
capillaries follow, might inhibit the movement of mobile xenobiorgs within
them—particularly if xenobiorgs had to slip past leukocytes traveling in the
opposite direction. On the other hand, as mentioned above, lymphatic capillaries
have no valves to block upstream migration of xenobiorgs.
Lymphatic pre-collecting vessels. Lymphatic capillaries coalesce to form precollecting vessels. Pre-collecting vessels resemble lymphatic capillaries in
following a tortuous course and in having permeable walls. However, they are
somewhat larger than lymphatic capillaries, are surrounded by irregularly
distributed smooth muscle cells, and include occasional one-way valves. These
latter two features enable them to pump lymph in the direction of larger
downstream vessels. The valves and the flow of liquid would oppose the
movement of motile xenobiorgs upstream (toward smaller vessels), although these
obstacles may be superable (see below).
Lymphatic pre-nodal collecting vessels. Lymphatic pre-collecting vessels
empty into pre-nodal collecting vessels. These pre-nodal collecting vessels are
sheathed in smooth muscle. They have three-layered walls and valves to direct
lymph flow in only one direction. These larger collecting vessels often follow a
tortuous and very complex path on their way to draining nodes.
Pre-nodal collecting vessels empty into one or more lymph nodes in series.
Eventually, the chain of lymph nodes ends, and the vessels become post-nodal
lymphatic vessels.
Human lymphatic vessels eventually drain into either the thoracic duct or the
right lymphatic duct. From there, lymph is pumped into the blood circulation.
Predictable patterns combined with haphazardness. The concentration of
lymphatic vessels varies between organs, and there are organ-specific differences
in lymphatic vessel structure. The two halves of a human body are not perfectly
symmetrical in lymph vessel organization, and there are clear differences between
individual people. Lymphatic vessels often bypass nodes that they would be
expected to join. The organization of lymphatic vessels appears to be a
combination of predictable patterns and haphazardness.
As mentioned above, the overall organization of the human lymphatic drainage
system resembles a tree, with lymphatic capillaries as the smallest twigs and the
thoracic duct or the right lymphatic duct as the trunks. However, close inspection
of human cadavers shows that many deviations from this pattern also occur.
The non-treelike and unpredictable branching of lymphatic networks might
complicate efforts to move xenobiorgs upstream through them. Blind alleys, in
particular might be problematic, because attempts to disperse xenobiorgs into
every branch of a network could inevitably maroon some xenobiorgs in culs-desac.
Lymph channels run parallel to blood vessels. An interesting feature of
lymphatic vessels is that they tend to run parallel (or anti-parallel) to arteries and
veins. Those that run parallel to arteries tend to be richer in oxygen than they
would otherwise be (due to diffusion of oxygen from the arteries)—a fact that
might improve the habitability to lymphatic vessels for xenobiorgs. (However, the
partial pressure of oxygen in lymphatic vessels appears generally to be adequate to
support aerobic xenobiorgs.)
Schemes to create new lymphatic vessels specifically to distribute xenobiorgs,
and attempts to simultaneously access tissues from both the lymph and the blood,
might benefit from the tendency of lymphatic vessels to parallel blood vessels.
Engineering of new lymphatic networks. Lymphatic networks can regenerate
when destroyed by injury. In addition, they can be remodeled: the new network can
differ significantly from the old. Hence, it might be possible to create new
lymphatic networks to access specific target organs, although this would of course
take time.
Lymph node positions and purpose. Most, if not all, lymphatic collecting
vessels encounter at least one lymph node before finally joining the thoracic duct
or right lymphatic duct and emptying into the blood. Often, lymphatic collecting
vessels encounter as many as 5 or more nodes. Humans have 500-600 lymph nodes
throughout their bodies. Nodes can differ in diameter by at least a factor of 5.
A major purpose of lymph nodes is to introduce the immune system to foreign
antigens. Antigens in tissue are captured by tissue-resident dendritic cells and
taken to the local draining lymph node, where they are presented to naïve T cells.
Lymph nodes are specialized to maximize encounters between antigens and T cells
that might recognize them
Lymph nodes and cancer metastasis. The lymphatic system is a major avenue
of cancer metastasis. Cancers specifically suspected of metastasizing through
lymphatic channels include malignant melanomas, oral cancers, breast cancers,
head cancers, and neck cancers, among others. Blocking the spread of cancers by
intercepting them within the lymphatic system is critically important to cancer
management. Doctors usually hope to intercept cancers at sentinel nodes. A
sentinel node is the first node along any collecting vessel that drains the primary
cancer site. Sentinel nodes are not always the nodes closest to the cancer because,
as mentioned above, lymphatic vessels can bypass nodes.
Mapping of lymphatic drainage near cancers. Mapping of the lymphatic
drainage system near a cancer such as a malignant melanoma can be critically
important to preventing the spread of that cancer. Normally mapping is attempted
with inert particles such as radioactive colloids and contrast agents such as
gadolinium. In some cases, the particles are phagocytosed by white blood cells and
taken to lymph nodes. However, xenobiorgs which carried either radioactive
tracers or contrast agent—and which were free to spread without limit—might well
be better mapping agents.
Killing of cancers within lymph nodes. Current techniques to prevent spread
of cancers through lymph channels are primitive. Typically, they involve removing
nodes and channels that are infected with cancer or likely to become infected—
leaving patients with the problem of lymphedema. However, eventually it might
become possible to engineer lymph nodes to trap and kill spreading cancer cells.
Xenobiorgs (either intracellular or extracellular) within the node could attack the
cancers directly or indirectly (by stimulating other cells to do it).
Lymph nodes seem to be promising places to waylay cancer cells, because they
seem designed to promote meetings between large numbers of cells. This situation
could provide a good way to survey populations of cells for cancer cells.
Engineering lymph nodes to control local allergy. The ability to engineer
lymph nodes might help control some types of allergy. Allergic reactions are often
restricted to specific sites such as lung, skin, or gastrointestinal tract—an may be
accompanied by activity in associated lymph nodes. Xenobiorgs within such lymph
nodes might interfere with allergy flare-ups.
Lymph node engineering might be aided by the fact that lymph nodes can be
transplanted.
The need for continued research. Cancers induce the formation of new
lymphatic vessels, and may then metastasize through those vessels. This problem,
and the need to stop it, guarantee that de novo lymphatic vessel formation will
continue to be studied.
An interesting finding is that increases in the amount of dissolved oxygen in a
tissue tend to suppress formation of new lymphatic vessels. Understanding of how
this happens might allow the same effect to be produced by substances other than
oxygen.
Nodes as injection sites. Lymph nodes may be good ports for injecting
xenobiorgs that are engineered to travel upstream (against the flow of lymph). This
idea is discussed in Chapter 9.
The flow of lymph. As described above, lymph begins as blood plasma. It
seeps into tissues from blood vessels, and thereafter is collected by the lymphatic
drainage system and returned to the great veins of the neck.
Rates of lymph flow vary between tissues.
One-way valves control lymph flow. Lymph flows in one direction only. This
is due in part to the fact that blood is under pressure, but unidirectional flow is also
enforced by two types of valves. The first type of valve (the primary valve) allows
fluid to enter lymphatic vessels from surrounding tissue, but prevents back-flow.
The second type of valve (the intra-luminal valve) exists within lymphatic channels
and allows flow only from smaller to larger vessels.
Enabling xenobiorgs to swim against the flow of lymph. In order to access
tissues by way of the lymphatic system, it will be necessary to move xenobiorgs
upstream—not only overcoming the current, but moving through the intraluminal
valves against the usual direction of flow. Chapter 9 discusses how this might be
done.
The flow of lymph through the drainage system is aided by rhythmic pumping
of the walls of larger lymphatic vessels. It has been reported that when pumping is
not necessary to maintain the flow of lymph in the normal direction, the
intraluminal valves remain wide open. This might be the situation most
advantageous for moving xenobiorgs upstream.
The diameters of lymphatic vessels and their pumping are influenced by
calcium ions, nitric oxide, substance P, acetylcholine, bradykinin, histamine,
catecholamines, endothelin-1, thromboxane, and prostaglandins. These, along with
externally applied pressure, an externally applied magnetic field, or localized
electric stimulation, might influence the flow of lymph the valves that control
lymph flow. Judicious application of these stimuli might allow xenobiorgs to swim
upstream.
Cancer metastasis is probably aided by lymphatic pumping. Moreover, some
cancers may produce chemical signals that inhibit this pumping and thus slow their
own spread. This is obviously of interest to cancer treatment, and guarantees
further investigation.
Moving xenobiorgs from lymphatic channels into surrounding tissues. The
final step in moving xenobiorgs into chosen tissues via the lymphatic drainage
system would for the xenobiorgs to exit the lymphatic capillaries into surrounding
solid tissue. There are at least three ways in which they might do this. First, they
might simply poke a hole in the lymphatic vessel wall and move through that hole.
Second, they might be able to move through the so-called primary valves that
allow fluid to flow into lymphatic capillaries, but not the other way. For this to
succeed, the xenobiorgs would have to open or pry apart the primary valves in
order to squeeze through, and could not be wider than the primary valves.
Third, xenobiorgs might be able to exit lymphatic capillaries via diapedesis.
Diapedesis is a mysterious process by which leukocytes (white blood cells) exit
blood-carrying capillaries into surrounding tissues either by squeezing between
endothelial cells or by moving through endothelial cells (both versions are known
to occur). Lymphatic capillaries are also lined with endothelial cells, and it appears
that leukocytes can traverse the endothelial cell lining in both directions.
At present, the idea that xenobiorgs might exit lymphatic capillaries by
squeezing between endothelial cells seems to be the most reasonable.
Use of Engineered Nematode Microfilariae as Medical
Agents (A Brief Summary of Chapter 10)
The need for xenobiorgs to move against the flow of lymph. Unlike the
blood vasculature, the lymphatic drainage system does not complete a circuit. A
xenobiorg cannot reach an upstream region of the lymphatic drainage system by
making a second pass through the system. A xenobiorg can reach an upstream
region only by crawling or swimming against the flow of fluid—and to do this, the
xenobiorg must be motile.
Carriers of single-celled xenobiorgs. Although single-celled xenobiorgs might
crawl or swim upsteam in the lymphatic drainage system, against the current and
despite the one-way valves, they would probably reach upstream regions more
efficiently if they were carried there. Chapter 10 describes a group of small,
multicellular organisms that might serve as carriers.
The organisms that Chapter 10 describes are the microfilariae (embryonic
larvae) of parasitic nematodes. Such microfilariae would have several advantages
as intra-lymphatic xenobiorgs.
Microfilaria are small. First, they are small enough to navigate lymphatic
capillaries.
Filarial nematodes inhabit the lymphatic drainage system. Second, although
microfilariae do not reside in lymphatic vessels, other life cycle stages of the same
species find and colonize lymphatic vessels. These may reside in lymphatic vessels
for years, resisting the immune system but causing no harm. The same abilities
might be activated within microfilariae.
Parasitic nematodes are good candidates to form the basis of a lymph-traveling
xenobiorg because such nematodes are the only pathogens known to inhabit the
lymphatic drainage system. The absence of other foreign organisms may, at least in
part, be due to the presence in lymph of large numbes of lymphocytes—immune
system cells likely to attack invading microorganisms.
Microfilaria are can seek or avoid a stimulus. Third, microfilariae are motile
and can seek chemical attractants. Like other nematode stages and/or species they
may also be able to seek physical attractants such as heat, and may be able to flee
chemical repellants―although no microfilariae are yet known to do either. The
ability to flee a repellant might be useful in dispersing microfilariae throughout a
lymphatic network. If no useful microfilariae have the desired abilities, genetic
machinery to support those abilities might be imported from other nematode
species.
C. elegans is a very useful model. Fourth, the parasitic nematode species that
would supply the microfilariae belong to the same phylum (Nematoda) as the bestcharacterized multicellular organism, Caenorhabditis elegans. Study of C. elegans
focuses on how it moves and how it decides to move. Powerful tools exist to study
C. elegans, and many of these might be applied to parasitic nematodes. Thus, the
behavior of microfilariae is probably programmable using well-understood
neurological principles.
Natural prokaryotic endosymbionts. Fifth, microfilariae carry prokaryotic
endosymbionts that may be useful in engineering and controlling them. These
endosymbionts (members of the Wolbachia genus) are also mentioned in Chapter
7.
Filarial nematodes warrant more research. Parasitic filarial nematodes cause
lymphatic filariasis. This disease affects more than 120 million people in the
tropics, and endangers more than 1 billion people. It causes elephantiasis, a
disfiguring and debilitating disease. The 3 parasitic nematodes that cause
lymphatic filariasis—Wuchereria bancrofti, Brugia malayi, and Brugia timori—
have been far less studied than the burden they impose warrants. Furthermore,
there is the danger that drug-resistant variants may emerge. Therefore, continued
research on these 3 species is likely.
A list of parasitic nematodes with microfilaria. Wuchereria bancrofti, Brugia
malayi, and B. timori are the best candidates to supply engineered microfilaria.
However, Chapter 10 discusses other parasitic nematodes that might supply
microfilaria that could be engineered. These include Loa loa (the African
eyeworm), three species of Mansonella, Onchocerca volvulus, and a number of
nematodes that parasitize non-human mammals.
Difficulty in culturing. Wuchereria and Brugia have the disadvantage that they
are difficult to propagate in laboratories. This is especially true of Wuchereria.
Chapter 10 discusses the techniques used, the progress that has been made, and the
progress that might be possible.
Physical signals that might control microfilariae. Engineered microfilaria
would be much more useful if their behavior within human lymphatic drainage
networks could be controlled. Physical signals to control xenobiorg behavior might
include sound, subsonic vibration, heat, cold, visible light, magnetic attraction, or
electromagnetic radiation.
Chemical signals that might control microfilariae. For a chemical to control
the movement of an engineered microfilaria, it would have to form a gradient that
the microfilaria could perceive and follow toward either greater or lesser
concentrations. Such a chemical could be applied to the skin in a patch, injected
subcutaneously, or injected into a lymph node.
Other chemical signals, such as signals for the microfilariae or xenobiorgs to
disperse, congregate, or secrete medicines, need only be perceived, but not
followed. These could be injected into the blood.
There are an enormous number of nematode species in nature, and many of
them respond to specific chemicals. The number of known chemical attractants for
nematodes is itself quite large.
Tools to analyze nematode anatomy and behavior. Chapter 10 describes the
progress that has been made, or seems possible, in analyzing filarial nematode
anatomy and behavior. The well-studied nematode Caenorhabditis elegans will
serve as a model. Green Fluorescent Protein and its derivatives are likely to be very
useful. Analysis of nematode structure and function has been automated so that
large numbers of individuals can be processed quickly with a large number of
assays. Optical tweezers aid the automation, as does bio-electrospraying. Rapid
sorting of many specimens can produce more homogeneous material for
experimentation, which can be very important in interpreting experiments
involving, for example, RNA interference. Ultrafast laser surgery is used to study
the effects of inactivating specific nerves.
Genetic engineering goals for filarial nematodes. If microfilariae are to serve
as the basis for lymphatic xenobiorgs, methods to genetically engineer the
microfilariae will have to be developed. Several types of change to microfilarial
genomes will be necessary. First, unwanted genes, such as those that irritate the
host or provoke an immune reaction, must be silenced. Second, the ability to move
toward certain chemical or physical cues and away from others will have to be
added. Third, automatic maneuvers to escape from entrapment (see below for a
longer discussion) will probably have to be programmed in. Fourth, microfilariae
must be given the ability to deliver medicines and perhaps living cells. Fifth, they
may need additional abilities to thwart the human immune response. Sixth, they
may need to be programmed to die on schedule or on cue.
Genetic engineering techniques for filarial nematodes. Chapter 10 describes
techniques that have been used to engineer the genome of Caenorhabditis elegans.
One of the most useful is “recombineering”, combined with the use of so-called
“fosmids.” A second is chemical mutagenesis, combined with high-throughput
sequencing. A third is transposon-mediated alteration, using a Mariner-class
transposon from the fruit fly Drosophila melanogaster. A fourth is genetic
engineering by microparticle bombardment. A fifth technique is targeted insertion
of DNA segments using engineered zinc finger nucleases, possibly combined with
Tat-mediated protein transduction. As many as possible of these will have to be
adapted for use on filarial nematodes.
The complication of trans-splicing. One important complication in
engineering the genome of Brugia malayi and related nematodes is that messenger
RNAs are processed by trans-splicing. The details of this trans-splicing have not
yet been fully worked out, and yet predictable expression of engineered genes will
depend on it.
The usefulness of RNA interference. Chapter 10 also describes another
important technique in genetic analysis of nematodes, the use of RNA interference.
In Caenorhabditis elegans, RNA interference can produce heritable gene silencing.
Unlike mutation, RNA interference can silence multiple copies of a gene, which is
a great advantage when an investigator hope to silence genes in diploid organisms
(such as nematodes) or to silence gene families.
RNA interference used on filarial nematodes. RNA interference also
succeeds in filarial nematodes, although it is less useful than in Caenorhabditis
elegans. There is reason to hope that RNA interference machinery from some other
species might be transferred to Brugia or Wuchereria. Caenorhabditis elegans
might be a good source of this machinery. Alternatively, the yeast Saccharomyces
castellii might be a source of simpler RNA interference machinery. It is also
plausible that controlling nutrition in filarial nematodes might influence their
susceptibility to RNA interference. In addition, engineered bacteria might invade
the cells of filarial nematodes and supply the machinery to support RNA
interference, the RNAs to induce RNA interference, or both.
Susceptibilitity to RNA interference in filarial nematodes tends to be stagespecific, with microfilariae not susceptible. However, it is reasonable to hope that a
way of inducing this susceptibility precociously might be found.
It is also possible that RNA interference will often be manifested only in in
progeny of the engineered worms, as is the case in Caenorhabditis elegans.
Analysis of progeny is difficult or impossible in such parasitic nematodes as
Brugia malayi, which must cycle through an insect vector.
Silencing of mRNA with artificial polymers. As alternatives or supplements
to RNA interference, oligomers incorporating unnatural bases can be used to
silence gene activity. These oligomers may incorporate “locked” nucleotides along
with phosphorothioate ester bonds. Alternatively, “morpholinos” may be used to
silence chosen genes. These alternatives are most useful when interference using
RNA fails because the RNA molecules used are too large to enter the target cells,
too susceptible to nucleases, or too easily dislodged from the target mRNA by the
translation machinery. Since microfilariae are tiny creatures and quite slender, it
might be relatively easy to insert oligomers into all of their cells by soaking.
Although RNA interference (described above) prevents mRNA from being
translated, RNA can also keep genes from being transcribed. This phenomenon is
called RNA-Induced Transcriptional Silencing (RITS), and requires doublestranded RNA with homology to the DNA to be silenced. In Caenorhabditis
elegans, silencing is heritable. RNA can also activate specific genes, perhaps by a
mechanism similar to RITS. These techniques might also be applicable to filarial
nematodes.
Online scientific resources. There are many online facilities dedicated to
sharing scientific information about Caenorhabditis elegans and other nematodes.
Chapter 10 evaluates a number of these.
Abilities that engineered microfilariae might need. Microfilariae that have
been engineered to navigate the human lymphatic drainage system may need a
number of special abilities. These might include the following:
First, as discussed above, engineered microfilariae would need the ability to
move upstream against the flow of lymph. In order to do this, they would of
course, need some means to determine which direction was “upstream.”
Second, also as discussed above, engineered microfilariae would need the
ability to traverse the valves that keep lymph moving in only one direction. Large
numbers of lymphocytes move through these valves, but engineered microfilariae
would have to traverse them in the opposite direction.
Third, engineered microfilaria might become trapped within the lymphatic
system. Chapter 10 discusses three kinds of trapping predicted to occur, the types
of harm they might do, and how they might be prevented or reversed.
Fourth, engineered microfilariae may have to carry either single-celled
xenobiorgs or human cells that carry intracellular xenobiorgs. In the appropriate
place, they will have to disgorge these, so that they can act.
As an alternative, engineered microfilariae may have to carry drugs that they
will disgorge in the proper place. They may then have to plug the lymphatic vessel
that they occupy in order to prevent the drug from diffusing away from its target
tissue.
Fifth, xenobiorgs may be useful in staining lymphatic vessels, so that complete
lymphatic networks can be outlined when viewed by magnetic resonance imaging
or some other imaging technique. This would be an enormous help in controlling
melanomas and other cancers. But, staining lymphatic vessels would require that
the xenobiorgs carry a stain that can be tracked by imaging.
Sixth, an early use of lymphatic xenobiorgs would likely be to attack and kill
adult filarial nematode parasites in infected patients. The adult stages of filarial
parasites resist drugs. Microfilarial xenobiorgs would be ideal for the task of
killing filarial adults, but of course would have to be engineered to navigate toward
their targets, sense when they had reached their targets, and then attack the targets
in some way.
Seventh, when microfilarial xenobiorgs had finished their assigned tasks, they
would have to be removed somehow. They might be engineered to die cleanly,
provoking no immune response. As an alternative, they might be engineered to
swim downstream in the lymphatic system until they enter the blood, and perhaps
eventually to burrow into the intestinal lumen and exit the body in the feces.
Chapter 10 discusses how this might be accomplished.
Induction of taxis in microfilaria. Although it is clear that microfilaria can
move, in most cases it is not certain whether they are capable of directed motion,
i.e. motion toward an attractant or away from a repellant. As Chapter 10 discusses,
our current knowledge of nematode locomotion indicates that every attractant can
be converted into a repellant and vice-versa.
Chapter 10 discusses one known case of microfilarial chemoattraction
(Onchocerca lienalis). These microfilariae might be engineered to perform the
tasks suggested in this chapter. Alternatively, and more likely, the machinery that
supports this chemoattraction might be genetically moved into Brugia or
Wuchereria and expressed in microfilariae.
Chapter 10 also discusses the taxis toward lymphatic vessels shown by filarial
nematodes such as Brugia pahangi. These appear to move between lymphatic
vessel types as they mature—a trait that might also be exploited.
Physical stimuli known to attract nematodes include light, heat, and vibration.
In addition, there are a large number of chemical attractants for nematodes that
exist in nature. Receptors and neural connections for many of these might be
moved into engineered microfilariae. Chapter 10 discusses some of the large
number of known attractions.
Nematode repellants also exist. Some are made by the nematodes themselves,
and may help disperse populations. A combination of attractants and repellants
might allow doctors to both herd and disperse engineered microfilariae within the
lymphatic vessels of human patients.
Methods to deliver chemoattractants might include drops applied to the eyes,
mists applied to the nasal passages and lungs, patches applied to the skin, pellets
injected under the skin, and continuous-feed reservoirs delivering attractant to a
tissue or lymphatic system. Chapter 10 discusses the problem of maintaining over
time a chemical gradient within the human body.
Control of taxis in nematodes. Chapter 10 discusses seven principles that
guide neural control over directed movement. In addition, it discusses sources of
confusion in the intepretation of behavioral experiments.
The opportunity and complication of learned behavior. Caenorhabditis
elegans adults can learn from their experiences, and this learning can be
surprisingly sophisticated. Microfilariae are much simpler than adult nematodes,
and it is not yet known whether any of them can learn. If so, this ability will
present both an opportunity and a complication for the medical control of
microfilarial behavior.
Crawling vs. swimming. Caenorhabditis elegans normally crawls, while
microfilariae normally swim. These are distinct behaviors, and comparisons
between the two nematode types should be made with this in mind.
Overshoot, circling, and aging. The behavioral peculiarities of overshoot and
circling can cause misinterpretation of experiments on nematode behavior unless
they are adequately accounted for. In addition, aging affects nematode behavior
and must be controlled as an experimental variable.
Computer modeling. Nematode behavior consists of a stereotyped actions
whose frequencies increase or decrease depending on conditions. Because of this,
nematode behavior can be convincingly modeled on computers and tested against
records of actual behavior. Chapter 10 describes the kinds of movements that
Caenorhabditis elegans performs and how this leads to directed locomotion.
Magnetic manipulation of microfilariae. Microfilariae might be guided to
targets within the human body by magnetism or electromagnetic radiation, if the
microfilariae contained an effective sensor. Magnetite contributes to magnetic
sensing in a number of species, including some bacteria, and is present in
Caenorhabditis elegans.
Chapter 10 discusses the possible use of magnetite as a magnetic guide in
microfilaria. Either the microfilaria themselves or their Wolbachia bacterial
endosymbionts might harbor the magnetite. Either way, it would be necessary to
transmit magnetic information to microfilarial neurons.
Engineered microfilariae and the human immune system. The general
question of how xenobiorgs can evade the immune response is dealt with in Chaper
20. However, Chapter 10 discusses certain problems specific to engineered
microfilariae.
Helminth parasites, a group that includes nematode parasites, steer the human
immune system away from a Th1-type (inflammatory) response and toward a Th2type (i.e. anti-inflammatory) response. To the extent that a Th2 response can be
induced locally, rather than systemically, Th2 induction might protect microfilarial
xenobiorgs without harming the host. If so, the entire set of Th2-inducing tricks of
a large group of human and animal parasites is available for genetic installation.
Chapter 10 specifically discusses the defenses that filarial nematode parasites
have against the human immune system. The defenses of greatest interest are those
that mask the parasite from the immune system without disabling the immune
system and those which engineering might restrict to limited regions of the body.
Defenses that non-filarial helminth parasites mount against the human immune
system are also discussed in Chapter 10. As with filarial nematode parasites
emphasis is on those that merely mask the parasite or which can be restricted to
small regions of the body.
Also discussed is the possibility that many people may have pre-existing
immunity to Wuchereria and Brugia nematodes.
Other uses of microfilariae. Although the main use of engineered
microfilariae is envisioned to be the transport of single-celled xenobiorgs in the
upstream direction within the lymphatic drainage system, engineered microfilariae
may have other uses as well. Chapter 10 makes the case that in the blood they
might secrete insulin or dissolve blood clots. Chapter 10 also elaborates on the
possiblity that engineered microfilariae might attack adult filarial worms within
lymphatic drainage networks, and on the possibility (mentioned above) that they
might delineate lymphatic drainage networks as part of an effort to prevent cancer
metastasis. Microfilariae might also slither into lymphatic capillaries, secrete
medicines, and block the capillaries so that the medicines would eventually diffuse
into surrounding tissues, rather than be drained away by the lymphatic drainage
system.
Entry into solid tissues by trans-endothelial migration. Most tasks done by
xenobiorgs would likely be done within solid tissues. I envision single-celled
xenobiorgs being delivered to solid tissues by engineered microfilaria in much the
same way that landing craft delivered soldiers to French beaches on D-Day.
However, the xenobiorgs will have to enter the surrounding solid tissues from
within lymphatic capillaries, and they will have to do it on their own—the
microfilaria will not be able to take them there. How might they do this?
There is a process known by several names (trans-endothelial migration,
extravasation, diapedesis), that I refer to as trans-endothelial migration. Transendothelial migration transports lymphocytes past the endothelial cells that line
blood vessels and into the surrounding solid tissues. In some cases lymphocytes
move between endothelial cells while in other cases they actually move through
endothelial cells—and in both cases, the cooperation of endothelial cells is
required.
At least with the endothelial cells that line blood vessels, migration can be in
either direction. This is presumed true also of the endothelial cells that line
lymphatic vessels—but has not been demonstrated.
Most of the anticipated value of using engineered microfilaria to swim upstream
in lymphatic drainage vessels lies in their presumed ability to transport cells that
can exit lymphatic capillaries without causing great damage. Establishing that
transported cells can in fact do this is a key early task in any project to access
tissues by way of the lymphatic drainage system.
Chapter 10 discusses this idea in detail.
Additional questions. Chapter 10 ends by raising several additional issues, a
few of which were mentioned previously.
After engineered microfilariae have accomplished their assigned medical tasks,
would it be necessary to remove them from the body. If so, how could it be done?
Also, it seems likely that microfilariae within the lymphatic drainage system will
sometimes become trapped. Would it be a medical necessity to free them, or could
they be allowed to die and degenerate in situ? If it were necessary to free them,
how could this be done?
How can engineered microfilariae be contained so that they do not spread from
person to person? If they were used in areas where filarial diseases are common,
they might be spread by mosquitos.
Brugia malayi is the most likely source of engineered microfilaria. Can
researchers create at least one strain of Brugia malayi that is easily cultured?
Can tissue of the more common filarial nematode Wuchereria bancrofti be
cultured? This might supply enough DNA to permit sequencing of the Wuchereria
bancrofti genome. This, in turn, might allow Wuchereria bancrofti to be used as a
source of engineered microfilaria, and might make Wuchereria adults more
vulnerable to attack by engineered microfilaria.
Can the components of systemic RNA interference by transferred from
Caenorhabditis to a strain of—for example—Brugia malayi? Can transposon
manipulation, recombineering, microparticle bombardment, and automated
analysis similarly be transferrred?
How much direct harm and indirect harm (by provoking the immune system) do
microfilariae do, and could any harm be reduced by genetic engineering?
Do microfilariae persist in the blood long enough to be useful as medical
agents? Can their tendencies to move between various blood compartments over
the course of a day be harnessed to concentrate them in specific blood regions?
Homing to Chosen Tissues By Way of The Blood (A Brief
Summary of Chapter 11)
♦ Homing Requirements Imposed by the Nature of Blood
Circulation
Homing via the blood vasculature. The human body has its own methods to
move specific types of cells to tissues where those cells are needed. These methods
do not use the lymphatic drainage system, as discussed in Chapters 9 and 10, but
instead use the blood vasculature. Exploitation of these methods is probably the
most promising way to move xenobiorgs to their targets. Moving of cells within
the body to specific target location is termed homing.
The requirements of homing via a circuital system. The blood vasculature
differs from the lymphatic drainage system in that the blood vasculature forms a
complete circuit. A blood cell can eventually visit any part of the body served by
the blood vasculature simply by traveling with the flow of blood. From the
standpoint of a physician who might want to move xenobiorgs to some target tissue
within a patient’s body, the circuital nature of the blood vasculature is both an
advantage and a disadvantage. The advantage is that xenobiorgs injected into the
blood anywhere in the body can eventually drift to almost any part of the body.
The disadvantage is that the xenobiorgs must somehow be “informed” that they
have arrived at their destination tissue and that they should exit the vasculature.
Two stages of blood-based homing. Movement of cells from a tissue of origin,
such as the bone marrow, to a destination tissue occurs in at least two steps. First,
the mobile cells must enter the blood, travel to their destination tissue, and exit the
blood. (This may be more than one step). Second, they must travel through the
extracellular matrix to their proper site of action.
Recognition between seeker and target cells is not enough. The simplest way
conceptually for cells to seek other cells is for the target cells to carry a set of
surface ligands and the seeker cells to carry the cognate receptors. Recognition
would occur only when there was a perfect match of all receptors and ligands.
However, seeker cells that never leave the blood would never encounter their
partners. Seeker cells that do leave the blood and filter through tissues might take a
very long time to encounter their partners. Something more is needed.
Recognition between seeker cells and endothelial cells. In fact, it is the
endothelial cells that line blood vessels in the target tissue that provide the
necessary ligands. Two types of molecules play key roles in the interactions that
lead seeker cells to exit the vasculature in a specific region; these two types of
molecule are selectins and integrins, along with the macromolecules that bind
them. Chapter 11 discusses the roles of selectins, integrins, and other
macromolecules in homing of circulating cells to specific tissues.
The processes by which solid tissues stimulate nearby endothelial cells to bind
circulating cells are discussed. The sequence of events from rolling adhesion to
tight adhesion to trans-endothelial migration is discussed. Distinct types of
seeker cells interact with endothelial cells in different ways; elucidating the reasons
for the differences might help optimize xenobiorg homing.
The role of chemical gradients. Most cells that can home to a specific organ or
tissue show in vitro chemotaxis toward at least one attractant molecule. However,
chemotaxis requires that the attractant molecule form a gradient and blood
circulation would be expected to mix and quickly destroy any chemical gradients.
Therefore, it seems likely that the gradients function during the second stage
(defined above) of homing.
♦ The Molecular Machinery of Homing
A list of involved macromolecules. Chapter 11 lists the macromolecules,
especially the ligands and receptors and chemoattractants, known to be involved in
homing within humans. The list is quite long.
Chemokines. Chemokines are a superfamily of small polypeptides, most of
which include 90-130 amino acid residues. They control the adhesion, chemotaxis,
and activation of many types of leukocytes. They are major regulators of leukocyte
traffic, and control leukocyte behavior under both normal and abnormal (e.g.
inflammation) circumstances.
Chemokines cause leukocytes to enter target tissues by inducing the leukocytes
to adhere to vascular endothelium. After they migrate through vessel walls and into
tissues, leukocytes move toward high local concentrations of chemokines.
Chemokines can be divided into four subfamilies: CXC, CC, CX3C and XC.
A great deal has been learned about the cell surface receptors that bind
chemokines and transduce a signal that reports the binding to the interior of the
cell. Some chemokines bind more than one receptor, and some receptors bind more
than one chemokine. Cells integrate information from multiple chemokines in
order to “choose” optimal behavior
Chemokines function in processes other than control of leukocyte movement.
They are also involved in angiogenesis (growth of new blood vessels) and
hematopoiesis. Any plan to use chemokines to control xenobiorg homing would
have to consider possible side-effects.
Selectins. Selectins are among the molecules that mediate recognition and
binding between leukocytes and endothelial walls. They mediate an early step of
leukocyte extravasation, reducing the speed of leukocytes as they roll along blood
vessel walls, which enables the leukocytes to move through the wall.
There are three selectin types. E-selectin is expressed in the endothelium that
lines blood vessel walls. L-selectin is expressed in leukocytes generally. P-selectin
is expressed in blood platelets and in endothelial cells.
Selectins participate in constitutive lymphocyte homing, inflammation and in
cancer metastasis. The inflammation may be either a desirable response to
pathogen invasion or an undesirable contribution to post-ischemic inflammation,
atherosclerosis, glomerulonephritis or lupus erythematosus.
Chapter 11 discusses selectins and the possible application of selectins to
xenobiorg homing.
Integrins. Integrins are another component of selective binding of mobile cells
to their targets. Integrins are heterodimers that consist of one α protein chain and
one β chain. Mammals, including humans, have 18 α and 8 β chains. Some of the
chains exist in distinct variants caused by alternative splicing; for example, there
are 4 variants of the β1 subunit. Through different combinations of α and β chains,
about 24 distinct integrin dimers form within the human body.
Integrins involved in leukocyte extravasation are present mainly on leukocytes,
and bind partners on the vascular endothelium. For example, integrin LFA-1
(Lymphocyte function-associated antigen 1; αLβ2) is present on circulating
leukocytes and binds ICAM-1 and ICAM-2 on endothelial cells. Integrin Mac-1
(Macrophage-1; αMβ2) is present on circulating leukocytes and binds ICAM-1 on
endothelial cells. Integrin VLA-4 (Very Late Antigen-4; α4β1) is present on
leukocytes and endothelial cells, and facilitates chemotaxis.
A signal that originates inside of a cell directs integrins on the cell surface to
switch from a low-affinity to a high-affinity binding conformation. Integrins
mediate firm adhesion of circulating cells to endothelial walls, a step that follows
rolling adhesion mediated by selectins.
Solutions to the problem of moving xenobiorgs to target tissues will very likely
exploit integrins. Chapter 11 discusses the possible use of integrins in homing
schemes.
The extracellular matrix. After exiting a blood vessel, a xenobiorg will have
to navigate the extracellular matrix in order to reach target cells. The extracellular
matrix greatly affects the structure, viability, and functions of cells that reside
within it. The extracellular matrix provides signals that affect adhesion, shape,
migration, proliferation/survival, and differentiation of cells.
The extracellular matrix includes proteins, proteoglycans (polysaccharides
attached to proteins) and glycosaminoglycans. Glycosaminoglycans are a class of
polysaccharides derived from hexosamine that form mucins when complexed with
proteins.
Intact, healthy extracellular matrix displays many signals that affect cells.
However, many other signals are visible to cells only after they have been exposed
by damage and degradation of the matrix. Fragments from from collagens I and IV,
elastin, fibronectin, laminins, entactin/nidogen, thrombospondin and hyaluronan, to
give a few examples, all mobilize inflammatory cells.
Some bacteria can bind the extracellular matrix through MSCRAMMs
(microbial surface components recognizing adhesive matrix molecules). This
binding is an early step in colonization and subsequent infection. These
MSCRAMMs might facilitate xenobiorg migration through the extracellular
matrix.
Leukocytes also adhere to various components of the extracellular matrix, such
as fibronectin, fibrinogen, vitronectin, collagen, entactin, and laminin-111. The
binding regions that mediate these adhesions might be used to create new proteins
that could aid the movement of xenobiorgs through extracellular matrix.
The protein SDF-1α (Stromal-Derived Factor 1α) has a strong affinity for
matrix glycoproteins. The responsible region of SDF-1α might also be used to
create new proteins that could aid the movement of xenobiorgs.
Constraints on homing process for xenobiorgs. The purpose of exploring
homing is to find plausible ways in which extracellular xenobiorgs or cells
harboring intracellular xenobiorgs could be directed to specific tissues via the
blood. Whatever process is chosen or designed must satisfy three constraints. First,
the process must work quickly and effectively. Second, the machinery must not
provoke an immune response. Third, the migrating cells must not be distracted by
other homing events that might occur at the same time.
Native machinery that is rarely or never used. The most desirable homing
process would exploit molecular machinery already present in humans (in order to
avoid an immune response), but which is rarely or never used. A type of cell
migration that occurred only prenatally and which relied on receptors and ligands
not used subsequently would be perfect. A type of cell migration that occurs only
in pregnant women would be somewhat less valuable, but nevertheless useful all of
the time in half the population, and most of the time in the other half. In contrast,
homing schemes that exploited the same machinery that leukocytes use to migrate
to inflamed areas or the same machinery that stem cells use to navigate to areas of
ischemic tissue might be undermined by actual inflammation or ischemia occuring
at sites other than the chosen target tissue.
Stem cell homing to ischemic tissue: an example. Chapter 11 explores in
depth the homing of stem cells to ischemic tissues. This is NOT an ideal candidate
for adapation to directed homing of xenobiorgs, as explained immediately above,
but has been analyzed extensively and paints an informative picture of how
episodic homing functions. Intervention, such as by increasing the concentration of
stromal-derived factor 1α (SDF-1α) can at least double the recruitment of stem
cells to injured heart in mice.
Low-energy shock waves also increase the recruitment of endothelial progenitor
cells to chronically ischemic hind limbs in mice. The mechanism appears to be by
the induction of Stromal-Derived Factor 1 and of Vascular Endothelial Growth
Factor, and may involve nitric oxide. Shock wave therapy has been used for
decades to break up kidney stones, and is both well investigated and well tolerated.
Immune cell homing to distressed tissue: an example. Immune cell homing
to Chapter 11 also explores the homing of immune system cells to wounds, to
diseased tissue, and to inflamed tissue. As with the homing of stem cells to
ischemic tissues, this is a questionable candidate for adaptation for direct homing
to xenobiorgs, because it might have to compete with genuine wounds. However, it
has been explored extensively and is informative.
Fibrocytes are circulating cells that migrate to new wound sites within a few
hours. The ligand CCL21 (Secondary Lymphocyte Chemokine) is produced at the
site of injury, and attracts fibrocytes by binding CCR7, a receptor on fibrocyte
surfaces. CCL21 also attracts fibrocytes in in vitro chemotaxis assays, which
indicates that fibrocytes locomote up gradients of CCL21. This suggests that the
CCL21 governs the migration of fibrocytes after they have migrated from the
blood into the wound.
Additional treatments that increase homing. Homing is a complex process
and the effectiveness of a homing strategy is likely to be influenced by many
subtleties that can only be revealed by extensive experimentation. As one example,
adipose-derived stem cells, if isolated by liposuction, are better able to bind the
adhesion molecules on endothelial cells if the stem cells are subjected first to
hypoxia. (The adhesion molecules on the endothelial cells are Vascular Cell
Adhesion Molecule 1, VCAM-1, and Intercellular Adhesion Molecule 1, ICAM-1.)
It also appears that at least some adhesion molecules on circulating leukocytes
are effective only if they are present at the tips of microvilli, rather than on the
planar cell body. This is potentially important for two reasons: First, a receptor
introduced into a cell by engineering might not function unless it were expressed at
the tips of microvilli. Second, there may be receptors on the surfaces natural
human cells that do not now function as receptors, but which could do so if moved
to the tips of microvilli.
Special inflammatory conditions. Medical conditions that are caused by the
invasion of specific tissues by the immune system may hold clues to homing.
Several of these, including vernal keratoconjunctivitis, liver inflammation,
hepatitis C infection of the liver, ectopic dermatitis, eczema, delayed-type
hypersensitivity, infection by Trichinella spiralis, multiple sclerosis, and Sjogren’s
syndrome are described in Chapter 11.
Organ-specific recirculation of cells. Many organs, or even tissues within
organs, may have cells within them that are free to move around but which are
retained within the organ or tissue. The retained cells might have any of several
purposes; as one example, they might be tissue-specific stem cells whose purpose
was to replaced dead cells as the need arose. The signals that retained mobile cells
within an organ or tissue would be ideal to mediate homing of xenobiorgs. Chapter
11 explores organ-specific recirculation of cells.
Lymphocytes have been reported to selectively recirculate through individual
organs, kept in place by their expression of adhesion molecules specific for the
target organs. It has been reported that α4β7 integrin mediates homing to the gut
(by binding the ligand MAdCAM-1). It does this in cooperation with receptor
CCR9 (which binds the ligand CCL25) and receptor LIGHT, which binds an
unknown ligand.
Antigenic memory cells return to the site of their initial antigenic stimulation.
This increases their chances of re-encountering the individual antigens to which
they were sensitized. It is thought that they were restricted to certain areas of the
body even before their stimulation.
In addition:
- αEβ7 is present on intraepithelial lymphocytes that reside in the small intestine
and mediates their retention at this location.
- It has also been reported that L-selectin mediated homing to the peripheral
lymph nodes.
- It was reported that lung-homing cells use the receptor PSGL-1 to bind Pselectin.
- Furthermore, T-cell binding to skin is mediated by CLA+ve binding to Eselectin.
- Neutrophils leave the bone marrow and then return, under the guidance of
chemokines and the receptors CXCR2 and CXCR4.
- Mature B cells carry the receptor CXCR5, and are attracted into lymphoid
follicles by CXCR5’s ligand CXCL13, which is expressed in the lumen of high
endothelial venules. The CXCR5-CXCL13 attraction also draws B cells to the light
zone of germinal centers.
- The ligand CXCL12 (also called SDF1α and mentioned above) attracts B
lymphocytes expressing the receptor CXCR4 to secondary lymphoid organs.
- Natural killer cells from blood versus intestine show clear differences.
- Mast cells from lung versus intestine show differences.
- E-cadherin expression on intraepithelial lymphocytes helps retain them in the
epithelium. Activation of the lymphocytes reduces their expression of cadherins
and allows them to change their location.
- Glial cells have a special ability to invade nervous tissue such as the brain
non-destructively. This has been suggested as a method of carrying medicines into
the brain, but might also carry xenobiorgs.
- Full binding of both B and T cells to the white matter of the brain requires the
presence of the Hermes-1 epitope of surface molecule CD44, and also requires the
presence of hyaluronate.
- T cells that home to epidermis express Cutaneous Lymphocyte Antigen
(CLA). CLA binds E-selectin, which is expressed on skin endothelial cells.
However, the binding of CCR4 to CCL17 mediates the arrest step of skin homing,
and CCR10 binding to CCL27 mediates the subsequent chemotaxis step of skin
homing.
Homing of cancers. Cancer and infectious disease may also be sources of
useful information about homing. It is well known that some cancers seem to
migrate preferentially to certain target organs. Breast cancer, for example,
metastasizes primarily to bone, lungs, liver and brain. Prostate cancer also
metastasizes to bone. These preferences in metastasis might to some extent reflect
the expression of specific receptors on cancer surfaces and specific ligands in the
organs metastasized to.
The influence of receptors and ligands on cancer metastasis is probably partly
obscured by other influences, however. One such influence is the flow of blood.
Another is the favorability of the target organ for cancer growth; cancers that
reside in bone stimulate bone dissolution, which releases growth factors that in turn
stimulate the cancer’s growth. Thus, the apparent preferential cancer migration to a
specific organ might have several causes.
One potential advantage of studying the migratory behavior of cancers is that
many thousands of new cases appear every year. There is unfortunately plenty of
material to examine, and systematic observations of large numbers of cancers and
the organs that they migrate to might reveal informative correlations between
structure and behavior.
Homing of infectious disease. Pathogens often preferentially invade specific
human organs. Although preferential colonization of a specific organ by a parasite
might reflect more than just homing, for the same reasons that preferential
colonization by a cancer might (discussed above), cases are known where the
surface receptors of a pathogen do control which organs are invaded.
The Thrombospondin-Related Anonymous Protein (TRAP) of Plasmodium
falciparum causes the parasite to home to the human liver. TRAP recognized
receptors on hepatocytes. TRAP is usually sequestered, and is only exposed when
the parasite contacts target cells such as liver cells. This probably protects it from
attack by the host immune system. Both the binding specificity for liver cells and
the sequestration of a binding protein until it is needed might be duplicated in
xenobiorgs or human cells carrying xenobiorgs.
An enormous number of interactions between parasites and surface proteins of
human cells are known, and the number is growing. Some of these interactions
involve viral proteins. For example, the human protein CD155 is a nectin-family
protein that poliovirus exploits as a receptor. Many of these might be exploitable
by xenobiorgs.
If scientists could accumulate a large repertoire of proteins that could bind
specific targets, they might be able to switch the proteins on the surface of a
xenobiorg frequently enough to evade any immune response.
Anti-adhesive surface molecules. There is also suggestive evidence that some
surface molecules on circulating leukocytes can interfere with binding to receptors
on the vessel wall (the endothelium). If these interfering molecules are deleted,
binding increases. It not clear how anti-adhesive molecules function, but there is a
chance that they could be used to increase the specificity by which an extracellular
xenobiorg or human cell carrying a xenobiorg bound to human target cells.
♦ Strategies by which Circulating Cells Find Target Tissues
Cooperative binding by clusters of cells. It has been suggested that cells of
the same type bind weakly to a target tissue but strongly to each other, that the
cells may form clusters whose aggregate binding to the target tissue is quite strong.
Cooperative binding of this type between different classes of cells might allow
xenobiorgs or cells carrying intracellular xenobiorgs to pick bind only cells
carrying a specific complement of surface ligands. Chapter 11 discusses this idea.
Subtle modifications to receptors. Changing a cell’s binding specificity may
involve producing new cell surface receptors, or removing old ones. However, it
may also involve conformational changes or other subtle modifications. The
holding of a native receptor in an inactive conformation by a xenobiorg could
reduce much, although not all, of the body’s usual immune response to non-native
molecules.
Shear flow is important. The flow of liquid past the region where a cell in the
blood binds the endothelial wall is quite important to binding. In some cases, shear
is necessary for successful binding and trans-endothelial migration. This will have
to be considered and modeled for xenobiorg homing to succeed.
Milling around of cells. B and T cells mill around in germinal centers, a
process that increases their chances of encountering antigen-presenting cells. This
milling around occurs within defined, circumscribed areas. The same ability might
be engineered into xenobiorgs (or human cells that contain intracellular
xenobiorgs). It could increase the likelihood that a xenobiorg could find the correct
cell type in a mixed tissue or an unmodified/unentered cell in a tissue where
xenobiorgs were intended to perform some action a single time on each cell of the
tissue. Chapter 11 describes the molecular machinery that supports this milling
around.
Discrimination of targets by binding in stages. A basic problem in targeting
cells to destination tissues is how to ensure that only the desired target is bound.
This is a serious problem in a body where the same recognition molecules appear
in many tissues. The easiest conceptual solution is to require simultaneous binding
by multiple receptor-ligand pairs for adhesion to occur. Multiple receptors on the
mobile cells would have to simultaneously bind multiple ligands on the target
tissue. However, in at least some cases, recognition by different pairs of molecules
is sequential rather than simultaneous.
When mast cell (a type of immune system cell) precursors home to the mouse
intestine, the circulating mast cell precursors express the adhesion molecules
CXCR2 and c-kit. When these interact with their respective ligands, they induce
phosphoinositide 3-kinase, which in turn increases the affinity of α4β7 integrin for
its endothelial ligands, MAdCAM-1 and VCAM-1. Both recognition stages (i.e.
before and after phosphoinositide 3-kinase induction) must succeed for the entire
recognition process to succeed.
Individual organs may have distinct compartments. The lung has at least
two distinct compartments which differ in their control of leukocyte extravasation
(i.e. binding to and then moving through blood vessel walls). The bronchial
compartment is part of the systemic circulation and therefore requires tethering for
adhesion to occur. In contrast, the alveolar bed is supplied by the low-pressure
pulmonary circulation, and tethering is less important.
Consistent with this, P-selectin is present in the bronchial circulation but not in
pulmonary capillaries. Moreover, blood T cells reportedly do not bind alveolar
tissue. Thus, there are crucial differences in leukocyte trafficking into the two lung
compartments.
Very likely, many organs consist of compartments and tissues that differ as
homing targets.
♦ Extravasation
Neutrophil extravasation. There may be multiple cell types that can home to
specific organs and multiple reasons that they might do so. However, one of the
best-studied processes is the homing of leukocytes to inflamed regions.
Neutrophils are the first cells to arrive and neutrophil extravasation has been much
studied.
Four main steps. Neutrophil extravasation can be divided into four sequential
steps: 1) rolling 2) activation by chemoattractant stimulus 3) arrest and adhesion
4) transendothelial migration. The first 3 steps produces signals that initiate the
next step.
Rolling. While in circulating blood, neutrophils usually do not float freely, but
attach loosely to the vessel endothelium by a low-affinity interaction between
selectins and carbohydrate. During inflammation, chemical signals act upon the
endothelium, inducing selectins E and P. These bind to carbohydrates on the
rolling neutrophil, but only slow the neutrophil down, without stopping it. New
selectin-carbohydrate interactions continually form, while old ones are ruptured by
the shear force of the blood circulation. The result is that neutrophils tumble endover-end along the vessel endothelium, a binding process called rolling.
Activation. As the lymphocyte rolls, it is activated by a number of chemical
signals; some of these are permanent features of the endothelial cell surfaces, but
others are secreted locally by cells involved in inflammation. Binding of these
chemical signals to receptors on the neutrophil membrane activates G proteins
within the neutrophil.
Arrest. Activation of the G proteins induces integrins on the neutrophil surface
to change their conformation. This greatly increases their affinity for Igsuperfamily adhesion molecules on the endothelium. The resultant binding
between neutrophil and endothelium is strong enough to arrest the rolling
neutrophil.
Trans-endothelial migration. Cells can move from the inside of a blood
vessel, through the wall and into surrounding tissue in either of two ways. One way
is to squeeze between endothelial cells, a process called paraendothelial
transmigration. The second way is to pass directly through an endothelial, cell, a
process called transcellular transmigration. More is known about paraendothelial
transmigration.
Paraendothelial transmigration. Circulating leukocytes usually arrest near the
boundaries between endothelial cells. A series of molecular changes lead to
severing of bonds between the VE-cadherin dimers that join adjacent endothelial
cells. The endothelial cells move apart and a gap forms between them. Leukocytes
exit the vessel through this gap.
The seal between adjacent endothelial cells quickly re-forms. Usually, there is
time for only one leukocyte to exit. The entire trans-endothelial migration process
takes only minutes.
The actin cytoskeleton of endothelial cells participates in transmigration.
Interesting structures that form during transmigration are “docking structures” and
the “transmigratory cup.” Chapter 11 discusses these and other parts of the
complex machinery that supports paraendothelial transendothelial migration.
Much of the genetic control over the transmigration machinery has also been
elucidated and is discussed in Chapter 11.
♦ Techniques to Manipulate Homing and Extravasation
Soluble blocking molecules. Both the human body and genetic engineers have
in many cases used soluble receptors or ligands as decoys to block productive
interactions between receptors and ligands on cell surfaces. It might in some cases
be possible to restrict the interactions of xenobiorgs to a few selected cell types
using engineered soluble decoy receptors. Because rapidly flowing blood tends to
wash soluble proteins away, this strategy would be more likely to succeed either in
regions of blood circulation where the rate of flow was very slow or in solid tissues
accessible by xenobiorg that had already exited the blood circulation.
Single-chain monoclonal antibodies might prevent receptor-ligand interactions
by binding non-productively to one or the other. These single-chain monoclonal
antibodies might be injected, or synthesized by xenobiorgs.
Because many signaling proteins incorporate sugar moieties, it is in some cases
possible to synthesize a signaling inhibitor simply by conjugating sugars to serum
albumin.
Also, of course, extracellular xenobiorgs derived from bacteria, and expressing
decoy receptors or ligands might make excellent blocking agents.
Artificial structures to guide homing. Chapter 11 also discusses the possible
creation of mechanical guides to support homing of extracellular or intracellular
xenobiorgs. These would presumably be constructed of biologically inert materials
coated with biologically active substances. Precedents, such as artificial “wound
chambers” have already been explored. It might be feasible to perforate some
organs with hollow microtubes that the organs would tolerate much as they tolerate
sutures.
It might be possible to guide xenobiorgs locally using electric current, magnetic
fields (if the xenobiorgs had within them material that responded to a magnetic
field), and chemical gradients. One promising gradient type is a gradient of
calcium cations.
The faux-germinal center formed by B cells in Sjogren’s syndrome is induced
by inappropriate expression of the chemokine CXC13. It is possible that doctors
might be able to induce formation of similar unnatural biological structures that
could guide homing and other activities of xenobiorgs.
Engineering of signal proteins. Hormones and other signaling proteins often
turn out to be multifunctional. As one example, insulin-like growth factor 1 (IGF1) is a chemoattractant or stimulator of migration for neuroblasts, macrophages
multiple myeloma cancer cells, and prostate cancer cells—but has many other
effects as well. In many cases the various functions of multifunctional
macromolecules prove to be separable. Hence, derivatives of IGF-1 might attact
cells or stimulate their movement without inducing the other effects of IGF-1.
An enormous number of chemoattractant and chemoreceptor proteins exist.
Shuffling of domains via recombinant DNA might create new chemoattractants
and new receptors that could efficiently move xenobiorgs into target tissues. Use of
existing protein sequences would be less likely to provoke an immune response,
although newly created boundary peptides could of course do this.
Lewis blood-group antigens have also been suggested as mediators of homing.
Receptors and ligands often consist at least partly of polysaccharides or other nonprotein molecules.
It may also be possible to change the receptor specificity of parasite proteins by
repeated rounds of selection; these proteins might then be used in homing schemes.
It might also be possible to borrow recognition domains from monoclonal
antibodies that recognize human proteins. Chapter 11 discusses these possibilities.
Manipulation of cation concentrations. Some proteins require cationic
cofactors such as Ca2+ Mg2+, Cu+, or Mn2+ to function. Such proteins can include
both enzymes and binding proteins; one example is attachment of circulating
neutrophils to E-selectin on endothelial cells, which requires Ca2+ . If the amount
of cofactor necessary to enable a protein to function can be manipulated by
engineering, it might be possible to arrange for a series of proteins to act in
sequence, simply by slowly raising (or lowering) the concentration of the cofactor.
Some cations, such as Ca2+ and Mg2+, often oppose each other functionally. For
example, binding between the integrin αVβ3 and fibronectin depends on Mn 2+ and
is reversed by Ca2+. Hence, physicians or xenobiorgs might manipulate cation
concentrations to influence xenobiorg binding specificities.
Other chemical influences on protein-protein binding. Other chemical
inhibitors of specific binding interactions include the sugar lactose, and the
artificial inhibitors BIO-1211 and AMD3100. Retinoic acid, in contrast, may give
local permission for the protein CD46 to induce CCR9.
Inhibitors of the signaling steps that follow homing receptor-ligand binding are
also known. These include PP2 529573 (a Src protein inhibitor), LY294002 (an
inhibitor of phosphatidylinositol 3-kinase), and pertussis toxin (a G-protein
inhibitor).
Stimulation of trans-endothelial migration. There are several methods to
increase trans-endothelial migration that might be used by xenobiorgs. Antibodies
to the protein VE-cadherin increase trans-endothelial migration, presumably by
preventing VE-cadherin from sealing the breach between endothelial cells after
one or more leukocytes have exited. Interleukin-1β also disrupts the links between
endothelial cells, allowing leukocytes to exit.
Antibodies that are attached to polystyrene beads can stimulate transendothelial migration by cross-linking the receptors ICAM-1 (Intercellular
Adhesion Molecule 1) and VCAM-1 (vascular cell adhesion molecule 1), which
are present on endothelial cells. Cross-linking is a very common way in which cell
surface receptors become activated.
Inhibition of extravasation. Control of homing might require that
extravasation in some parts of the body be minimized, while extravasation in other
parts of the body is encouraged. Monoclonal antibodies against the proteins Eselectin, L-selectin, P-selectin, and P-selectin glycoprotein ligand-1 (PSGL-1)
reduce monocyte binding to experimental surfaces. A monoclonal antibody to
VCAM-1 (not attached to polystyrene beads as in the paragraph above) reduced
adhesion of melanoma B16 cells to cultured hepatic sinusoidal endothelium cells.
Hydrogen peroxide can contol extravasation. In a system involving adhesion
of melanoma cells to hepatic sinusoidal endothelium, hydrogen peroxide (H2O2)
promoted adhesion and invasion by inducing VCAM-1. The in vivo concentration
of H2O2 could be manipulated by the addition of superoxide dismutase, which
increases H2O2, and of catalase, which destroys H2O2.
A sensitive assay for H2O2 within living cells exists.
Cell-to-cell spread. As discussed in Chapter 7, xenobiorgs can spread directly
between adjacent host cells.
Parabiosis. Parabiosis is the natural or artificial joining of two individual
organisms (generally of the same type) that includes exchange of blood. Artificial
parabiosis for medical research was introduced in the 1860s, was used most
frequently in the period from 1960-1980, and usually involves joining of rats or
mice.
Parabiosis was instrumental in the discovery of the the obesity-related satiety
hormone leptin and the discovery of parathyroid hypertensive factor.
Heterochronic parabiosis, the joining of animals of different ages has shown that
biological aging is due to both to intrinsic changes in tissue and to changes in
circulating blood factors. Parabiotic experiments have revealed striking differences
in mobility between mobile blood cell types.
Experiments with parabiotic animals will show whether preferential
colonization of an organ by mobile cells (including cancer cells) is due to
peculiarities of blood circulation or to greater susceptibility of the organ. Homing
competition experiments using parabiotic animals may help elucidate the relative
importance to homing of various ligands, attractants, and (perhaps) repellants in
target tissues. If there are natural diffusible stimulators or suppressors of homing
generally, experiments with parabiotic animals may reveal them.
It is also true that removal of cells from a mammalian body and transfer of
those cells to an artificial environment can change the phenotype and behavior of
the cells. The reason may be shear stress (discussed above), changes in hormonal
mileau, or something else. Transfer of cells between organisms by parabiosis may
avoid such changes.
As an experimental technique, parabiosis has fallen into disuse in the last few
decades, but seems poised for a comeback. The existence of tools such as
fluorescent marker proteins, in vivo flow cytometry, and sophisticated medical
imaging makes possible many interesting experiments that were not feasible in the
past.
The need for a defined system. A big first step toward mastery of homing
would be to reconstitute a homing interaction using defined components and to
show that these components together are sufficient to mediate a homing
interaction. The mouse airway system is a promising venue for such a reconstituted
interaction. Chapter 11 discusses this idea.
A list of reagents. Chapter 11 also includes a list of specialized reagents, such
as monoclonal antibodies and small interfering RNAs that are available for homing
research.
♦ Confounding influences
Damaged endothelia. A number of influences can confound attempts to
understand homing, and could probably confound attempts to use homing for
medical purposes. One such influence is damaged endothelia, which may occur in
the elderly. Atherosclerosis, the deposition of fatty substances on the inner lining
of arterial walls could interfere with extravasation of blood-borne cells in arteries,
although the majority of such extravasion occurs in venules.
Cytokines and physical influences. The exposure of cells to cytokines or other
signaling molecules, or to physical influences, can often cause them to lose their
ability to home to a target. In some cases, simple purification of cells from a mixed
population can have this effect. For this reason, scientists trying to engineer
xenobiorgs so that they can home to a selected target might be wise to begin with
phenotypically stable cells that cannot home, rather than to rely on native cells that
are capable of homing, but whose behavior can mysteriously change.
Shear stress. One physical influence that has powerful effects on the homing
behavior of cells is shear stress. The flow of liquid past cells that are attached to a
vessel wall produces shear stress on the cells. This shear stress has complex effects
on binding and extravasation: shear stress sometimes seems to be necessary for
binding and extravasation, sometimes seems to hinder those processes, and
sometimes seems to have no effect. Shear stress can also greatly affect
differentiation of cells (such as stem cells) that are capable of differentiation. Shear
stress is a critically important variable that attempts to engineer homing must
consider. Cells can experience shear stress not only in the blood vessels of the
body, but also while they are outside the body, such as when they are growing in a
culture dish and the medium around them is swirled.
Hyperthermia. In rats, whole-body hyperthermia alters lymphocyte homing. It
increases lymphocyte migration to the bone marrow and skin and prolongs their
transit through these organs. It also inhibits homing to the lymphoid organs (lymph
nodes, Peyer’s patches, and spleen). The effect persists for about 16 hours after the
end of hyperthermia. In rats without adrenal glands, whole body hyperthermia did
not induce migration to the marrow, but did increase it to the skin.
An effect of this sort might complicate induced homing in people running a
fever, or with adrenal abnormalities. On the other hand, doctors might use local
hyperthermia to alter homing for some beneficial purpose.
Blood dialysis. Blood dialysis is necessary in patients with insufficient kidney
function. However, dialysis decreases the number of blood neutrophils to well
below normal and reduces expression of at least two neutrophil integrins: Mac-1
(integrin αMβ2) and gp150. It also decreased the expression of Leu-8, the human
neutrophil peripheral lymph node homing receptor, and CD43 the major
sialoglycoprotein in leukocyte homotypic adhesion.
Dialysis might complicate attempts to control homing of xenobiorgs or human
cells containing xenobiorgs. Xenobiorgs might clump, stick to endothelia without
extravasating or extravasate in the wrong tissue.
♦ Step-By-Step Development
Two early goals. A belief that runs throughout this e-book is that progress is
fastest in any technological field when modest amounts of progress produce
commensurate rewards. Before xenobiorgs can be engineered to home to chosen
tissues, at least two related goals may already have been achieved, and may serve
to propel progress in homing of xenobiorgs.
Homing of particles carrying traditional drugs. The first such technology is
the homing of traditional drugs. Although drugs are usually injected as free fluids
into blood or muscle, they can also be injected encased within carrier particles.
One such carrier particle is the liposome, an artificial vesicle whose outer surface
is a lipid bilayer. Another carrier particle is the micelle, an aggregate of surfactant
molecules within an aqueous fluid. Many variants of both liposomes and micelles
have been tested as drug-carrying agents and other particle types have been
evaluated as well.
The main purpose of sequestering drugs within particles such as liposomes is to
prevent the drugs from diffusing freely throughout body fluids and instead to keep
the drugs concentrated until they can be delivered to a small region of the body
where they are needed. Delivery of the drugs to target organs will require homing;
this homing will probably encounter the same obstacles as does homing of body
cells, and may exploit much of the same biological machinery.
Experience with homing of drug-carrying particles may lead the way to homing
of xenobiorgs. It is also plausible that the next logical step would be to use
microbes such as engineered bacteria or to use engineered human cells to deliver
drugs to target organs. The tools and experience gained from this might pave the
way for homing of more sophisticated xenobiorgs.
Remodeling of the extracellular matrix. Remodeling of the extracellular
matrix occurs continually. This remodeling consists mainly of collagen removal
and replacement. Understanding of extracellular matrix remodeling is important
for several reasons. First, collagen-based materials used in tissue engineering must
be fused with existing collagen, which requires collagen remodeling. Second,
remodeling is important in wound healing, and optimal wound healing may
sometimes required medical manipulation of matrix remodeling.
Third, many diseases involve inappropriate collagen remodeling. These
diseases include cardiomyopathy, spinal injury associated ulcers, scleroderma,
osteoporosis, osteogenesis imperfecta, osteoarthritis, gingival diseases and
numerous metastasized cancers. Amelioration of these diseases would benefit from
a clear understanding of extracellular matrix remodeling.
For the above reasons, extracellular matrix remodeling is likely to be studied.
However, an understanding of the extracellular matrix may help xenobiorgs
navigate the extracellular matrix. Furthermore, if local staging areas must be built
to organize xenobiorg access to tissues, those staging areas may well be
constructed of collagen.
A good first step in extracellular matrix repair and remodeling would be to
replace aged fibroblasts with more youthful counterparts. Fibroblasts are the main
contributors to extracellular matrix remodeling. Although such experiments would
be easiest to do using syngeneic mice, they might eventually be possible in humans
using induced pluripotent stem cells.
One of the earliest objects of controlled extracellular matrix remodeling would
likely be skin. Skin is easily accessible and relatively forgiving of mistakes. The
skin is subject to wounds, burns, accumulated ultraviolet exposure, and other
conditions that need effective treatment. Moreover, the appearance of skin is of
enormous psychological and social importance to people.
Although many of the enzymes and signals that regulate extracellular matrix
remodeling are known, much more remains to be learned.
Repnumi Regeneration and Senescence (A Brief Summary
of Chapter 12)
The purpose of this chapter. Many of the remaining chapters of this e-book
are devoted to the concept of Repnumi rejuvenation. Chapter 12 discusses one
aspect of Repnumi.
Although the purpose of Repnumi would be to counteract aging, aging could
also frustrate Repnumi. Chapter 12 discusses what aging is, both at the level of the
entire human body and at the level of individual cells—and what Repnumi would
have to do to reverse aging. Chapter 12 also discusses how aging might affect
Repnumi, and how Repnumi will have to cope with the effects of aging. In order to
discuss these issues, it is necessary to explain the basics of what Repnumi would
involve and of what aging is.
♦ What Repnumi Would Involve
What Repnumi would be. As discussed above, Repnumi would be the in situ
rejuvenation of human tissues by removal of aged nuclei, mitochondria, and other
damaged components and their replacement by younger counterparts.
Circulating engineered human cells as Repnumi donors. Replacement nuclei
and mitochondria would have to be delivered somehow to target cells. For reasons
discussed immediately below, this delivery probably could only be mediated by
engineered human cells, acting within the body.
Repnumi could not be performed by human surgeons. There are more than
13
10 cells in an adult human body, and most of them are hard to access. If a team of
human surgeons could somehow replace the nuclei and mitochondria in one cell
per second, complete replacement of all the nuclei and mitochondria in an adult
human body would require more than a million years. Repnumi cannot be
performed by human surgeons.
Repnumi could not be performed by nanotech robots. Only living cells
could be expected to maintain donor nuclei and mitochondria in a living and useful
state. There is no hope that in the foreseeable future nanotech robots will be
developed that can store isolated human nuclei and mitochondria so that they
remain alive and useful. Moreover, nanotech robots could not be created in enough
numbers to process 1013 cells. Repnumi cannot be performed by nanotech robots in
the foreseeable future.
Repnumi could not be performed by non-human cells. Although it is
possible to imagine that human nuclei and mitochondria might be carried to their
targets by non-human cells (such as giant amoebae for example), it is hard to see
how such a scheme could actually succeed. Neither nuclei nor mitochondria are
inert objects; instead, they exchange material with their surroundings. Nuclei, in
particular, both regulate and are regulated by their surrounding cytoplasm. Nonhuman cells are unlikely candidates for Repnumi donors.
Only human cells in the proper state of differentiation could donate the
necessary nuclei and mitochondria. However, the human donor cells might also
have to be engineered extensively, and/or carried to their target tissues by other
cell types entirely. This issue is discussed in Chapter 18.
Repnumi would probably require intracellular xenobiorgs. Repnumi would
unavoidably be a complicated process under elaborate control. Donated nuclei and
mitochondria would have to enter the target cells at about the same time that the
target cells were deprived of their original nuclei and mitochondria. The donated
nuclei, and perhaps the donated mitochondria, would have to match the
differentiated state of the target cell. Repnumi would have to incorporate
safeguards to exclude nuclei and perhaps mitochondria that were in an inappopriate
state of differentiation. This would require transmission of information about the
internal state of the target cell to the outer surface of the target cell.
It seems very likely that much of the activity of Repnumi would have to be
directed by intracellular xenobiorgs. Only intracellular xenobiorgs could accurately
assess the differentiated state of the target cell and transmit this information
somehow to the cell surface. Only intracellular xenobiorgs could remove the nuclei
and mitochondria from their host cells at exactly the right time. Only intracellular
xenobiorgs could perform other necessary tasks within the target cell, such as
removal of oxidized proteins and other accumulated garbage.
♦ What Aging Is
Chapter 12 assesses aged humans as a target for Repnumi from two points of
view. First, it discusses the whole human body as an object upon which Repnumi
will have to be performed. Second, it discusses aged human cells and the
conditions within those cells.
Chapter 12 discusses what is known about the consequences of putting youthful
nuclei and youthful mitochondria into aged cells, aged tissues, and an aged body.
The aging process summarized. Aging in somatic cells consists of many
processes, including damage to nuclear DNA, damage to mitochondrial DNA,
changes in telomere length, changes in regulatory proteins, and accumulation of
intracellular garbage. Chapter 12 summarizes cellular aging.
Young nuclei in old cytoplasms. Chapter 12 also discusses the consequences
to individual cells of combining a youthful nucleus with an aged cytoplasm. Such
experiments have been done in mammals and in single-celled organisms such as
Paramecium and Acetabularia.
The aged human body as a target of Repnumi. Engineers seeking to design
Repnumi would have to consider a number of questions concerning the effects of
aging on the human body. First, they would need to understand all of the effects of
aging on the human body, because an old person consisting entirely of rejuvenated
cells might still retain some undesirable features of being old. They would need to
assemble a complete list of the changes that they hoped to make and have a clear
idea of how they intended to effect each change.
Some adverse changes, such as atherosclerosis, vascular degeneration, joint
degeneration, and Alzheimer’s dementia might not be repairable simply by in situ
rejuvenation of every cell. What can be done about that?
In addition, Repnumi engineers would have to understand the effects of aging
on the biological systems that they hoped to manipulate to perform Repnumi. Does
arteriosclerosis affect trans-endothelial migration of homing cells? What are the
effects of smoking, alcohol, diabetes and the passage of time?
Some human tissues such as the liver are capable of considerable regeneration;
in these tissues, it might be adequate to perform Repnumi on only one cell in every
hundred, if the remaining cells could be killed and the rejuvenated cell induced to
proliferate to replace the dead cells. On the other hand, some tissues (especially
neural tissues) cannot regenerate at all and each of their cells might have to be
subjected to Repnumi. Some tissues that can regenerate in young people may lose
that ability in the elderly due to factors outside cells; how will calculations about
Repnumi be affected by that?
Aged human cells as targets of Repnumi. The second goal of Chapter 12 is to
assess aged cells as targets for Repnumi.
The aging of people is thought to be mainly a consequence of the aging of
individual human cells. The aging of human cells is thought to result from two
processes working together. The first process is ongoing DNA damage, which
sometimes remains unrepaired, and which at other times is repaired in a way that
produces somatic mutation. The second process is a series of changes that reduce
the ability of cells to multiply. The second process is thought to be the body’s way
to control cancers and other instances of delinquent cells that somatic mutation
produces, and which would otherwise run wild. The second process is a way of
containing the damage from the first process.
Inactivation of delinquent cells. As mentioned above, the ongoing somatic
mutation that occurs with aging produces delinquent cells. Although cancer is the
most well known type of delinquent cell, other types are known and many more
may exist. In some delinquent cells, mutated mitochondria export chemically
reduced compounds that poison neighboring cells. Other delinquent cells are virus
sources. In addition, one can imagine that somatically mutated cells might become
exporters of digestive enzymes, exporters of signals that disrupt the tissues around
them, exporters of substances that block capillaries, and so on.
Cell delinquency is an important part of the aging process. Detecting and
silencing or eliminating delinquent cells would be one important task assigned to
xenobiorgs. This might be done by either extracellular or intracellular xenobiorgs,
but if it were done in conjunction with Repnumi rejuvenation, intracellular
xenobiorgs would most likely be involved.
Counteracting of supracellular senescence-acceleration. It has been
suggested that aging may be accelerated by processes such as micro-inflammation.
These destructive processes occur within whole tissues, at the supracellular level.
They might continue even if every cell in the tissue was rejuvenated, like a fire can
continue even if partly-burned ashes are replaced by fresh fuel.
Little is known about the role of supracellular processes in aging. However,
xenobiorgs seem to be logical agents to use against them. This could be done in
conjunction with Repnumi rejuvenation or separately.
Replacement of nuclei and mitochondria. The most important part of
Repnumi, as discussed above, would be the replacement of aged nuclei and
mitochondria with fresh counterparts. Possible sources of suitable nuclei and
mitochondria are discussed in Chapters 13 and 14. How the Repnumi process
might accurately assess the differentiated state of target cells is discussed in
Chapter 16. Methods to remove nuclei and mitochondria from target cells are
discussed in Chapter 17. Methods to insert new nuclei and mitochondria are
discussed in Chapter 18.
Self-organization of rejuvenated cells? In a tissue undergoing Repnumi, it
might not be necessary to replace every differentiated nucleus with a youthful
exact counterpart. Cells in developing tissues are capable of great selforganization, and mature tissues may retain some of this capability. Chapter 19
discusses the current state of knowledge about reprogramming of cells within
mature tissues.
Removal of intracellular garbage and repair of other defects. Although
replacement of nuclei and mitochondria is probably a prerequisite for rejuvenation
of aged cells, other steps might be needed as well. Full rejuvenation might require,
for example, removal of clumps of oxidized proteins that unaided cells cannot
remove. In addition, aged cells might be poisoned by oxidized lipids, reduced
lipids, reactive oxygen species, reactive nitrogen species, and many other possible
toxins.
Intracellular xenobiorgs might be engineered to remove toxic compounds from
target cells. The removal might take the form of chemical degradation or it might
take the form of sequestration. In removal of toxins by sequestration, an
intracellular xenobiorg could engulf or absorb toxins, then exit its host cell, travel
through the blood to the intestine and exit into the intestinal lumen.
A proposed sequence of events. Chapter 8 summarizes the steps necessary for
Repnumi rejuvenation and suggests a procedure that might be followed to
accomplish all of the steps.
Age-ravaged tissues appear to regenerate. The aging process degrades whole
tissues as well as individual cells. This poses a critically important question: even
if the individual cells that compose a tissue could be rejuvenated, would this
restore the tissue itself to a youthful and healthy state? Or would the tissue remain
senesced even when composed of youthful cells? Might not Repnumi rejuvenation
induce destructive tissue remodeling, or even uncontrolled growth of some cells,
i.e. cancer?
This example may illustrate the problem: If a person were to cut his hand
slightly with a razor blade, the wound would eventually heal—perhaps leaving a
permanent scar, or perhaps not. However, if a person were to truncate his arm at
the wrist using a guillotine, the hand would not grow back. The cells at the end of a
person’s wrist either do not carry or cannot access the information needed for them
to form a new hand.
Now, suppose that a person slightly crushes his hand, using a powerful clamp—
perhaps breaking a few bones, but not greatly deforming the hand. In this case, the
hand would probably heal normally.
However, if the crushing of the hand were to increase, there would come a point
at which too much damage had been done, and full restoration was impossible
(without reconstructive surgery). The point I am making is that cells of a crushed
hand do not possess the information needed to restore the original healthy hand.
It is conceivable that the effect of aging on a human tissue is like the
guillotining or severe crushing of a hand: that it leaves the tissue unable to restore
its original condition, even if the cause of the aging is removed. Rejuvenation of
human tissues does not occur in nature, and there may be no darwinian reason for
severely aged tissues to be able to restore themselves even when composed of
youthful cells.
However, one extremely important experimental result suggests that senesced
tissues may in fact be able to repair themselves. Mice with defective telomerase
genes have shortened telomeres (structures at the ends of chromosomes) and show
signs of premature aging, including sterility and brain disease. Restoration of
telomerase activity restores normal telomeres, increases the size of internal organs
(spleen, testes, brain), and reverses many of the effects of the premature aging.
The mice with restored telomerase activity do not live longer than do normal
mice, and shortened telomeres are only one part of the aging process. Hence, these
mice are not perfect models for aging. Nevertheless, the telomerase-deficient mice
do suffer the ravages of age, and recover from those ravages when their cells
regain telomerase and thus competence to proliferate and perform their normal
functions.
Chapter 18 discusses rejuvenation of tissues.
Repnumi on the whole human body. I predict that Repnumi rejuvenation will
involve some very painful learning. In the beginning, rejuvenation will be of only a
few select tissues, probably beginning with skin and heart. This will probably
spark great public interest and financial support, and the number of rejuvenable
tissues will increase until eventually the entire human body is rejuvenable.
However, during this period there will be many mysterious deaths of supposedly
rejuvenated people, and perhaps arguments by some observers that nature cannot
really be manipulated to give people more than their allotted years.
I foresee at least two problems. First, uneven rejuvenation may place unhealthy
stress on tissues that have not been rejuvenated. As one example, we can imagine
how dangerous it would be to have a 20-year-old body with an 80-year-old heart.
Second, tissues that appear to be homogeneous will turn out to contain multiple
cell types, all necessary for proper tissue function. Doctors and scientists who were
unaware of the tissue’s complexity might replace it with homogeneous tissue that
functioned poorly.
Stem Cells and Repnumi Rejuvenation (A Brief Summary of
Chapter 13)
The connection between Repnumi and stem cell therapy. Chapter 13
discusses stem cells. There are three reasons for including a chapter on stem cells
in this book. First, stem cells are excellent candidates to supply the youthful nuclei
and mitochondria in specific differentiation states that Repnumi would require.
Second, stem cells are good candidates to replace aged, diseased, or damaged
tissues. Third, the ability of intracellular xenobiorgs to analyze, report, and
preserve a cell’s differentiated state, which would be necessary for Repnumi
rejuvenation, would also be useful in the understanding and manipulating stem
cells.
The advantages of pluripotent stem cells. Rejuvenation using stem cells
would probably have several advantages over Repnumi. First, there would be
fewer steps, since nuclei and mitochondria would not have to be moved from a cell
of origin to a destination cell. Second, the replacement cell would not inherit any
damage from an aged cell. Third, tissue replacement using stem cells is a more
mature technology (at least for now).
Rejuvenation of skeletal muscle might be accomplished entirely using youthful
satellite cells (muscle stem cells) with no need for Repnumi.
The advantages of Repnumi. Rejuvenation using Repnumi would have several
advantages over stem cell therapy, also. First, nuclei and mitochondria would
receive molecular guidance from the cytoplasm of the destination cell, directing
them how to integrate themselves into a functioning tissue. Second, with Repnumi
there is no need to kill and dispose of cells that are to be replaced. Third, Repnumi
could preserve the cytoplasmic organization of target cells, which might be
important in preserving memories in rejuvenated brain tissue.
Cooperation between Repnumi and stem cell therapy. Repnumi and stem
cell therapy might be used together in a rejuvenation strategy. As suggested above,
each might be superior in rejuvenating specific target tissues.
Repnumi used to create stem cells. Repnumi would depend upon new
techniques of transferring nuclei and mitochondria between cells. These techniques
might be used to transfer nuclei and mitochondria with well-preserved genomes
from tissues that cannot be induced to form pluripotent stem cells into the
cytoplasms from pluripotent stem cells.
Xenobiorgs and stem cell preservation. Chapter 12 focuses on the difficulties
of creating and preserving pluripotent human stem cells. Intracellular xenobiorgs
might both turn differentiated cells into induced pluripotent stem cells and preserve
that pluripotency in the face of all the environmental factors that can compromise
it.
Xenobiorgs and isolation of pure stem cell populations. It also has been
argued recently that populations of supposedly homogeneous stem cells can in fact
contain many subpopulations. If so, it may be necessary to isolate homogeneous
subpopulations. Intracellular xenobiorgs might do this by reading the internal state
of individual cells, by relaying that information to the cells surface where it can
direct cell sorting, and by preserving the cell’s internal state.
In vivo manipulation of stem cells. Chapter 12 also discusses attractants for
stem cells, such as electric fields, biochemical gradients, and so on. These might be
used to maneuver stem cells within the human body.
Distinguishing between replacement and assistance. Stem cells can
regenerate tissues in either or both of two ways. The first, replacement, is to
replace injured or malfunctioning cells within the tissue. The second, assistance, is
to release factors that stimulate the tissue to repair itself, oppose apoptosis, or limit
damage in some other way. Distinguishing between these possibilities is difficult,
and investigators have at times been mislead. Repnumi might help distinguish
between the two, since Repumi would be expected to rejuvenate tissues by the
replacement but not by assistance.
Marking of stem cells. As mentioned above, in the discussion on intracellular
xenobiorgs and adipose tissue, intracellular xenobiorgs could mark stem cells, and
thereby help track their contributions to differentiated tissues. A serious problem
plaguing stem cell research is that differentiation is a cooperative phenomenon. A
single, isolated stem cell may not differentiate in response to a stimulus, even
though it would differentiate if surrounded by other cells. In many cases, it is
unclear which cells give rise to differentiated structures. Intracellular xenobiorgs
expressing green fluorescent protein might mark either all the progeny of a given
cell, or only certain types of progeny.
Sources of Genetically Normal Donor Nuclei (A Brief
Summary of Chapter 14)
♦ Basic Assumptions
Chapter 14 discusses a problem faced by Repnumi regeneration. This is the
problem of obtaining donor cells that are genetically normal.
Repnumi donor cells should be genetically normal. Repnumi is envisioned to
consist mainly of replacing the nuclei and mitochondria of aged cells with more
youthful counterparts. However, there would seem to be little value in replacing
genetically degraded nuclei and mitochondria with genetically degraded
counterparts. Indeed, if the replaced cells were genetically heterogeneous, and thus
largely able to compensate for each others’ defects, replacing their nuclei and
mitochondria with homogeneous defective nuclei or mitochondria might be
disadvantageous. Hence, using genetically normal starting cells is likely to be
highly desirable for Repnumi regeneration.
(For the same reasons, using genetically normal cells is likely to be highly
desirable for stem cell therapy.)
Many donor cells could be created from a single precursor cell. The
discussion in Chapter 14 assumes that it will be possible to create a large supply of
Repnumi donor cells from a single precursor cell, or from a small number of
precursor cells. This presumption is based on the fact that an entire adult human
can develop from a single fertilized zygote. Elaborate genetic manipulations that
would not be feasible on large populations of cells might be carried out to create a
single precursor cell.
Obtaining normal mitochondria should be easy. The discussion in Chapter
14 also assumes that obtaining normal mitochondrial genomes will not be difficult.
The sole chromosome in human mitochondria consists of about 16,600 base pairs,
encoding 37 genes. Individual humans appear to tolerate great heterogeneity of
mitochondrial genomes, and it would seem that nearly everyone who was born
with normal mitochondrial chromosomes must still have a large supply of
satisfactory ones. If not, it should be possible to borrow mitochondria from another
person without risking tissue rejection discussed below. Instead, Chapter 14
focuses on the nuclear genome.
Several possible strategies might allow doctors to replace the nuclei of aged
human tissues with genetically normal counterparts.
♦ Autologous Repnumi
Searching for genetically normal cells. The person being rejuvenated by
Repnumi might also be the person who supplied the donor cells, in which case the
Repnumi would be termed autologous. However, a person in need of Repnumi is
likely to be elderly (or to have some genetic disease), which raises the question:
how can such a person supply a normal genome to use as starting material to create
a population of donor cells?
In autologous Repnumi, it would be necessary to examine the genomes of many
cells of the patient in hopes of finding at least one that is genetically normal. This
might be quite difficult.
It should be relatively easy to spot cells with karyotypic rearrangements.
However, it would be far harder to identify cells with point mutations in genes and
control regions.
Do some tissues include more genetically normal cells? It would be wise to
start by examining tissues that have a high proportion of cells with genomes
unchanged from the original zygote. It seems likely that some tissues will retain a
higher proportion of genetically unchanged cells than do others, and that cells with
low metabolic activity, low exposure to the environment, and which have not
proliferated much, will be the least changed—however, this is still conjecture.
Experience will tell us.
Frequent somatic mutations. It may also turn out that in each person, some
mutations are repeated in a most somatic cells. These would be mutations that
occurred during development. Cells harboring those mutations might be poor
Repnumi donors, and yet it might be difficult to find cells without the mutations.
Again, experience will tell us.
Aged myoblasts still function. One encouraging sign is that myoblasts that are
useful in repairing diseased human heart tissue can be harvested from people who
are as old as 91 years. This does not prove that such cells are genetically normal,
but it shows that at least some of the cells are normal enough to function properly.
How carefully to screen donor cells. Doctors and engineers seeking to
implement Repnumi would have to decide how thoroughly to examine prospective
donor nuclei and how much genetic change is acceptable. Most of the human
genome consists of DNA that can be changed with no ill effect, and it would
probably not be worthwhile to examine these sequences or to include them in
evaluations of donor nuclei.
Assembly of a genome from multiple sources. It is also conceivable that a
genome used to originate a population of Repnumi donor cells could be assembled
from chromosomes of different sources. Since chromosomes can be sorted,
chromosomes might originate from different cells of the Repnumi patient.
♦ Repnumi with Engineered Heterologous Nuclei
The use of well-characterized standard donors. Heterologous nuclei—nuclei
from some person other than the Repnumi recipient—could also be used as
Repnumi donor nuclei. This would have one huge advantage: it would not be
necessary to find an unchanged genome for each Repnumi recipient. It might be
possible to maintain reserves of human stem cells that had been thoroughly
characterized, which could be the progenitors of differentiated nuclei used in
Repnumi.
Changing a recipient’s personality. However, use of heterologous nuclei
would have two serious disadvantages. The first, mentioned here briefly, is that
heterologous nuclei might tend to change a person’s personality if used to
rejuvenate some cells such as brain cells and perhaps some endocrine cells.
The danger of tissue rejection. The second disadvantage of heterologous cells
is that they would provoke a strong immune response and tissue rejection unless
measures were taken to prevent that. One way of preventing an immune response
would be to alter the genes of heterologous cells so that they matched the host
genes. This, in turn, might be done by any of several methods.
Patient chromosome 6 replaces donor chromosome 6. Most of the proteins
that frustrate tissue transplantation attempts are encoded by genes in the Human
Leukocyte Antigen (HLA) region, which is part of human chromosome 6. It might
be possible to replace chromosome 6 of donor cells with a copy of chromosome 6
of the Repnumi patient. Since the donor tissue would be diploid, two copies of
chromosome 6 would have to be replaced. This would be difficult, and would have
the disadvantage that any defective genes on chromosome 6 of the Repnumi
patient would be retained in the rejuvenated tissue. It also would not replace any
immunogenic genes on other donor chromosomes.
Patient HLA region replaces donor HLA region. An alternative would be to
replace only the HLA regions of the Repnumi chromosome 6 donor. Since the
HLA region is is in the middle of the short arm of chromosome 6, replacement of
the HLA region would require using controlled gene conversion to create two
unique endonuclease sites on either side of the HLA region, cutting loose of the
donor HLA region, and ligation of one of the patient’s HLA regions into the
chromosome. This would also be difficult, would retain any faulty genes within the
Repnumi patient’s HLA region, and would not replace any immunogenic genes
anywhere in the donor genome outside the HLA region.
Donor alleles are converted to patient alleles. Another alternative would be to
use controlled gene conversion. Individual HLA alleles could be converted from
the donor sequence to the recipient sequence. Gene conversion under the control of
intracellular xenobiorgs is discussed in Chapter 7. The disadvantage to this
approach is that many alleles, both inside and outside the HLA region would have
to be converted.
♦ Repnumi by Tolerization to Heterologous Nuclei
Conditioning of a patients immune system. It might be possible to use
heterologous nuclei in Repnumi rejuvenation, but to condition the Repnumi
patient’s immune system to tolerate the foreign proteins that would be introduced.
Considered a priori, this is probably the best strategy. However, inducing the
human immune system to tolerate foreign tissues is a skill that has not yet been
mastered.
Also, as mentioned above, replacing the nuclei of some cells with heterologous
nuclei might alter a patient’s personality.
Assessing and Reporting the Differentiated States of Human
Cells (A Brief Summary of Chapter 15)
♦ Basic goals
Intracellular xenobiorgs report host cell differentiated state. Chapter 15
discusses the task of determining the differentiated state of target cells for
Repnumi regeneration. Generally, Repnumi should endeavor to replace target cell
nuclei with nuclei in exactly the same differentiated state—and the same may hold
true for target cell mitochondria, if mitochondria undergo tissue-specific
differentiation (this question is discussed in Chapter 15).
Although some information about a target cell’s differentiated state will be
provided by its position in the body and its surface antigens, information from
inside the cell will probably also be necessary for its full identification. The best
candidates to get this information are intracellular xenobiorgs. Chapter 15
discusses this idea.
Intracellular xenobiorg methods to gather and report information. Chapter
15 discusses ways in which intracellular xenobiorgs might gather information
about the differentiated state of cells and also ways in which they might report that
information to experimenters. Briefly (the issue is discussed below) criteria that
reveal a cell’s differentiated state could include: expression of specific proteins,
activation of specific proteins, expression of coding and non-coding RNAs,
changes in chromatin arrangement, changes in cell shape and polarity, changes in
the positions of organelles, changes to the cytoskeleton, changes in the identity and
arrangement of surface antigens, and changes in composition and position of
membrane lipids.
Xenobiorgs might judge spatial relationships by using complementary proteins
that produce a signal only when they are very close together. Methods of reporting
the information could include manufacture of fluorescent proteins that could be
inspected, secretion of chemicals or macromolecules that could be detected, and
changes in the sequence of bacterial DNA.
Intracellular xenobiorgs might record events over time. It might be possible
to engineer intracellular xenobiorgs not only to collect information about a cell’s
differentiated state, but to record that information so that researchers can later
access it. Intracellular xenobiorgs might read and record changes in a cell’s
differentiated state or changes in expression of individual genes so that the
sequence of changes could later be deduced. Furthermore, it might be possible to
simultaneously record chemical or physical stimuli supplied over time by
researchers so that the sequence of changes in differentiated state can be matched
to an objective clock. Chapter 15 discusses schemes to do this.
The example of cancer stem cells. Chapter 15 discusses schemes for
implementing such molecular record-keeping in intracellular xenobiorgs, and also
discusses an investigative specialty where it could be very useful. This
investigative specialty is the evolution of cancers, and in particular the question of
so-called cancer stem cells.
♦ Analyzing evolution of cancers
Detection of very early-stage cancers. As Chapter 15 discusses, intracellular
xenobiorgs may be able to display the spatial organization of individual cells. If so,
they might show the loss of polarity in normal cells that accompanies a shift to a
precancerous or cancerous state, and thus reveal precancerous or cancerous cells.
Moreover, because the cells adjacent to wounded tissue reorient their polarity in
response to the wound, the cells near a small cancer might reveal it. This would be
especially true if the cancer secreted metalloproteinases, an essential component of
metastasis.
Hence, intracellular xenobiorgs might reveal early events in experimental
cancers and might also reveal the existence of cancers in human patients. However,
most (but not all) methods by which intracellular xenobiorgs would display the
spatial organization of individual cells would require that an observer be able to
see the cell.
Tracking of cancer cells. Intracellular xenobiorgs represent another way to
label cancer cells, which in turn allows them to be tracked. Intracellular xenobiorgs
might label cancer cells more efficiently and with less selection than does labeling
by incorporation of genes into the nuclear genome of the cancer cells. Labeling
would probably be with Green Fluorescent Protein or one of its derivatives.
Labeling of cancer cells with fluorescent protein has already revealed useful
information, such as the fact that, at least in mice, liver resection reduces the
number of circulating liver cancer cells and early metastases.
Cancer stem cells defined. Cancers are heterogeneous; they include many
different cancer cell variants. It has been proposed that some cancer cells are much
more dangerous than others because they are more likely to produce viable
metastases—and may be the only cancer cells that can metastasize. These
extraordinarily dangerous cancer cells—which may or may not really exist—are
termed cancer stem cells.
Cancer stem cells are defined by their ability to self-renew indefinitely and to
spawn the full range of cell types present in the original cancer. However, in
addition, strong parallels have been noted between cancer stem cells and normal
stem cells of the type that differentiate into normal specialized tissues.
Chapter 15 describes the cancer stem cell theory and its alternative, the
stochastic theory—and discusses evidence for and against both.
Cancer stem cells and cancer treatment. The cancer stem cell theory has
important implications for cancer treatment. Therapies that involve induced
differentiation of cancer stem cells, selective silencing of cancer stem cell genes,
targeting of cancer stem cell surface antigens, and use of toxins selective for cancer
stem cells might be worthwhile if the cancer stem cell theory is valid, but would
certainly fail otherwise.
Unsolved problems involving cancer cells. Although cancers may begin their
existence as antigenically distinct entities, and therefore be vulnerable to the
immune system, they lose their antigenicity and vulnerability by several different
mechanisms. They acquire several different kinds of resistance to anticancer drugs.
They become more virulent, by multiple processes involving multiple genes. Some
melanomas develop special features to aid their own spread, such as vasculogenic
mimicry and the epithelial-mesenchymal transition.
Cancers change rapidly both through genetic and regulatory changes. Moreover,
cancer cells sometimes fuse with non-cancer cells.
Potential contributions of intracellular xenobiorgs to cancer research. As
the above summary indicates, cancers are poorly defined cell populations with
many subtypes. They change rapidly, usually toward more aggressive, treatmentresistant, and deadly forms. They may or may not depend on cancer stem cells for
metastasis and spread. There is a great need for new tools to dissect and elucidate
cancer behavior.
Intracellular xenobiorgs might make several contributions to cancer research.
First, the ability of intracellular xenobiorgs to read and report the internal state
of cancer cells might allow, for the first time, isolation of pure cancer
subpopulations. The cells could be marked with a colored or fluorescent protein,
or marked antigenically and then stained. Purified cancer cell subtypes would be
isolated by fluorescence activated cell sorting or by antigen-antibody affinity.
Second, as mentioned above, intracellular xenobiorgs might enable researchs to
mark both cancers, cancer sublines, and non-cancer cells. This could allow
researchers to monitor in developing cancers fusions between two or more cancer
cells, and also fusions between cancer and non-cancer cells.
Third, intracellular xenobiorgs might kill certain cancer subpopulations while
sparing others. This selective killing might include cancer cell fusions with other
cell types. Selective killing or sparing of individual cancer cell types may be the
best way to learn the importance of each.
Fourth, intracellular xenobiorgs might prevent the expression of specific genes
or small groups of genes within cancers. Several methods might be used, including
RNA interference, targeted mutation, and secretion of single-chain antibodies. This
could enable researchers to understand the contributions that individual genes
make to cancer aggressiveness.
Fifth, intracellular xenobiorgs might record changes in differentiated state that
occur as cancers evolve. Much of the confusion surrounding cancer stem cells
might be resolved if differentiation of one cancer cell type into another could be
monitored and quantified. Chapter 15 presents a number of imaginary scenarios
that illustrate how the ability of intracellular xenobiorgs to record conditions within
cancer cells could unravel the mystery of cancer stem cells.
Intracellular xenobiorgs might record many aspects of a cancer cell’s history.
These could include visits to various organs or tissues within the body.
Intracellular xenobiorgs might report—for example—whether specific cells in the
blood had ever exited the blood supply, entered the extracellular matrix, and then
reentered the blood.
It might be possible for intracellular xenobiorgs to be engineered to record
external chemical or physical signals supplied by researchers. These would be
recorded in addition to biological stimuli of the types described above, and would
serve as a clock.
Schemes by which intracellular microbes might sense and record conditions
within their host cell are discussed in Chapter 15. The ideas involve either
controlled DNA rearrangement or controlled asymmetric segregation of replicons
within dividing xenobiorgs. Bacteria have an internal “immune system” that
records recent non-fatal bacteriophage infections and synthesizes antisense RNA to
those viruses; this might be adapted to record host cell conditions.
Sixth, intracellular xenobiorgs could mark individual cancer cells subtypes in
ways that would allow metastatic cancer colonies to be analyzed. Cancer cells
colonize susceptible hosts at low rates (surprisingly). One proposed explanation is
that colonization requires interaction between distinct cell types within the cancer,
and that this interaction occurs infrequently. Intracellular xenobiorgs that marked
individual cancer cell subtypes could confirm or refute this suggestion, which
might open way to new treatments to prevent the deadly interaction.
Seventh, intracellular xenobiorgs might provoke an immune system attack
limited to specific cancer subtypes. They could do this by synthesizing some
especially immunogenic protein that would provoke a strong immune response:
either an antibody response, a cytotoxic response, or both. Limiting the response to
especially dangerous cancer subtypes might maximize the immune response’s
effectiveness while minimizing collateral damage.
The above techniques could be used in combination, of course.
♦ Monitoring, manipulating and making use of the differentiated
states of individual cells within tissues
Showing and manipulating differentiated states in healthy cells. As
explained above, Chapter 15 discusses how intracellular xenobiorgs might reveal
and manipulate the differentiated states of cancer cells. However, as explained
below, Chapter 15 also discusses how intracellular xenobiorgs might reveal and
manipulate the differentiated states of cells in healthy or apparently healthy tissues.
Organs such as the brain and the immune system consist of many different cell
types that must cooperate and communicate for the organ to function properly. A
full understanding of how healthy organs function, and how they can become
diseased, requires a full characterization of each cell type. However, it is very
difficult to accurately characterize individual cells, especially in solid tissues.
Instead, investigators are often limited to average measurements on bulk tissue, or
must resort to tissue disaggregation methods very likely to change the
characteristics that they hope to observe. Intracellular xenobiorgs might
characterize individual cells, even when those cells are tightly joined to other cell
types.
Differences caused by infectious disease. One cause of differences between
cells within a tissue is infectious disease. Infection of individual cells drastically
alters the behavior of those cells, and cells within an organ are likely to be infected
unevenly. The different cell types within an organ may respond differently to
infection, and the autocrine-paracrine signaling that is a constant feature of tissues
may be altered by infection. And, of course, infectious disease activates and
attracts the immune system. By reporting both the state of infection and the state of
differentiation of individual cells within a tissue, intracellular xenobiorgs might
clarify the process of infection.
Differences caused by circadian rhythms. A second cause of cell variation is
biological rhythms, especially circadian rhythms. Circadian rhythms influence
immune system function and vaccination efficiencies. Circadian clocks exist in
most if not all human tissues, and yet can be disrupted and desynchronized by
many environmental and behavioral factors. This generally has bad consequences,
such as exacerbation of rheumatoid arthritis. Intracellular xenobiorgs could reveal
much more about the circadian behavior of individual cells, and eventually might
treat rhythm disorders.
Isolation of rare cell types and removal of unwanted cells. The human body
is composed of at least 210 distinct cell types. There may be additional distinctions
that are not yet recognized, and there are cell types of great interest that exist only
transiently, such as immune cells that are active at the initiation of a vaccination.
Chapter 15 discusses a number of valuable and rare cell types that intracellular
xenobiorgs might help isolate. These include memory B cells, B10 cells, type 2
myeloid dendritic cells, pancreatic stem cells, primordial germ line cells, and
functional oocytes from aged females. In addition, intracellular xenobiorgs might
facilitate the removal of antigen-presenting cells from transplanted bone marrow
and thus reduce graft-versus-host disease.
Revelation of unexpected heterogeneity. Repnumi rejuvenation will require
accurate reconstruction of tissues and organs. Ideally with every cell nucleus and
mitochondrion will be replaced by a more youthful version of itself, but in the
same differentiated state. To accomplish this, doctors and scientists must know and
recognize all of the differentiated cell states that exist in the human body.
As mentioned above, biologists recognize some 210 distinct cell types in the
human body. However, biologists typically classify cell types as distinct based on
their appearance or by the antigens they display. There may be hidden differences
between cells that have indistinguishable appearances and antigen sets. Potential
examples include cells that have rearranged their chromatin in preparation for
changes in gene expression and cells that have repositioned their organelles as part
of a change in function. This idea is further discussed below.
Chapter 15 discusses examples of surprising heterogeneity in seemingly
homogeneous tissues. These include mRNA variation in macrophages,
transcriptional heterogeneity in blastocysts, heterogeneity in hepatic stellate cells,
heterogeneity in pancreatic β cells, and microRNA heterogeneity in mammary
epithelial cells.
Explanations of unexpected heterogeneity. The examples of heterogeneity
discussed might have several explanations. One is that the cells that differ are in
fact permanently, heritably different. However, intermittent transcription,
biological rhythms, cooperative regulation between cells, and injury to some cells
are also plausible explanations. Chapter 15 evaluates all of these.
Useful criteria for classifying cells. One clue to a cell’s identity, of course, is
its position within the body. Other criteria may include: the presence and location
of internal and cell surface proteins, rearrangement of the cytoskeleton, changes in
the position of the nucleus and other organelles, the amount and intracellular
location of various RNA species, changes in chromatin accessibility or
conformation, variations in the intracellular distribution of ions and small
molecules, presence of various enzyme small-molecule products, and variations in
charge across the cell membrane.
Intracellular xenobiorgs might be engineered to report and also to alter any of
the criteria listed above. In addition, intracellular xenobiorgs might mark specific
cell lineages over multiple generations, and thus allow researchers to learn whether
differences between cells are permanent and heritable. Chapter 15 discusses the
technical details of these possibilities.
Alteration of differentiated cell types within tissues. Results obtained using
other methods suggest that intracellular xenobiorgs could change the differentiated
state of individual cells within a solid tissue. This could reveal the contribution of
individual cell types to the functioning of the tissue they reside in.
Keeping Track of Which Cells Have Been Subjected to
Repnumi Rejuvenation (A Brief Summary of Chapter 16)
The need for organized Repnumi. Repnumi rejuvenation will be most
effective if all of the cells in a given tissue undergo Repnumi at least once, but few
if any cells undergo Repnumi more than once. Ensuring that this happens will not
be a trivial problem, and will probably require two procedures: marking of cells
that have been processed and organization of Repnumi activity into a “wave.”
Most target tissues will be solid. First, it should be noted that almost all, but
not all, Repnumi targets will be solid tissues, rather than fluid tissues such as
leukocytes or semen. This is because most fluid tissues exist temporarily and are
derived from solid-tissue progenitor cells. However, there are a few exceptions
such as long-lived memory B cells.
Repnumi on fluid tissues. The best method to systematically perform Repnumi
on mobile cells is probably to waylay them at places they are likely to pass
through. For example, the best place to waylay long-lived lymphocytes may be
lymph nodes or germinal centers. In these areas the body already has procedures to
maximize contact between lymphocytes and antigen-presenting cells and these
procedures might be adapted to find cells slated for Repnumi rejuvenation.
Repnumi in multiple stages. Repnumi rejuvenation of solid tissues will
probably occur in two or three stages. The first stage will be the seeding of the
tissue in question with intracellular xenobiorgs. The second stage will be Repnumi
replacement of the seeded tissues, largely directed by the intracellular xenobiorgs.
The third, and optional, stage will be removal of the intracellular xenobiorgs from
the tissue.
Seeding of target tissues by intracellular xenobiorgs. The first stage, seeding
of the target tissue with intracellular xenobiorgs, could be accomplished by
saturation. If a saturation scheme is used, xenobiorgs derived from bacteria will be
delivered to the target organ by injection or by one of the other methods (via the
lymphatic drainage system or the blood supply) discussed in Chapters 9-11. A high
multiplicity of infection of mobile xenobiorgs will be present and will enter target
cells at random. Intracellular xenobiorgs may spread directly from cell to cell,
since some bacterial parasites of cell can do this. Those that enter a target cell that
has already been seeded with an intracellular xenobiorg will perceive a chemical
signal and self-inactivate.
Invasion of a target cell by multiple xenobiorgs is probably safe, provided that
the xenobiorgs are not themselves harmful and provided that superfluous
xenobiorgs can be quickly inactivated. This would make infection by saturation
with a high multiplicity of infection a viable strategy.
Repnumi replacement. The second stage, Repnumi replacement, probably
could not be accomplished by saturation with a high multiplicity of infection. The
nuclei and mitochondria would have to be delivered by specialized cells that would
also remove the pre-existing nuclei and mitochondria (how this might be done is
discussed in Chapters 17 and 18). Delivery of multiple nuclei to target cells would
likely be harmful.
A temporary signal protein on the target cell surface. Instead, the
intracellular xenobiorg(s) already present within a target cell would probably
secrete a surface protein; this surface protein would signal to the outside that the
cell was ready for Repnumi. A special carrier construct outside the target cell
would recognize this surface protein, link to the target cell, receive the existing
nucleus and mitochondria from the target cell, and replace them with youthful
counterparts. When this was done, the xenobiorg within the target cell would cause
the surface protein to be resorbed by the target cell so that no further acts of
Repnumi could occur.
Avoiding an immune response. Many different surface proteins might be used
to signal that a target cell was ready for Repnumi rejuvenation. It would be better if
the host were immunologically tolerant of the surface protein, although a local
suppression of the immune response might be possible. In principle, any human
cell surface protein not normally present in the target tissue could be used. A
protein that appeared only before birth or only before adulthood and disappeared
thereafter might be best.
One of the first steps of Repnumi rejuvenation in actual patients would
probably be a test for an immune response to the signal protein or proteins used in
Repnumi. A signal protein that induced an immune response would be replaced by
one that did not.
Avoiding the need for a signal protein. It might be possible to avoid using
surface signal proteins if Repnumi were performed on only a small fraction of the
cells within a tissue. If the number of Repnumi-performing constructs were much
lower than the number of target cells, the frequency of multiple acts of Repnumi on
the same target cell might be neglibly low.
Presumably, some means would have to be found to kill and remove the cells
that were not rejuvenated by Repnumi. This might have to be done slowly, to allow
the rejuvenated cells to proliferate and expand into the evacuated space. In this
way, the entire tissue might be rejuvenated.
A traveling “wave” of Repnumi activity. It might be easier to control and
monitor the process of Repnumi rejuvenation if the process occurred in only one
part of a tissue at a time. This might be accomplished in a number of different
ways. Schemes might involve chemical gradients, magnetic fields, sound, or
electromagnetic radiation.
Removal of Original Nuclei and Mitochondria from Target
Cells of Repnumi (A Brief Summary of Chapter 17)
Repnumi vs. replacement of the entire cell. Repnumi rejuvenation would
require removal of the original nuclei and mitochondria from target cells. Chapter
17 discusses how removal might be accomplished.
However, there may be many cases where it would be preferable simply to
replace the entire original cell with a youthful one. There is already a wellestablished, natural, and safe process to remove the original cell: apoptosis.
Apoptotic cells shrivel, with their nuclei, mitochondria, toxic oxidized proteins,
viral parasites, and other contents largely contained. They are then phagocytosed
by their neighbors or by macrophages.
The advantages of Repnumi are that it would preserve existing intercellular
connections, and preserve any cell-specific information stored as cytoplasmic
RNA. These considerations might be important only for neurons and perhaps
myocytes (muscle fibers).
Clean removal of nuclei and mitochondria. In any case, a key step in the
proposed process of Repnumi rejuvenation would be to remove both the original
nucleus (or nuclei) and the original mitochondria. Not only must both the original
nuclei and mitochondria be eliminated, but elimination must be safe. The
machinery that eliminates the original nuclei and original mitochondria must not
damage their replacements. Moreover, debris must not be left outside the cell that
could provoke provoke an immune response or cause other damage such as fouling
the kidneys or blocking microvasculature. Thus, the original nuclei and
mitochondria must be removed cleanly.
Extrusion during erythrocyte maturation. The human body already has a
procedure for eliminating the nucleus and mitochondria from cells; both are
extruded during the maturation of erythrocytes. Chapter 17 explains our current
knowledge of the extrusion processes and discusses how parts of it might be
adapted to remove the original nuclei and mitochondria from cells.
The danger of extruded nuclei and mitochondria. If allowed to remain free
in the extracellular space or to migrate into the blood, large numbers of free nuclei
or mitochondria might prove toxic to a patient undergoing Repnumi. In post-natal
humans, erythrocytes form in bone marrow, and in the marrow macrophages or
other cells in the marrow may scavenge the expelled nuclei and mitochondria.
However, such scavenging would probably not occur during Repnumi unless
specifically engineered to occur.
Selective use of apoptosis machinery. Apoptosis is a process of cellular
suicide that degrades nuclear DNA. The molecular machinery of apoptosis is very
complicated, and some parts of the apoptosis machinery might be adapted to
degrade a cell’s nuclear and mitochondrial DNA while leaving the cell intact.
Whether such selective use of the apoptosis machinery is possible, and whether a
useful residual cytoplasm could result remain to be determined.
Manipulation of cytoskeleton to expel nuclei and mitochondria. It might be
possible to manipulate the cytoskeleton of a cell undergoing Repnumi so that
nuclei and mitochondria were expelled into a waiting macrophage or other receiver
cell. The engineered cell that donated youthful nuclei and mitochondria might also
scavenge the original nuclei and mitochondria, or donation and scavenging might
be done by other engineered cells.
Proteins that compact original nuclei. Compaction of the chromatin before
removal of the chromatin might be useful to Repnumi. If so, intracellular
xenobiorgs might synthesize some protein to compact the host cell nucleus. Many
proteins, human and non-human, are candidate compactors. These include the
proteins that compact chromatin in the early stages of erythrocyte formation
(mentioned above).
Insertion into Cells of Donated Nuclei and Mitochondria in
Repnumi (A Brief Summary of Chapter 18)
Methods to deposit donated nuclei and mitochondria. For Repnumi to
succeed, some method must be found to deposit nuclei and mitochondria within
target cells. One possibility is that the nuclei and mitochondria will be delivered
within a small cell that will disintegrate and release the replacement nuclei and
mitochondria within the enucleated target cell. An alternative is that nuclei and
mitochondria will be passed directly from donor to recipient cell via a pseudopod
that joins the recipient cell and then severs its connection with the donor. Chapter
18 discusses these alternatives.
Adaptation of fertilization machinery. It might be possible for an intracellular
xenobiorg to briefly create a structure on the surface of a target cell that mimics an
ovum ready for fertilization. The incoming nuclei and mitochondria would be
carried by a much smaller cell that would enter by the same means that a sperm
enters an ovum.
Adaptation of the immunological synapse. Pairing of target and donor cells
might be accomplished by adaptation of the molecular machinery that mediates the
so-called immunological synapse. An immunological synapse forms when either
cytotoxic T lymphocytes or natural killer cells pair with cells that they are
programmed to kill.
Adaptation of viral and other fusion proteins. Nature has produced many
proteins that can cause the membranes of separate cells to join. Once cells are in
contact, these proteins can fuse them permanently, so that the two cells eventually
form a single larger cell. A plausible task for such proteins would be to fuse a cell
containing a Repnumi replacement nucleus and replacement mitochondria with a
second cell that had lost its nucleus and mitochondria.
Proteins that might be adapted to fuse cells include the hemagglutinin protein of
the influenza virus, the F (fusion) protein of any of several viruses (measles,
parainfluenza, mumps), and the proteins that form the HIV virological synapse—
which allows HIV-1 to spread directly between T cells. Other candidate proteins or
protein regions include the gp63 protein of Leishmania donovani, the leucine-rich
repeat region of internalin protein of Listeria monocytogenes, and the molecular
machinery that moves Bacillus anthracis spores into dendritic cells.
The above proteins are all derived from human parasites, and thus operate
successfully within the human body. However, other proteins might be tried,
including some that are not from parasites. One such protein is the EFF-1 protein
of the free-living nematode Caenorhabditis elegans, which enables C. elegans cells
to fuse.
It might not be necessary to use a natural protein. Although it is still a very
active area of investigation, the chemistry of peptide-mediated cell-cell fusion is
largely understood. Accordingly, many peptides and proteins could be engineered
that would promote the cell fusion step of Repnumi exactly as doctors and
scientists wished it to go.
A tool to monitor cell fusion. One particularly useful tool to monitor cell
fusion is the divided enzyme. By divided enzyme, I refer to an enzyme protein
divided into two parts, and which can only function when the two parts are brought
together.
For laboratory experiments, where human observers and mechanical
instruments can monitor cell fusions, the divided protein of choice is Green
Fluorescent Protein or its artificial derivatives. Green fluorescent protein (a product
of the jellyfish Aequorea victoria) fluoresces naturally, but can be divided into
portions that only fluoresce when brought together. This is an excellent tool with
which to notice, confirm, and analyze cell fusions.
On the other hand, fluorescence deep within the body, during an episode of
Repnumi rejuvenation, would probably be of little value in controlling Repnumi.
Instead, however, many divisible enzymes produce chemical products that could
be used to control Repnumi. Some of these are human enzymes, while others are
not. Some already exist as subunits that must cooperate, while others would have
to be divided using genetic engineering.
The problem of mitochondrial heteroplasmy. Mitochondrial heteroplasmy is
the presence in the same cell of multiple distinct mitochondrial genomes. Humans
and other mammals have evolved biological processes that sharply limit
mitochondrial heteroplasmy.
Although some degree of mitochondrial heteroplasmy is normal and not
dangerous in humans, some forms of mitochondrial heteroplasmy are very
dangerous, and the reasons for this are not completely understood. Repnumi might
increase the mitochondrial heteroplasmy in rejuvenated cells and could be
dangerous for that reason. Chapter 18 discusses mitochondrial heteroplasmy, its
dangers, and how Repnumi rejuvenation could avoid those dangers.
The problem of cytoplasm transfer and epigenetic change. Epigenetic
changes are heritable changes in gene expression not caused by an alteration in
DNA sequence. (Changes can be inherited from parent cell to daughter cell and
sometimes from parent organism to progeny organism.) In some cases, fusing of
cytoplasms from distinct genetic backgrounds may epigenetically change cells.
Chapter 18 discusses this potential danger and how Repnumi rejuvenation might
avoid it.
Protection of Xenobiorgs from the Immune System (A Brief
Summary of Chapter 19)
Subjects covered. Chapter 19 discusses a large part of the human immune
system, and how xenobiorgs might be shielded from it. Chapter 19 discusses the
so-called “humoral” and “cellular” immune systems, based on antibodies and
cytotoxic T-lymphocytes, respectively. The discussion also includes so-called
“innate” immunity, provided that the involved components are outside the cell
occupied by the parasite that is the immune system’s target.
Toll-like receptors not covered. The part of the immune system that Chapter
19 does NOT discuss is the part that detects intracellular invaders and responds by
attacking the invader within lysosomes and by altering expression of host cell
genes. In other words, Chapter 19 does not discuss most host defenses based on
Toll-like receptors and similar proteins. These are discussed in Chapter 7.
Extracellular xenobiorgs and antibodies. Extracellular xenobiorgs would be
derived from pathogens of either humans or non-human mammals. These
pathogens might be bacteria, unicellular eukaryotes or perhaps (see Chapter 10)
parasitic nematodes. Whatever their origin, they would have to evade the human
immune system. A large part of the immune response to extracellular xenobiorgs
would consist of antibodies.
Defenses against antibodies. As elaborated on below, the most promising antiimmune defenses for extracellular xenobiorgs are those that extracellular
pathogens already use. These are heavily oriented toward defeating antibodies.
They include large changes in surface antigens that can outrun the antibody
immune response, and small changes in surface antigens that can frustrate socalled affinity maturation of antibodies. They also include exploiting the antibody
immunodominance phenomenon to focus the host’s antibody response on
antigens whose blockage will not harm the parasite. In addition to these passive
defenses, extracellular pathogens interfere with specific components of the
immune response.
Immune system killing of xenobiorg host cells. In contrast to extracellular
xenobiorgs, intracellular xenobiorgs would be vulnerable to an antibody response
only if they synthesized some new antigen that appeared on the surface of their
host cell. However, intracellular xenobiorgs would have to evade killing of their
host cells by cytotoxic T lymphocytes or natural killer cells.
Defenses against killing of host cells. The question of how cancers evade the
immune system is much more relevant to intracellular xenobiorgs than to
extracellular xenobiorgs. Cancers evade the host immune system both passively
(by making themselves invisible to the immune system) and actively (by
interfering with specific components of the immune system). The evasion methods
are elaborated on below.
Blocking of apoptosis. The cytotoxic immune response to cancers and
pathogen-infected cells depends on induction of apoptosis by either cytotoxic Tlymphocytes or natural killer cells. The use of intracellular xenobiorgs to block (or
stimulate) apoptosis is discussed at length in Chapter 7. However, it should be
mentioned here that blockage of externally induced apoptosis at a very early stage
could protect intracellular xenobiorgs from the cellular immune system.
Spatial restriction of immunosuppression. Both xenobiotherapy and
Repnumi rejuvenation are likely to be restricted to small regions of the body at any
given time. Protection of these processes from the immune system should also be
local, since immunosuppression throughout the body imposes very high costs. A
number of schemes to restrict immunosuppression to small regions of the body are
elaborated on below. However, Chapter 19 also discusses a few general principles.
Rapid degradation of immunosuppressants. In general, immunosuppression
is likely to be more sharply focused if the molecular agents that mediate it are
created in large amounts where they are needed, but decay rapidly. Thus, unstable
mimics of natural immunosuppressants may be very valuable.
Two-component immunosuppressants. It may also be possible to sharpen the
focus of immunosuppression by using two-component immunosuppressants.
Imagine that suppression of local immunity is mediated by two agents, A and B,
synthesized at the same location. A and B have moderate affinity for each other,
and must act together to be effective. Unless A and B have high affinity for each
other, they will diffuse away from the their site of synthesis as independent
molecules. At any given point, their combined concentration will be the product of
their individual concentrations, and diminish much more rapidly with distance
from their site of synthesis.
In any case, localized immunosuppression is a long-sought goal in
transplantation medicine. That fact ensures that research into the subject will
continue, and improves the chances that any progress made will find quick clinical
use.
♦ Adaptation of Protections for Embryos and Neonates
The maternal immune system of mammals would attack developing embryos
and fetuses unless some biological feature existed to block that attack. Moreover,
as it turns out, there may be a need to block excess immune system activity in
neonates—and this is done via nursing milk.
Progesterone. Progesterone is required for immunological tolerance of an
mother mammal for a fetus and acts locally. Progesterone, or some derivative of
progesterone, might be exploited to shield local regions of xenobiotherapy or
Repnumi.
Protein immunosuppressants. It least in mice, soluble factors from the
placenta can block the effector stages of maternal antipaternal cell-mediated
immunity. Both cytotoxic T-lymphocytes and natural killer cells are blocked.
Because these factors are sensitive to trypsin, they are presumed to contain
proteins.
Humans may have similar factors, or alternatively, factors from mice might
function in humans. Such factors, or rapidly-degrading variants of them, might
protect regions of xenobiotherapy or Repnumi.
Immunomodulators in colostrum. Colostrum is a yellowish liquid secreted by
the mammary glands of female mammals a few days before and after the birth of
their young. Neonates have immature immune systems, and colostrum is
accordingly rich in immune factors. Nevertheless, colostrum also contains
immunomodulators that may protect the neonate from overstimulation by
numerous environmental antigens.
The immunomodulators in colostrum might be adapted to protect regions of
xenobiotherapy or Repnumi. Depending on their nature, rapidly-degrading variants
of them might be created and used.
♦ Prevention of Immune Cell Homing
If leukocytes cannot reach areas of xenobiotherapy or Repnumi, or even if only
some category of cells (such as effector cells) is excluded, the likelihood and
strength of any adverse immune reaction will be reduced.
♦♦ Blocking of leukocyte extravasation
Inactivation of endothelial cells by intracellular xenobiorgs. Inflammation
and other immune responses occur when cells at an infection site signal chemically
for assistance. Endothelial cells on nearby blood vessels (usually post-capillary
venules) arrest traveling leukocytes and induce them to enter the local tissue. If
these steps were to fail, inflammation and other immune response would not occur.
Hence, the simplest approach to preventing immune system attack on
xenobiotherapy or Repnumi might be to seed local endothelial cells with
specialized intracellular xenobiorgs that would block arrest of traveling leukocytes.
When these specialized xenobiorgs were no longer needed, they could be
inactivated and discarded somehow. Chapter 19 discusses this.
Shielding of the endothelial surface by cells. It might be possible for
leukocyte/xenobiorgs to arrest at an area of inflammation, flatten themselves
against the vessel walls, and physically block access to the endothelium. Whether
this would unacceptably constrict the size of the blood vessel in which it occurred
would depend on that vessel’s diameter.
Molecular inhibitors of homing arrest. A number of substances inhibit the
arrest stage of leukocyte homing. Some are synthesized by parasites, others are
synthesized by cancers, and still others are artificial. While such inhibitors are
worth investigating, any use of them to protect a limited region of the body from
immune system action faces a basic difficulty: the flow of blood. Soluble
molecules secreted into the blood, and unanchored cells carrying such molecules
on their surfaces, would be quickly carried away.
Circulating leukocyte/xenobiorgs might arrest at a region of inflammation and
implant inhibitory molecules into the outer membranes of the endothelial cells.
Such as strategy would succeed only if the endothelial cells did not internalize the
implanted molecules, and did not react in some other unacceptable way such as by
changing their shape or detaching from the vessel wall.
Chapter 19 discusses some candidate inhibitors.
Mucin AgC10 from Trypanosoma cruzi. Trypanosoma cruzi is a human
parasite that causes Chagas disease, and L-selectin is an adhesion molecule present
on monocytes. Mucin AgC10 from Trypanosoma cruzi interferes with L-selectinmediated monocyte adhesion to endothelium. Mucin AgC10 seems to cause
shedding of L-selectin from the monocytes.
Mucin AgC10 linked to a membrane anchor might be implanted by circulating
leukocyte/xenobiorgs into endothelial cells near an area of xenobiotherapy or
Repnumi, and protect that area from infiltration by monocytes.
Synthetic compound SLX. P-selectin is an adhesion molecule present on
endothelial cells, and sialyl-LewixX is a natural ligand of P-selectin. SLX, a
synthetic analog of sialyl-LewisX, blocks binding P-selectin to sialyl-LewisX. This
action by SLX enables it to block rejection of transplanted tissue in rats.
Attachment of SLX to a suitable anchor and implantation into endothelial cell
membranes might inhibit P-selectin. In this case, adhesion molecule (P-selectin)
and its inhibitor would reside on the same membrane. For the P-selectin and the
SLX moiety to interact, the SLX might need to be connected to the membrane by a
flexible molecular stalk.
Because SLX is a synthetic molecule, and presumably cannot be made by cells,
the active molecules would have to be pre-loaded into the circulating
leukocyte/xenobiorgs.
♦♦ Blocking of homing after extravasation
After leukocytes exit the blood vasculature and enter tissues, they usually must
travel through the extracellular matrix to reach the site of inflammation—or in this
case the site of xenobiotherapy or Repnumi rejuvenation. Although as a way of
preventing this, it would probably be more effective to block leukocyte
extravasation, it still might be worthwhile to block their movement after
extravasation.
Blocking of CD44-fibronectin binding. Fibronectin is a protein that is part of
the extracellular matrix. Fibronectin is thought to guide leukocytes within tissue
after they extravasate. CD44 is the leukocyte receptor that binds fibronectin, and
antibodies to CD44 can block adhesion of lymphoid cells to fibronectin.
The monovalent single-chain antibodies that xenobiorgs could synthesize and
export would seem to be ideal for blocking the fibronectin-CD44 interaction.
(Monovalent antibodies would be superior to divalent natural antibodies, because
they would not cause the leukocytes to aggregate.) Presumably anti-CD44
antibodies, anti-fibronectin antibodies, or both could be used.
♦♦ Clues from human diseases
Diabetes. Human diabetics have weakened immune systems. In non-obese
diabetic mice, diabetes compromises the homing function of the endometrial
endothelium. The effect of diabetes on the immune system is sure to be
investigated further for its own sake, and might reveal additional information that
could temporarily inactivate the endothelium in selected parts of the body.
Clues from cancers. Tumors can reduce the numbers of type-1-polarized
effector T cells at the tumor site, which probably contributes to their escape from
immune surveillance. This effect, if controllable, might protect areas of
xenobiotherapy or Repnumi rejuvenation from immune system attack.
Cancers that overexpress the endothelin B receptor shield themselves from
infiltration by anti-cancer T cells. Artificial overexpression of the endothelin
receptor might also thwart immune system infiltration of areas of xenobiotherapy
or Repnumi.
Squamous cell cancers can also down-regulate E-selectin in their vicinity, an
activity that shields them from the immune system. E-selectin is present on the
endothelial cells of blood vessels that have been activated by cytokines. E-selectin
participates in leukocyte arrest. If it were known how squamous cells downregulate E-selectin, the effect might be adapted to shield areas of xenobiotherapy
and Repnumi.
♦♦ Disabling immunity in part of the body
It is still not clear to what extent various leukocyte types are spatially restricted
in the body. (Marking of leukocytes with intracellular xenobiorgs might help
answer that question.)
If leukocytes are restricted to specific regions, it might be possible to protect
areas of ongoing xenobiotherapy or Repnumi rejuvenation from immune system
attack without disabling immunity in other parts of the body. If this is true, several
immunosuppressants described below might be useful.
The task will not be easy. Localized immunosuppression has been a constant
goal of organ transplantation medicine. However, extracellular and intracellular
xenobiorgs might facilitate the local release of immunosuppressants.
Blocking egress of lymphocytes from lymph nodes. Fingolimod is a drug
derived from the fungus Isaria sinclairii. Fingolimod is a sphingosine analog, and
prevents the egress of lymphocytes from secondary lymphoid tissue. It is an
agonist of the sphingosine-1-phosphate receptor, and as such prevents T-cell egress
from thymus into blood, T-cell egress from lymph nodes and Peyer's patches into
lymph, and B-cell egress into lymph. Fingolimod might protect specific parts of
the body from being recognized as foreign by the immune system.
Preventing purine biosynthesis in B and T cells. Mycophenolic acid
reversibly inhibits purine biosynthesis in cells. This selectively prevents the growth
of B and T cells, since other cell types can recover purines via a separate salvage
pathway and escape the drug’s effect. Mycophenolic acid is harvested from
Penicillium stoloniferum or P. echinulatum, and hence might be made and secreted
by xenobiorgs. Mycophenolic acid might be useful in creating pockets of immune
deficiency within the body.
Influenza virus neuraminidase. Influenza virus neuraminidase may create a
local immunodeficiency in lung mucosa. The neuraminidase may remove sialic
acid from B and T lymphocytes residing in mucosa and may thereby cause them to
home to bone marrow, away from mucosa. One can imagine redirection of immune
system cells in other contexts by either removing chemical moieties from them or
adding chemical moieties to them.
♦ Passive Protection Against the Immune System
Extracellular xenobiorgs would probably be derived from human or animal
pathogens that operate outside of cells. Such pathogens have methods to evade the
immune system that can probably be transferred between species.
There are two basic strategies for immune system evasion by extracellular
xenobiorgs. The first strategy is passive evasion: not disabling the immune system,
but dodging the antibodies. The second strategy is to disable immune system
function by blocking the action of some critical component. Each strategy has
advantages and disadvantages.
The first strategy, passive evasion, has the advantage that it will probably not
weaken the immune system’s functioning generally; it will not reduce the immune
system’s effectiveness against other pathogens or cancer cells. However, it has the
disadvantage that the immune system may remain aroused and active against an
“infection” that it is unable to destroy. This continuing arousal of an immune
response is generally unhealthy.
The second strategy, actively disabling immune function, has the advantage that
it may silence an immune response. However, it has the disadvantage that it may
weaken the immune system’s ability to fight other pathogens or cancers. In
addition, the body’s regulatory systems are so interconnected, that disabling part of
the immune system may have unforeseen consequences.
Passive and active strategies to evade the immune system are discussed below.
♦♦ Passive evasion of antibodies
A passive strategy: radical antigenic variation. One strategy that an
extracellular pathogen can use to evade the host antibodies is to continually replace
surface antigens with new and different surface antigens. By the time the host is
able to mount an antibody response to an invader, the invader has a new antigenic
profile.
In actual infections, it is generally just a minority of the pathogens that shift
their antigenic profile in time to escape destruction. This accounts for the recurring
symptoms characteristic of some infectious diseases. However, extracellular
xenobiorgs might be engineered to shift their antigenic profiles en masse, either in
response to a signal or according to a schedule.
Antigenic variation over time in Trypanosoma brucei. The African
trypanosome, Trypanosoma brucei, causes sleeping sickness. Trypanosoma brucei
is a single-celled eukaryote that lives free in the human bloodstream.
T. brucei trypanosomes are encased in millions of copies of a single surface
glycoprotein. These surface glycoproteins shield other invariant proteins of the
trypanosomes from recognition by the human immune system. Other proteins on
the trypanosome surface, such as ion channels, transporters, and receptors probably
could not change their antigenic profile without also altering or losing their
function.
The surface glycoproteins are highly immunogenic; they induce an antibody
response that kills trypanosomes carrying them. However, the surface
glycoproteins—which are called Variable Surface Glycoproteins—are replaced
with new and antigenically different variants with a frequency of about 0.1% per
cell division. Trypanosoma brucei populations with the host are large enough to
ensure that individuals expressing new Variable Surface Glycoproteins are always
present. Because it takes several days for an immune response against a given
Variable Surface Glycoprotein to develop, the immune system is always at least
one step behind.
The method by which Trypanosoma brucei evades the host immune system
allows the great bulk of the trypanosome to be periodically killed. This results in
recurring waves of parasites in the blood. Very likely, xenobiorg populations that
were protected by antigenic replacement would instead be engineered to replace
their surface antigens en masse, as suggested above.
Antigenic variation in Anaplasma phagocytophilum. The gram-negative
bacterium Anaplasma phagocytophilum causes granulocytic anaplasmosis in
humans and related diseases in other mammals. The bacterium is obligately
intracellular. However, although the bacterium spends all or almost all of its life
within human cells, it seems to have features whose purpose is to evade humoral
immunity.
The most abundant protein in Anaplasma phagocytophilum is Msp2, encoded
by a multigene family of at least 22 paralogs in the Webster strain genome and 52
or more paralogs in the HZ strain genome. (Paralogs are distinct genes descended
from a duplicated common ancestor.) Antigenic diversity among the Msp2 genes is
increased by gene conversion. Presumably, this antigenic diversity confuses
humoral immunity.
These results argue that even intracellular xenobiorgs might need to escape
humoral immunity, and might benefit from schemes to confuse the humoral
immune system.
Antigenic variation in tandemly repeated peptide sequences. Plasmodium
falciparum causes the most dangerous form of malaria in humans. During an
infection, Plasmodium falciparum is present both within human cells and free in
the blood.
Most of the protein antigens of Plasmodium falciparum contain short sequences
that are extensively repeated in tandem arrays. In most cases, the tandem peptide
repeats of an array vary in sequence. This variation may interfere with affinity
maturation, a process by which antibody-producing cells “learn” to produce
antibodies with increasingly higher affinity for a given antigen.
Surface proteins that included carefully designed arrays of tandem repeats
might protect extracellular xenobiorgs by blocking the generation of high-affinity
antibodies.
Immunodominance of epitopes. In epitope immunodominance, an immune
response is mounted against only a few of the epitopes present on a parasite. If the
antibody response to an immunodominant epitope for some reason fails to kill a
parasite, the entire antibody response may fail.
An antibody response to an immunodominant epitope may fail to kill a parasite
because the epitope is frequently replaced (see above), because high-affinity
antibodies to the epitope cannot be created (see above), because the epitope is shed
into the surrounding medium to act as a decoy, or because the parasite resists
antibody-mediated killing.
Shielding of parasites. Trypanosoma cruzi, the causative agent of Chagas
disease, has a trans-sialidase that transfers sialic acid from the environment to the
parasite’s mucins. These mucins entirely cover the parasite surface. The sialyated
mucins protect the parasite against complement-mediated lysis as long as lytic
antibodies are not also present.
Extracellular xenobiorgs might make use of any of these passive methods to
evade host antibodies. However, as noted above, all of these methods would allow
an antibody response to the xenobiorg to develop.
♦♦ Passive evasion of the cellular immune system
It might be possible to passively evade cellular immunity as well as humoral
immunity. Cellular immunity involves the presentation of small fragments of
proteins on cell surfaces. Small fragments that are recognized as foreign induce
cytotoxic T lymphocytes to destroy the presenting cell.
The machinery that degrades proteins into fragments and the machinery that
presents those fragments on the cell surface both have biases. Many potential
peptide fragments are never created and some peptide fragments tend to
outcompete others in binding to the presentation machinery. These biases might be
exploited by genetic engineers to minimize the presentation of foreign peptides on
the surfaces of cells infected by xenobiorgs.
It might be possible to redesign xenobiorg proteins to resist degradation into
peptides that strongly provoke cytotoxic T lymphocytes. It might be possible to
flood the host cells with proteins or peptides that will exclude provocative peptides
from the antigen presentation machinery.
♦ Blocking the Action of Immune System Components
The human body has elaborate safeguards to prevent autoimmunity. Moreover,
human pathogens and successful human cancers have adaptations to thwart an
immune response. Genetic engineers who wish to suppress the immune system can
borrow these features or invent new ones. Chapter 19 proposes schemes to disable
immune system machinery in order to protect areas of xenobiotherapy and
Repnumi rejuvenation from immune system attack.
♦♦ Harnessing the body’s protections against autoimmunity
The thymus gland. The human thymus gland is the organ in which most selfreacting T cells are deleted. Ways to manipulate the thymus gland or thymus tissue
to induce immunologic tolerance are discussed in Chapter 19.
Treg cells. In the periphery, tolerance to self antigens is mediated mainly by the
CD4(+)CD25(+)FOXP3(+) subset of regulatory T cells (Treg cells), which can
suppress the activity of autoreactive T cells that have escaped deletion in the
thymus. Artificial stimulation of this population’s numbers or activity might
protect areas of xenobiotherapy or Repnumi.
Mesenchymal stem cells. Mesenchyme-derived stem cells are candidate agents
to reverse Parkinson’s disease. Mesenchymal stem cells inhibit nearly all immune
responses that depend on cell-cell contact and release soluble factors that suppress
immunity. These might be especially valuable in protecting intracellular
xenobiorgs from CTL killing of their host cell.
Human galectin-3. Galectins are a class of lectins present in mammals, and
which bind β-galactoside sugars. Humans have 10 distinct galectins. Unlike most
lectins, galectins are soluble rather than membrane-bound. Human galectin-3
opposes T cell receptor activation. Human galectin-3 or some derivative thereof
might shield xenobiotherapy or Repnumi from cell-mediated immunity.
♦♦ Decoy components
gp130. Melanoma cell lines release soluble gp130, a potential antagonist of all
cytokines of the IL-6 family. gp130 is part of all interleukin-6 receptors, and the
soluble protein is probably created by proteolysis. Soluble gp130 protects some
melanomas from inhibition by IL-6.
Soluble receptors for tumor necrosis factor. Malignant gliomas continually
shed soluble receptors for tumor necrosis factor. These soluble receptors may
block immune system function and spare their parent cancers.
The above two examples indicate that inactive decoy components can block
critical immune system interactions. Chapter 19 discusses additional examples
where pathogens or cancers protect themselves by releasing decoy immune system
components, and discusses opportunities for genetic engineers to use the same
principles to protect regions of xenobiotherapy or Repnumi from the immune
system.
♦♦ Immunity-blocking machinery from pathogens
CMV homolog of IL-10. Human cytomegalovirus (CMV) encodes a
homologue of interleukin-10 that inhibits maturation of dendritic cells and reduces
their functionality. Dendritic cells are antigen-presenting cells, and are important
components of the mammalian immune system. The homologue encoded by CMV
prevents dendritic cells from secreting interleukin-12, and thus blocks several
important immune-system functions.
If the CMV-encoded homologue’s effects could be limited in its effects to a
local region, it might protect xenobiotherapy and Repnumi from interference by
the immune system.
Serpins. Serine protease inhibitors (serpins) expressed by many parasites from
viruses to parasitic nematodes can inhibit immune responses. The vaccinia
(cowpox) and myxoma (rabbit pox) viruses, and microfilariae of the parasite
nematode Brugia malayi, express serpins that inhibit immune responses.
Nef protein of lentiviruses. Virus-infected cells are usually destroyed when
fragments of viral proteins are presented on the host cell surface by the Major
Histocompatibility I system, and the cell is consequently destroyed by cytotoxic T
lymphocytes. This prevents the infection from spreading to healthy cells.
Many viruses protect themselves from this process by downregulating the
MHC-I presentation machinery.
The Nef proteins of primate lentiviruses (including the viruses HIV-1 and HIV2) downregulate expression of cell surface receptors that are important for immune
system function. Among the receptors downregulated are CD4, CD28, the T-cell
antigen receptor, and the class I and class II major histocompatibility complexes
(MHC-I and MHC-II).
Some Nef proteins, although not those from HIV-1 and HIV-2, downregulate
the chemokine receptor CXCR4. The Nef proteins downregulate CXCR4 by
downregulating its endocytosis. This strongly inhibits migration of lymphocytes
toward SDF-1, the CXCR4 ligand. Nef proteins, or portions thereof, might locally
shield xenobiotherapy or Repnumi.
Mycobacterium. The Mycobacterium genus of bacteria includes the pathogens
that cause tuberculosis and leprosy. Mycobacterium antigens down-regulate the
chemokine receptor CCR5. CCR5 is thought to function in the inflammatory
response to infection.
Staphylococcus aureus. Staphylococcus aureus is a Gram-positive bacterium,
and a dangerous human pathogen. S. aureus thwarts human the human immune
system in multiple ways.
Staphylococcus aureus superantigen-like protein 7 binds IgA and complement
C5 and inhibits IgA-FcαRI binding and serum killing of bacteria. S. aureus
superantigen-like protein 10 binds human IgG, as does S. aureus protein A.
S. aureus coagulase clots plasma and coats the bacteria, which probably prevents
them from being phagocytosed. In addition, the bacterium sythesizes 4 separate
proteins to block complement activity.
S. aureus superantigen-like protein 10 binds the chemokine receptor CXCR4,
and prevents it from interacting with the chemokine CXCL12. CXCL12 is also
called Stromal cell-derived factor-1, and strongly attracts lymphocytes.
An interaction between a Staphylococcus aureus toxin and class II MHC
products on antigen-presenting cells can inhibit co-stimulatory activity and thus
impair clonal expansion of T cells specific for bacterial antigens.
Other pathogens. Humans have many viral, bacterial, and eukaryotic
pathogens. All of these have ways to subvert the human immune system, and many
may be adaptable to act within just part of the human body. Chapter 19 discusses
what is known.
♦♦ Immunity-blocking machinery from cancers
Cancers often produce tumor-specific antigens. Recognition of these antigens
by the immune system would in many cases destroy the cancer—but often the
cancer escapes.
Ovarian cancer MUC16. Many cancers lose their MHC-I peptide-presentation
machinery.The body has Natural Killer cells which destroy human cells that have
lost their MHC antigen presentation machinery. However, the ovarian cancer
antigen MUC16 inhibits the cytotoxicity of Natural Killer cells and downregulates
CD16, a key Natural Killer cell control protein. This helps the cancer escape
control.
Squamous cell carcinomas can recruit FOXP3(+) regulatory T cells that induce
self-tolerance. (Mice lacking FoxP3 regulatory T cell migration develop severe
skin inflammation.) Squamous cell carcinomas also produce prostaglandin E2,
which is a potent inhibitor of the cellular immune response.
Adenosine. Adenosine occurs in the interstitial fluid of solid tumors at
concentrations that can inhibit cell-mediated immune responses in tumor cells.
Extracellular adenosine inhibits T lymphocyte activation and effector function,
including T cell adhesion to tumor cells and cytotoxic activity, by signaling
primarily through A2a and A3 adenosine receptors on the surfaces of T cells. A2a
adenosine receptor signaling has also been implicated in adenosine-mediated
inhibition of cytokine production and cytotoxic activity by activated natural killer
cells.
Other means, such as single-chain monoclonal antibodies, might be found to
stimulate the same receptors that extracellular adenosine does.
Adaptation of tumor escape mechanisms. Tumors have many means to
escape destruction by the immune system. Many are known, and very likely others
remain to be discovered. At least some may act locally and be adaptable to
defending small areas of xenobiotherapy and Repnumi rejuvenation.
♦♦ Engineered immune system inhibitors
Our understanding of the immune system is comprehensive enough to enable
engineers to design immune system inhibitors that do not exist in nature. These
inhibitors will include monoclonal antibodies, decoy receptors for ligands, ligands
that either block or overstimulate receptors, and targeted toxins that kill specific
immune system cells. Several examples already exist.
Xenobiorgs probably cannot be engineered to synthesize and export classic
four-chain antibodies, or at least doing so will not be easy. However, as Chapter 19
discusses, in many cases xenobiorgs could make single-chain mimics of such
antibodies.
Infliximab. Psoriasis is a chronic skin disease caused by inappropriate activity
of the immune system. The engineered monoclonal antibody Infliximab, which
binds and inhibits soluble and membrane-bound tumor necrosis factor, gives longterm relief against psoriasis. Infliximab is also approved to treat Crohn’s disease,
ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, and ulcerative
colitis.
Antibodies to LFA-1 and ICAM-1. Lymphocyte function-associated antigen
1 (LFA-1; also termed integrin αLβ2) binds intercellular adhesion molecule 1
(ICAM-1). Together LFA-1 and ICAM-1 mediate contact between antigenpresenting cells and T-lymphocytes. Antibodies to either molecule can prolong
tolerance to allografts in mice.
Localized release of single-chain antibodies near regions of xenobiotherapy and
Repnumi rejuvenation might prevent an immune reaction from developing. In
principle, this is better than thwarting an immune reaction that has already started.
Killing activated T cells. It might also be possible to kill activated T cells.
Mice were protected from experimental autoimmune encephalitis by a construct
that fused the ligand CXCL10 to a truncated (but still effective) version of a
diphtheria toxin. This construct bound the receptor CXCR3 and killed cells
expressing CXCR3, reducing their infiltration into areas of inflammation.
Although this is probably less desirable than inactivating the T cells would be, it
might be effective.
The same principle might be applied to antibody-producing B cells.
Repnumi Rejuvenation of the Central Nervous System (A
Brief Summary of Chapter 20)
♦ Repnumi rejuvenation and the special problem of
individualized nuclei
Repumi rejuvenation and memory. Repnumi rejuvenation of the brain may
face a special obstacle. The brain is the seat of memory, and memory is an
enormously important part of a person’s identity. If mental memory, like
immunological memory, involves somatic changes in DNA sequences,
replacement of brain cell nuclei with unrearranged nuclei of the same type could
weaken or erase a person’s memories.
Similarly, unique regulatory changes (e.g. involving DNA methylation) may
occur in the nuclei of individual brain cells and contribute to memory. These
changes could not easily be duplicated by replacement nuclei during Repnumi
rejuvenation.
Repnumi rejuvenation and personality. Moreover, even if memory itself does
not involve changes in brain cell DNA sequences or regulation, such changes may
occur over time, affect brain functioning, and contribute to personality.
Replacement of brain cell nuclei with fresh nuclei of the same type could erase
these contributions to personality.
DNA methylation within the brain seems to play a role in multi-factorial
psychiatric disorders. In at least one case, hypermethylation of brain DNA
excerbates psychosis, while hypomethylation tends to reverse psychosis.
Interestingly, overall methylation in brain DNA increases with age.
Neurons and glial cells. The brain includes two main cell types, neurons and
glial cells. Of the two, neurons are thought to contribute much more to brain
function and consciousness. However, both may be important, and both may
accumulate DNA changes or epigenetic changes that affect their function.
Repnumi and non-brain parts of the nervous system. Also, other parts of the
nervous system might contribute to consciousness, and might also undergo DNA
rearrangements or epigenetic changes that affect their function.
I refer to the nuclei of brain or other nervous system tissue that are important to
identity and which have undergone individualized and unpredictable changes as
individualized nuclei.
Strategies to preserve memory and personality. If we do not want to erase
memory or peculiarities of personality, we must either replace aged genomes with
more youthful genomes that duplicate all important genetic and epigenetic changes
of the of the original genomes, or we must learn to restore the original genome to a
youthful state by repairing it.
Repair of some regions, replacement of others. So far, only a little is known
about the changes that occur in brain cell genomes over time and even less is
known about their significance. Many of the changes that occur may be
undesirable or inconsequential. Whole chromosomes are sometimes lost from brain
cells; is this beneficial in some way, or is it merely an accident?
If mental memory involves DNA sequence changes or epigenetic changes, but
follows the same pattern as immunological memory, the relevant changes may be
limited to just a few regions of the genome. It might be possible to isolate and
repair just those regions, while replacing the rest of the genome.
Neural identity and protocadherins. An important part of neural cooperation
may be the assigning of an individual identity to each neuron. The protocadherin
protein family, which mediates many connections between neurons and other
neuron behavior (such as apoptosis) has been suggested to give individual neurons
a unique identity. Protocadherins are known to mediate neural self-avoidance.
Repnumi rejuvenation would have to maintain the sequences and expression of the
protocadherin genes within each neuron.
Although the contribution of the protocadherin genes is not known, an
analogous gene in the fruit fly Drosophila melanogaster (Down syndrome cell
adhesion molecule, DSCAM) can form 38,016 splicing isoforms. This is allows
every neuron in the fly to display a unique set of DSCAM proteins on its cell
surface. DSCAM interaction stimulates self-avoidance mechanisms that are
essential for normal neural circuit development.
Human protocadherin genes are largely clustered in the on chromosome 5 in
band 5q31. This might make it possible for an invading xenobiorg to excise and
sequester only that region, while replacing the rest of the genome. However, other
protocadherin genes, scattered throughout the genome, might also have to be
preserved individually.
DNA rearrangement in developing brain. At least one DNA region
undergoes rearrangement and yields circular DNA in mouse brain during
embryogenesis. The DNA has sequence similarity to a region in the human
genome.
The immune system. The human immune system also incorporates a kind of
“memory” based on DNA rearrangements. The need to preserve individualized
immune system nuclei is probably much less than the need to preserve
individualized brain nuclei would be. People who have become immune to certain
diseases and wish to retain that immunity might simply be re-vaccinated.
However, preserving the memory of long-lived immune system cells, such as
memory B cells, might be worthwhile. Repairing some parts of genome of memory
B cells, while replacing the rest could serve as a model for the same manipulation
on neurons.
A much more serious problem attending Repnumi rejuvenation of immune
system tissue would be prevention of autoimmunity.
♦ Repnumi rejuvenation or cell replacement in the brain:
additional considerations
Immunological privilege. Some neurons also immunologically privileged. This
might allow xenobiorgs to operate within them with less danger of provoking an
immune response.
Problems posed by neuron structure. Neurons are exceptionally large,
elongated, compartmented cells. Hence, removing and replacing all mitochondria
or mitochondrial genomes within them will be exceptionally difficult. Ridding
them of intracellular junk using intracellular xenobiorgs may also be difficult.
Cell replacement as an alternative. It seems likely that replacing brain cells
using neural stem cells will be much easier than would be replacing their nuclei
and mitochondria using Repnumi. Therefore, a key question is to what extent
replacement of lost brain tissue using stem cells can substitute for Repnumi
rejuvenation. During the course of an adult life, the brain loses about 1/3 of its
neurons. And yet people usually retain many of their memories, their personalities,
and their sense of self. Replacing the lost one-third of neurons would presumably
not interfere with those same things.
Neural stem cells. The subject of how to create, preserve, and administer
human neural stem cells to treat human brain injury is under intense investigation.
Isolation of neural stem cells. Human neural stem cells are not too difficult to
isolate. They can be created from non-neural cell types such as skeletal muscle
cells, bone marrow mesenchymal stromal cells, and others. Some human neural
progenitor cells can be isolated from the nasal mucosa, even the mucosa of people
with a neurodegenerative disease (Parkinson’s).
Neural stem cells ameliorate damage. Human neural stem cells will migrate
toward injured brain tissue. Human neural stem cells can ameliorate ischemic
stroke in animal models and can ameliorate Parkinson’s disease in rats. They can
improve learning and memory in animal models of human brain diseases such as
Alzheimer’s disease, and rats with other forms of induced brain damage. It is still
not known whether they can restore pre-existing memories that appear to have
been lost via some process such as Alzheimer’s disease.
Potential contributions by intracellular xenobiorgs. Intracellular xenobiorgs
might contribute a great deal to neural stem cells. They could induce differentiated
cells to dedifferentiate into neural stem cells. They could preserve such cells in the
proper state, and direct the transformation of neural stem cells into desired cell
types at the desired site.
Intracellular xenobiorgs could ensure that genes essential for neural survival,
such as the presenilin gene, continue to be expressed. Intracellular xenobiorgs
could could also ensure that helpful gene products such as secretoneurin are
expressed by neural stem cells, even if those stem cells would not normally do so.
Secretoneurin is a neuropeptide. It reduces damage from stroke and increases
neural plasticity. It attracts stem cells to areas of damage, and promotes formation
of new blood vessels. This might be delivered by engineered human cells, or by
intracellular or extracellular xenobiorgs.
It is also possible, as discussed in Chapter 7, that intracellular xenobiorgs could
preserve much nervous tissue by blocking apoptosis. Neural tissue is quite
sensitive to apoptosis, and inappropriate apoptosis greatly magnifies brain damage
from stroke, Alzheimer’s disease, Parkinson’s disease and so on.
Facilitated Telepathy: The Wildest Possibility (A Brief
Summary of Chapter 21)
The most amazing use to which xenobiorgs might be put would be to facilitate
telepathy. Facilitated telepathy, should it come into existence, might alter human
existence as much as has the printing press.
Definition of facilitated telepathy. Facilitated telepathy would be the use
engineered devices to read the state of a person’s individual brain cells and to
influence the state of those same brain cells. The purpose would be to transmit
thoughts to and from a person (or animal).
I prefer the term “facilitated telepathy” to “telepathy” because telepathy has
long been a staple of science fiction and is often portrayed as being mediated by
something other than ordinary physical phenomena such as light,
electromagnetism, sound, and so on. As discussed below, facilitated telepathy
would very likely be mediated by light, by radio waves, and by magnetic fields—
and would employ powerful computers.
♦ The technology of facilitated telepathy
Marking of individual neurons. In normal human brains, each neuron is
thought to be uniquely marked by a combination of protocadherin variants
(discussed in Chapter 20). Intracellular xenobiorgs might also mark neurons
individually in a way that could be detected from outside the skull (see below).
Intracellular xenobiorgs might mark neurons periodically along their length,
and might also mark the various parts of neurons individually. Neurons have
structure, with axons, dendrites, the soma, and smaller structures; xenobiorgs
might mark these as all belonging to the same neuron, but distinguish the neuron
region as well.
Although neurons are the dominant contributors to brain function, other cell
types are present as well, and might also contribute. Intracellular xenobiorgs might
also mark these.
♦♦ Optogenetics
Optics plus genetics. Optogenetics is a new and very powerful set of
techniques for exploring brain function. Optogenetics combines optics and
genetics, and enables researchers both to control and monitor individual neurons.
Optogenetics allows recording of events on the millisecond time scale, which is
essential to analysis of neuron behavior.
Light sources. Optogenetics has a number of essential components. First, there
must be light source such as an optical fiber or a light-emitting diode. Lightemitting diodes have the advantage that they can draw their power from a wireless
source, which enables researchers to avoid tethering an animal.
Sometimes a window. Second, if the experimental animal has a skull, there
will have to be a zirconia window or other arrangement to let light in. (The animals
investigated so far include monkeys, rats and mice—which have skulls—and fruit
flies and nematodes, which do not.)
Light-sensitive proteins. Third, the critical reagents for optogenetics are lightsensitive proteins. These consist of opsin proteins (usually microbial) attached to
effector domains. The fused proteins can excite neurons, silence neurons, or report
information from neurons. They can raise or lower the levels of signal
biochemicals such as cyclic AMP and inositol triphosphate. Using the fused
proteins, researchers can achieve considerable control over the internal
biochemical state of brain cells.
Tissue-specific expression. A critical advantage of the light-sensitive proteins
used in optogenetics is that their expression can be restricted to chosen tissues
using tissue-specific gene promoters or other methods of tissue-specific gene
expression.
Instruments to read and record data. Fourth, there must be ways of reading
and recording the experimental results. Recording devices could include devices to
monitor animal behavior, devices to read electrical activity within parts of the
brain, devices to monitor changes in the flow of blood to parts of the brain, devices
to monitor activity of individual neurons, and devices to read and record many
other types of information.
Xenobiorgs could relay light to the brain. Xenobiorgs, either intracellular or
extracellular, might enhance optogenetics in a number of ways. First, they might
relay light signals from the exterior of the brain to the interior. Currently, light
must either be restricted to brain tissue near the brain surface, or must be carried to
deeper areas via implanted fiber optics. A class of fluorescing xenobiorgs stationed
either within or between cells could relay light signals throughout the brain. This
would require sophisticated mechanisms to emit light upon receiving light, and
then to quench the signal. One possibility is that intermittent magnetic fields
covering the brain might provide alternating permissive and non-permissive
conditions that would quench light relays after they had spread a signal throughout
the brain.
Xenobiorgs could relay light from the brain. Second, one type of xenobiorg
might read experimental information deep in the brain and then relay that
information via a second type of fluorescent xenobiorg to the brain surface, where
it could be recorded.
Sophisticated manipulations. Third, because xenobiorgs would be derived
from microbes (probably bacteria), they could be engineered to perform more
sophisticated tasks than is possible with ordinary genetic engineering of nuclear
genomes. For example, a xenobiorg within a nerve axon might be engineered to
report nerve impulses as they pass by, but to stop such reporting if cyclic AMP
levels rise above a certain level. This could limit reporting to a certain class of
neurons that interested experimenters.
Determining the order of steps. Light-activated proteins that perform steps in
a multistep process can also be used to determine the order in which those steps are
performed. If a step performed by protein A comes before a step performed by
protein B and the proteins are activated briefly in that order, the process will occur
in full. If the proteins are activated in reverse order, the process will stop after step
A but before step B.
Transferability between animal strains and species. Fourth, because
xenobiorgs would be a type of module, they could easily be transferred—for
example—between strains or species of mouse. They might not behave exactly the
same in each host type, but they would probably allow greater standardization than
does ordinary genetic engineering.
Requirement for multiple xenobiorg types? Facilitated telepathy based on
optogenetics might require multiple types of xenobiorg to perform distinct
functions. Intra-neural xenobiorgs and extracellular xenobiorgs (stationed at neural
junctions) might be used individually or together. Relaying of light signals through
the brain, translating light signals into chemical or electrical impulses and vice
versa, and reading the state of neurons are all distinct tasks and might require
separate xenobiorg types.
In the long run. In the long run, xenobiorgs activated by light, and which
report by light, might form the basis for facilitated telepathy. It has already been
used for purposes such as altering cocaine dependence in mice.
♦♦ Functional Magnetic Resonance Imaging
fMRI detects changes in blood flow. The second most powerful technique for
monitoring brain activity is functional magnetic resonance imaging (fMRI). fMRI
monitors ongoing brain activity by detecting associated changes in blood flow. The
physical phenomena used in the process are radio waves and magnetic fields.
Noise must be removed. fMRI has its limits. The data that it gathers generally
consists of a signal of interest corrupted by much noise. This noise must be
removed by statistical techniques.
BOLD fMRI. The most widely used version of fMRI, Blood-oxygen-level
dependent (BOLD) fMRI, can localize brain activity to within millimeters, but has
time resolution of no better than a few seconds. This level of time resolution is far
too crude to capture all neural activity.
Successes of fMRI. Despite these limits, fMRI allows researchers to deduce
what a human subject is viewing at any given time. fMRI may also allow
investigators to determine whether a human subject has seen a particular scene
before, for instance a crime scene. fMRI may also allow investigators to determine
whether a human subject telling the truth, and perhaps even to deduce a human
subject’s intentions.
Contributions from xenobiorgs. Intracellular or extracellular xenobiorgs
within the brain might increase the spatial resolution and time resolution of fMRI.
Efforts to develop better biomarkers than BOLD are constantly being made.
Physical characteristics within the brain that might be reported include
temperature, acidity/alkalinity (pH), calcium levels, levels of almost any
biochemical, neuronal magnetic field, and the Lorentz effect.
Functional magnetic resonance imaging and optogenetics might be used
together.
♦♦ Magnetite
There may already be a way in which neurons receive information directly from
the environment without involvement of sensory organs. Magnetite (Fe3O4) is a
ferrimagnetic iron oxide present in many species and produced biogenically.
Geomagnetic field sensing. In many species with deposits of magnetite, it is
not known whether the magnetite has a function. However, in other species it is
known to function in navigation via geomagnetic field sensing. The best known
example of this is magnetotactic bacteria, which produce chains of magnetic
particles that act as a ‘compass needle’ orienting the organism as it swims.
Magnetite connected to neurons. Magnetite has a direct neurological
connection in the rainbow trout, Oncorhynchus mykiss. It may also be involved in
navigation of homing pigeons.
An effect of electromagnetic radiation. Magnetite is present in the nematode
Caenorhabditis elegans. This organism produces heat shock proteins in response to
mobile phone-type electromagnetic field exposure—an effect thought to involve
the magnetite. Heat shock proteins are generally produced in response to stress.
Stimulating the brain with rapid bursts of magnetic energy can be used to treat
major depression. The magnetic energy itself is generally safe, but not without
effect.
Magnetite and Alzheimers disease. Magnetite may accumulate in humans
with Alzheimer’s disease.
Magnetite and facilitated telepathy. Since some natural bacteria can create
and harbor magnetite, xenobiorgs might also be engineered to do so. If
electromagnetic fields that affect the magnetite can be used safely around the
human brain, magnetite might form another means by which instruments outside
the skull could interact with brain cells.
♦♦ Movement of prosthetic limbs
Considerable progress has already been made in enabling humans and other
animals to control external devices using just their thoughts. A rat can make a
robotic arm push a lever, a monkey can play a video game and a person with
quadriplegia can pick up a bottle of coffee and sip from it, all simply by thinking
about the action. So far, there has been less progress in giving prosthetic limbs a
sense of touch.
♦♦ Exchange of information between humans and machines
Human-machine and human-machine-human exchanges. Assuming that it
is achieved, facilitated telepathy will involve exchanges of mental information
between humans and machines. For example, an engineer might telepathically ask
an electronic calculator to solve a complex mathematical problem on the fly as the
engineer ponders how to design something. In some cases, the exchange between a
human and a machine will be followed by an exchange between the machine and a
second human—with the end result that thoughts are exchanged between people.
The need for adaptation and mapping. It is already clear, however, that
human brains differ slightly. In the present, this fact complicates attempts to use
experience from one brain to decode signals from another, and in the future it may
complicate facilitated telepathy in at least two ways. First, time and effort may be
required of of both machine and human for a machine to learn to communicate
with a given human’s brain, and the machine will not communicate well with a
second human without another long period of learning. Second, in order for
thoughts to be transferred between humans, information will have to be mapped
from one brain to another—and unless the mapping process is very obvious and
straightforward, it seems likely to produce heated controversy.
Human-animal exchanges. People will, of course, try similar information
exchanges between humans and non-human animals. The required mapping will
certainly not be straightforward, and will certainly cause controversy.
♦ The sociology of facilitated telepathy
The final part of Chapter 21 speculates on the social effects that facilitated
telepathy might have.
♦♦ The social effects of human-to-machine facilitated telepathy
A large library of knowledge and skills. If facilitated telepathy works well
technically, people may gain automatic and quick access to a large library of
knowledge and skills that would otherwise be too awkward for them to find. One
can imagine a tourist walking confidently through the streets of an unfamiliar city.
Thanks to a small computer implanted in her skull, and which communicates with
her brain, she knows exactly where she is going, where the city’s points of interest
are, where the nice restaurants are, and where the bad parts of town are.
The tourist is not wearing a watch, but she knows exactly what time it is. When
people speak to her in a language that she has never heard, after a few moments’
delay, she understands the gist of what they are saying. If she is uncertain about the
laws of the country she is visiting, after thinking about the subject for a few
moments, she has the answer to any questions she may have.
When she reaches her hotel room, a thought strikes her. She realizes that the
currency exchange rate between her native country and the country that she is
visiting has changed, and that she can now afford to spend a bit more for lunch
than she had planned. This changes the restaurant that she will have dinner in.
While she is in her hotel room, she replays the dreams that she had the previous
night. She has a strong interest in her own dreams, and does not believe the
psychologists who maintain that they are nothing more than a meaningless
byproduct of memory processing during sleep. However, she discovers that her
previous night’s dreams were jumbled and meaningless, and reluctantly admits to
herself that her own dreams are beginning to bore her. Instead, she amuses herself
by mentally experiencing a comedy show that she is fond of in her home country,
but which is not broadcast in the country she is visiting.
Thanks to the device in her skull, the tourist also has an excellent memory for
her own experiences. She never forgets the name of a person she has met, although
she may have to pause a moment for the information to appear. If someone asks
her what she was doing exactly three years ago, to the day and minute, after
thinking about if for a few seconds, she can give a correct answer.
The device in the tourist’s skull informs her that there has been a major
earthquake in her home country, but that all of her relatives and friends are safe.
The device tells her that despite her fondness for red-colored clothes, she would
look better to most denizens of the country she is visiting if she wore blue rather
than red—and she decides to follow this advice when dressing for dinner. The
device also tells her that she would make a better impression on strangers if she
were bolder and more confident—but she ignore this, knowing that the device has
been wrong in the past.
♦♦ The social effects of human-to-human facilitated telepathy
American society (and probably most societies) gives each person the right and
responsibility to direct his or her own life. Society generally favors increases in
choices and opportunity, even when it is clear that some people will make unwise
choices and end up worse off than if they had never been given a particular
opportunity. Thus, although facilitated telepathy is likely to spawn both good and
evil, society is likely to embrace it if it is medically safe.
Retrieving of archived memories. It would be nice if Americans and other
people could experience what was in the mind of Abraham Lincoln as he signed
the Emancipation Proclamation. This is not possible, of course, because Lincoln
was dead long before facilitated telepathy became possible. However, later events,
such as the first human visit to the minor planet Pluto might be recorded and
available over the Internet.
Sharing of recent memories. If thoughts can be read, transmitted and
broadcast over the Internet to interested viewers, many experiences such as the
triumphs and failures of great athletes may be made available to the world. Internet
dating sites may allow users to post “thoughtcasts” along with photographs and
videos.
It was Oscar Wilde who said, “The public have an insatiable curiosity to know
everything, except what is worth knowing." People who wish to experience the
thoughts of celebrities, along with watching or reading about their antics, may find
opportunity to do so.
The thoughts of animals. People will surely try to experience the emotional
states of animals, both of industrial animals and of pets. Most likely, we will
discover that animals are much more like us than we currently think—although this
idea will be contested by those who claim that animal sensations change their
subjective effect when reproduced in the context of a self-aware human brain.
The result will probably be a great increase in animal rights. People may stop
eating animals, and instead get meat, milk, and leather from cultured cells. Since
neurobiology is the discipline that will make facilitated telepathy possible, and
neurobiological research depends on the continual sacrifice of experimental
animals, neurobiology may be come the first scientific discipline to commit
suicide.
We may also discover that animals have their neuroses and psychoses. I suspect
that most wild animals will turn out to be psychotic.
Facilitated telepathy and abortion. People will also try to experience the
sensations that a developing fetus experiences—and the results may change
society’s judgment about abortion.
Increased self-awareness. Some people will record and analyze everything
about their lives, perhaps try to preserve it all for posterity, and in many cases will
post it all on the Internet. There will be a glut of such information, much of it
repetitive. It will turn out that most of our souls are much the same.
On the other hand, detailed recording and analysis of people’s behavior and
thoughts may make possible a degree of self-examination that has never before
been possible. At least some people will learn to behave more rationally and
efficiently.
Unfamiliar experiences. People may mentally experience things that they
would never try in real life—from exotic eroticism to race car driving, skydiving,
and BASE jumping.
Better communication. I suspect that people’s thought processes are usually
more sophisticated than their attempts to communicate. This has the pleasant side
effect of allowing most people to think of themselves as brighter than average—
since they compare what they are thinking with what other people are saying.
However, it impedes communication, and facilitated telepathy may remove that
impediment.
The danger of mind hijacking. Facilitated telepathy will also have its dangers.
The first big question that has to be answered is whether an interloper could take
control of a person’s mind via facilitated telepathy. As Chapter 21 discusses, many
parasites alter the behavior of their animal hosts, to their own advantage and to the
disadvantage of their hosts. Could a mind hijacker do the same in humans?
Could a person resist? One possibility is that since the human mind has never
been exposed to facilitated telepathy, it will have no natural defenses against it. A
contrasting possibility is that the human brain is composed of neural centers that
compete to control a person’s behavior, that wise and responsible desires are
constantly fending off unwise and irresponsible desires, and that these defenses
would be adequate to resist attempts at mind hijacking.
The dangers of subtle biases. My guess is that we need not worry that a man
engaged in facilitated telepathy will suddenly grab a kitchen knife and fatally stab
the person next to him. Instead we should worry about the implantation of
subliminal biases for or against certain political viewpoints, and against certain
religious or racial groups, for example. Perhaps we should also worry that
telepathically experiencing the pleasures of such things as heroin and cocaine
might lead people to experiment with those substances.
Pleasure delivered directly to the brain. In the 1950s, experiments seemed to
show that mammalian brains have “pleasure centers”, whose electrical stimulation
causes an animal intense pleasure. Rats with metal electrodes implanted into their
nucleus accumbens will repeatedly press a lever which activates this region, will
do so in preference over eating and drinking, and will eventually die from
exhaustion.
The true relationship between brain organization and the experience of pleasure
is now known to be far more complex than it seemed. However, there is still the
danger that facilitated telepathy might enable pleasure to be delivered directly to
the brain, without mediation by sensory organs, and that this might become
addictive.
Social “brainworms”. So-called “bookworms”, people who spend large parts
of their lives reading, are a familiar part of society, and there are certainly people
addicted to television. If libraries of memories accumulate that are accessible over
the Internet, a subculture may develop of people who spend their days wandering
through those libraries—reliving the lives of other people, rather than leading their
own. Perhaps they will be called “brainworms”, a modern counterpart to the oldfashioned bookworm. It may not be a healthy existence.
Recorded thoughts and social media. If thoughts can be stored and
transmitted over the Internet, websites that allow people to upload and download
thought recordings will probably arise. Presumably, these will be much like social
media sites such as YouTube, Facebook, and Twitter are now. The library of
recorded mental passages will soon exceed the amount that any one person could
experience.
Presumably, most recorded mental passages will attract little attention, while
some will “go viral”, briefly attracting enormous attention. Many extreme states of
mind and many extreme experiences will be posted. There will be mental
pornography, including much that is violent and depraved. The ability of ordinary
people to post thoughts on the Internet, particularly if it can be done anonymously,
is likely to provoke at least one serious First Amendment (free speech) crisis.
Fakery. As with photographs posted on the Internet, there will probably be
many posted fake mental experiences. Computer software designed to aid such
fakery—ThoughtShop if you will—may be marketed.
Breaches of privacy. Many people may choose technology that allows them to
receive thoughts, but not to send them—because sending thoughts by facilitated
telepathy might compromise the sender’s privacy, perhaps without the sender
knowing it. It might be possible to glean passwords and other information that
would facilitate identity theft from unwary senders.
Senders might inadvertently reveal sexual desires and experiences that they
would prefer not to, as well as past experiences involving illegal drugs, income tax
evasion, and all manner of misdeeds.
If machinery is developed that allows people to record and upload their
thoughts, it may have to have reliable editing abilities if it is to be widely used.
Selective deletion of memories. Facilitated telepathy may give psychiatrists
the ability to selectively delete memories in patients. Conceivably, machinery
might be developed that would give ordinary members of the public that ability to
do this on their own—although this seems likely to be proscribed by law.
Allowing people to delete their own memories could create forensic difficulties,
as criminals delete memories of their crimes.
It would be interesting to learn whether people who selectively deleted
unpleasant memories were in fact happier than if they did not. I certainly doubt that
they would be wiser. They might be in brighter spirits, but they would probably
have more bad surprises, and find themselves unable to relate to many other
people.
Pleasureless psychiatric syndromes. Strange pleasureless psychiatric
syndromes might develop. Many people develop a taste for so-called film noir,
movies that depict life in a very bleak and cynical light. Many teenage girls cut
themselves or starve themselves, seemingly through a desire to blot themselves out
of existence. Other people try to erase their consciousnesses by use of alcohol or
drugs. In a similar vein, the ability to selectively delete memories may inspire
some people to deliberately deprive themselves of all positive sensations and
feelings.
Altering of marriages and friendships. Facilitated telepathy could reveal a
great deal about people to each other. If people have an emotional “flavor”, those
who perceive that flavor will develop likes and dislikes. Some married couples
may decide that they should not remain married, while in other cases, unexpected
romances may blossom.
People may be able to see themselves in another’s thoughts. People often
dislike photographs and videos of themselves, and they may not like what they see
through the eyes of a friend.
Direct inculcation of knowledge. An additional ability that facilitated telepathy
may provide is direct inculcation of knowledge or skill into a student’s brain,
bypassing the eyes and ears. I doubt that direct inculcation of knowledge, should it
be possible at all, will be a matter of a person going to sleep and waking up
knowing how to solve problems in calculus. It seems more likely that it will be an
interactive process where a person tries to learn by traditional means, and that their
efforts are supplemented and guided by facilitated telepathy.
If direct inculcation of knowledge becomes possible, it will raise a very
controversial question: Should it ever be used on children? To pose an extreme
question, should be used routinely in public and private schools? To pose another
extreme question, should babies be born knowing that certain hazards of modern
life can kill them?
More reasonably, should parents decide what is best for their children? If
parents who are ambitious for their children want them to benefit from direct
inculcation, should society allow it? What if the children themselves want it?
Should we ban all xenobiotherapy simply because it might lead to this
dilemma? Should we allow medical xenobiotherapy, but ban all facilitated
telepathy? Should be allow facilitated telepathy in consenting adults, but ban it it
children?
Personally, I hope that xenobiotherapy of children in any form—including
facilitated telepathy—is limited to procedures deemed medically necessary: the
prevention of childhood cancers, for example. The decision on how much—if
any—of this new technology to adopt should be made by people when they are
adults.
One more thing. The above possibilities are speculative; facilitated telepathy
may produce them or may not. However, if thoughts can be exchanged over the
Internet, there is one affliction we will suffer with near certainty… Get ready for
telepathic spam.
Remaining Safety Issues (A Preview of Chapter 22)
Chapter 22 will discuss safety issues concerning xenobiotherapy and Repnumi
rejuvenation that were not discussed in preceding chapters.
Uneven Repnumi rejuvenation. One issue that may be discussed in Chapter
22 is the danger of strengthening some bodily tissues via Repnumi rejuvenation
while leaving other senesced. An obvious mistake would be to give a person the
lung function and skeletal muscles of a 20-year-old, while leaving him or her with
the heart of an 80-year-old.
Hormone changes and Repnumi. Among the facets of this issue that warrant
attention are the declines in levels of hormones such as growth hormone that occur
in the elderly. Such declines are usually thought to be an adaptation to senescence
rather than a cause of it, but they are likely to complicate Repnumi, which would
rejuvenate tissues unevenly.
Cryptic tissue heterogeneity. A second issue that may be discussed in Chapter
22 is the possibility of hidden heterogeneity in supposedly homogeneous tissues.
95% of the hormonal interactions that occur in the human body occur at very close
range, and many apparently homogeneous tissues may consist of groups of
cooperating tissues. Failure of Repnumi practitioners to recognize such instances
and to rejuvenate all cells with appropriately differentiated nuclei could cause
enormous harm.
Repnumi with genetically altered nuclei and mitochondria. A third issue
that may be discussed in Chapter 22 is the potential for Repnumi rejuvenation
using nuclei and mitochondria that are not only more youthful, but are also
genetically altered. It may be possible to use replacement nuclei and mitochondria
that will senesce less slowly than did the original nuclei and mitochondria, for
example.
Abuse of xenobiotherapy and Repnumi. A fourth issue that may be discussed
in Chapter 22 is the potential for abuse of xenobiotherapy and Repnumi
rejuvenation by athletes and others who may seek to boost the performance of their
bodies or brains. Any number of things might be tried from the use of intracellular
xenobiorgs to boost muscle growth to the replacement of human muscle
components with components from stronger species such as chimpanzees, gorillas,
or leopards.
Interaction of xenobiotherapy and contagious disease. A fifth issue that may
be discussed in Chapter 22 is the possibility that methods to evade the human
immune system may be transferred from xenobiorgs to human pathogens. The
specialized features needed for pathogens to move from person to person are so
elaborate that there is virtually no chance that well-constructed xenobiorgs could
mutate into disease agents. However, the possibility that xenobiorgs might donate
their abilities to evade the human immune system to genuine pathogens is more
serious. Ways to prevent this are discussed.
All of these issues will be discussed in this e-book, perhaps in Chapter 22 or
perhaps in other chapters.
Step-By-Step Implementation of Xenobiotherapy and
Repnumi (A Preview of Chapter 23)
A key theme of this e-book and this project is that technological progress is
fastest when modest efforts produce commensurate rewards. In other words, when
spending a modest amount of money and time yields a reasonably quick profit
people are more likely to invest. Initial efforts produce knowledge, facilities, and
reagents that can accelerate further efforts.
As one example, progress in cybernetics has been faster than progress toward
nuclear fusion. I believe that this is due in part to the fact that a 40% increase in
computer speed can be very lucrative, while spending enormous sums and many
years to move nuclear fusion from 40% of the way to plasma ignition to 60% of
the way is not.
Here is an example of how a simple Repnumi success might lead to more
complex attempts. Diabetic foot disease affects 15% of the 200 million people with
diabetes. Rejuvenated endothelial progenitor cells within a diabetic foot might
reverse diabetic foot disease. This would be a valuable and simple application that
could lead the way to Repnumi rejuvenation of more complex tissues.
As another example, both xenobiotherapy and Repnumi rejuvenation will
probably require homing techniques to carry necessary biological reagents to their
desired site of action. However, before this is done, these same homing techniques
may be developed and used for the much simpler task of moving conventional
drugs to a desired site of action.
Although the step-by-step approach is emphasized throughout this book,
Chapter 23 will coordinate ideas about how progress toward one goal can
accelerate progress toward other goals.
Clearing of Sclerotized Tissue, Proliferation of Rejuvenated
Cells, and Redifferentiation of Differentiated Cells (A
Preview of Chapter 24)
Chapter 24 discusses facets of Repnumi rejuvenation not discussed in previous
chapters.
Removal of sclerotized tissue. One biological problem that Repnumi
rejuvenation will face is that aged tissue is often replaced by sclerotized, noncellular tissue. Presumably, it would be better to remove this; but how could it be
done? By surgery? By specialized cells that secrete metalloproteinases?
How are other abnormal tissues, such as scar tissue, sometimes resorbed?
Proliferation of rejuvenated cells. For Repnumi rejuvenation to succeed, it
may not be necessary to replace the nucleus and mitochondria of every senesced
cell. Replacing the nuclei and mitochondria of a minority of cells, and allowing
those cells to proliferate and replace their neighbors might be sufficient.
New techniques may be found to induce rejuvenated cells to proliferate.
Recently, differentiated cardiac cells have been induced to proliferate and
regenerate, something that had previously been impossible. Chapter 24 will discuss
what is known about
Redifferentiation of cells. In addition, it may not always be necessary to
replace a given nucleus with another nucleus of exactly the same type. In mice, at
least, pancreatic α cells can differentiate into β cells when β cells populations are
depleted. Knowledge of how human cell populations can remain in balance via
redifferentiation could simplify Repnumi.
The Lifting of an Ancient Curse: The Social Consequences
of Repnumi Rejuvenation (A Brief Summary of
Chapter 25)
Since Repnumi rejuvenation does not exist yet, we cannot be sure that it is
possible, or how quickly it may be developed. However, it seems likely to be
phased in slowly.
As I have suggested in other chapters, Repnumi rejuvenation would probably
start with tissues that were exceptionally easy to engineer, such as the skin and
perhaps the endothelial cells in the feet of people with diabetes. It might then
spread to tissues of exceptional importance, such as the heart.
♦ The Economics and Politics of Repnumi Rejuvenation
The economic effects. A full-body course of Repnumi rejuvenation, if and
when such becomes possible, is likely to be very expensive. Since, most candidates
for Repnumi rejuvenation would be elderly, the costs might be partly offset by
reduced Medicare costs. However, it seems likely that if society pays for this
procedure, the recipients will probably have to forfeit their Social Security benefits
until such time as they become physically decrepit. It is also possible that Repnumi
will be funded by large loans that will have to be paid back, much as college
educations are often funded.
There would seem to be two major economic benefits to Repnumi rejuvenation.
The first is that enormous amounts of practical experience are lost when experts of
any kind retire from their jobs—and Repnumi could greatly reduce such
retirements. The second is that since most money spent on healthcare is spent on
physically aged people, healthcare spending might come down.
Economists argue over whether deficit spending is good for a modern economy
over the long run, but there is no doubt that deficit spending can produce great
prosperity in the short term. In both the American Civil War and in World War II,
war was good for the American (or Union) side, despite so many resources being
devoted to destruction. Advocates of rapid, universal implementation of Repnumi
rejuvenation (if and when it becomes possible) will probably argue that it is an
enormous investment well worth going into debt for.
The political effects. The ability to rejuvenate people is a prize that most of the
public would probably desire. It is probably a cause big enough to motivate
millions of people. For Americans it might become the next Manhattan Project or
Space Program. For Russia, it might be another Great Patriotic War. For China, it
might be a new Long March. However, unlike those other great national efforts,
the development of xenobiotherapy and Repnumi could involve international
cooperation.
One political disadvantage to both xenobiotherapy and Repnumi rejuvenation is
that both will be based on Genetically Modified Organisms. There is much public
opposition to GMOs—some based on sound considerations of public health and
environmental protection, and some based on blind superstition.
I believe that while genetic engineering as a technology should go forward, it
should incorporate six basic principles:
- Reliable containment
- Public disclosure of all relevant technical information concerning a given
GMO, including DNA sequences
- Independent third-party testing for safety and efficacy
- Extinction of costs, so that the price of a GMO is reduced to the cost of
storage and distribution, once the initial development costs are defrayed.
- GMO use should leave no permanent chemical residues in the environment.
This final consideration applies more to agricultural GMOs than to medical
ones, but might become a consideration if xenobiorg manufacturing is
considered.
- All genetic engineering should be implemented in a way that respects as far as
possible the Right of Perpetual Veto on the part of the public. Chapter 25
discusses what I mean by this.
An interesting question is whether rejuvenated old people would become more
concerned about the future of the Earth. Would their attitudes about issues such as
public financing of college education and global warming be changed by the
knowledge that they likely be alive to deal with the consequences of present-day
decisions?
Repnumi will be limited to rich nations and rich people in poor nations. Since
the same can be said of most modern blessings such as access to adequate food,
adequate housing, sanitation, clean water, adequate medical care, civil rights, and
civil liberties, perhaps nothing will change. On the other hand, perhaps we will
finally see a determined effort by humanity to bring all of world civilization up to
modern standards.
♦ The Social Consequences of Rejuvenation and Xenobiotherapy
Yoppies. In the 1960s and 1970s, American society produced the Yippies,
countercultural activists of the Youth International Party. Later, it produced the
Yuppies, Young Urban Professionals. In that same vein, Youthful Older People
may be called Yoppies. In any case, I will use that term here.
The erotic lives of Yoppies. If it genuinely restores youthfulness to the elderly,
Repnumi will profoundly affect the cycle of sexual attractiveness which is so
important in our culture. Today, most people are at the peak of attractiveness in
their twenties and thirties. Repnumi will restore physical attractiveness to the
elderly.
On average, Yoppies may be more attractive than younger people. Yoppies will
have more money and the many advantages that money brings—including
cosmetic surgery.
Youngsters are often awkward, often unsure of themselves, and often
desperately unhappy. In contrast, older people are better established, more
accepting of themselves and of the world. Older people have had more time to
develop intellectually and to have mastered the social graces—although not all
older people have done so.
An interesting question that can only be anwered by experience is whether
Yoppies will be overcome with surging sexuality and behave as foolishly as
adolescents. Yoppies will have an experienced adult’s knowledge, memories, and
psychological maturity; but how much will they be influenced by them?
If decades of experience in living confer intellectual, financial, professional,
and social advantages, male and female Yoppies may face different erotic
pressures and opportunities. While most young women would be satisfied with a
handsome man who was rich, successful, wise, powerful, capable, and polished,
many young men prefer women who do not make them look and feel silly by
comparison. This might make male Yoppies more successful with younger women
than female Yoppies would be with younger men. On the other hand, this latter
prejudice could change, as indeed the relationship between the sexes has already
changed greatly during the 20th and 21st centuries.
Repnumi may turn out to be very destructive of existing marriages. Experts will
probably recommend that if married partners are near the same age, they they
should undergo the procedure together. Even marriages that seem solid might not
survive the rejuvenation of only one partner.
For some people, Repnumi rejuvenation may alter marriage itself. A cynic
would say that marriage in old age is largely an association for mutual support
between unattractive and physically impaired people who are used to each other,
will tolerate each other, and trust each other to some extent. A cynic would say that
old married couples remain together for financial reasons, from fear of loneliness,
and because of the physical dangers of living alone.
I am sure that many long-standing marriages are much more than this, but there
is probably an element of these motives in most marriages between old people—
and Repnumi will remove most such motives. As a result, some people who
undergo Repnumi rejuvenation may eventually replace marriage with a loose
association of erotic friends. They may acquire a clutch of former lovers that they
are on good terms with. In time, this may become a social norm.
The social lives of yoppies. In the world of today, people tend to self-segregate
by age. There are two reasons for this. First, members of a given age cohort have
the same history, and often the same situation—they can relate to each other.
Second, members of a given age cohort tend to be in the same physical state:
juvenile, young adult, middle aged, and elderly. Repnumi rejuvenation will remove
this latter constraint on the intermingling of people; it will be interesting to see
whether this increases socializing between different age groups.
Post-Repnumi depression. As wonderful as youthfulness is, it is not heaven. A
new psychiatric syndrome, post-Repnumi depression, may develop. Even people
who are delighted with the results of Repnumi will probably experience a sense of
ennui and letdown—a feeling that there is no point to endless social and sexual
gymnastics.
The depression will result in part from a re-recognition that youth and beauty do
not themselves guarantee happiness. Another part of the depression will probably
be a devaluation of the activities of youth.
The financial lives of yoppies. The elderly develop strong apprehensions about
outliving their financial resources—in part because they realize that they cannot
outlive those resources for very long, in part because they prefer to order their own
lives, and in part because they do not want to become an undignified burden on
other people or on society. If Repnumi rejuvenation restores health, physical
ability, and mental ability, it may relieve older people of the need to pinch pennies.
It will be interesting to see whether Repnumi rejuvenation changes people’s
behavior with regard to financial investment. Will people begin to invest in
securities that will not yield a return within what would have been a normal
lifespan without Repnumi?
Repnumi rejuvenation and young people. Although Repnumi rejuvenation
would seem irrelevant to children, it may affect them powerfully. For one thing,
the ratio of adults to children may increase. This may decrease the economic and
political importance of the young.
If Repnumi rejuvenation leads couples to have fewer children, or to increase the
time between them, this would alter the average environment in which children are
raised. A child’s ordinal position in a family affects the child’s intelligence. As
birth order increases, IQ decreases—with first borns having especially superior
intelligence. The most widely-accepted explanation is that first borns receive more
attention and resources from parents and are expected to focus on task
achievement, whereas later borns are more focused on sociability. This suggests
that Repnumi rejuvenation may make children academically brighter but less adept
at socializing with their peers.
If children are lucky, they are born into a paternalistic and maternalistic world.
They are valued, taken care of, protected, and guided. Adults seem, by comparison
with children, wise, knowledgeable and powerful. Adults are pillars of stability,
and hopefully of goodness, in a child’s world. Adults make things good, and they
make things fair. They teach children right from wrong.
As children grow older, they learn first that adults are not perfect, and
eventually that adults are just ordinary people. One criterion of having grown up is
the ability to see one’s parents as other adults.
The process of seeing adults as peers will probably take much longer when
Repnumi becomes common. Every child will be surrounded by adults that can
remember back decades or even centuries, and who will seem to be familiar with a
whole world of obscure things.
In the working world, younger people might take much longer to reach
positions of seniority. On the other hand, the guidance of more experienced people
might provide many younger people with an increased sense of security.
Repnumi and international relations. Repnumi will be limited to rich nations
and to rich people in poor nations. It may increase the incentive, already large, for
people in poor nations to migrate to rich ones.
Repnumi would increase people’s well being, and can be considered a form of
wealth. Rises from poverty to a middle class status usually reduce birth rates.
Repnumi will probably produce a consensus that society cannot allow each person
to reproduce 100 times over a 500-year period, and in countries with universal
access to Repnumi, birth rates may fall drastically.
In poor nations—where children ar the only practical form of social security
and children’s labor is needed—people have large families largely due to economic
necessity. However, the 21st century is likely to see a worldwide campaign to raise
people’s standard of living everywhere to acceptable middle class levels.
Environmental constraints will probably make birth rate reduction a part of this
campaign, despite the heated controversy this will arouse. Repnumi may play a
part in this, perhaps as an inducement for people to have smaller families.
Repnumi and loss. Even if physical aging can be reversed, people will still die,
of course. But even if people did not die, the things that people live for would
continue to die. Enthusiasms, friendships, loves, hates, loyalties, marriages, faith,
cherished beliefs, fashions, social mileaus, neighborhoods, institutions, political
causes, and great projects of all kinds would continue to die even if people
themselves did not.
Repnumi cannot change this, and for many Yoppies that discovery will be quite
poignant. An early death at least allows some of our illusions to outlive us.
Perhaps society's response to a greatly lengthened life span will have to include
procedures for the orderly and civilized burial of marriages and other institutions
that were supposed to last forever, but in fact cannot—as well as a general
recognition that all such commitments are ephemeral.
Repnumi and social responsibility. Human existence is largely shaped by two
types of possession. The first type of possession is possession of things are less
important than the person who owns them. Such things may include money,
material objects, valuable skills, interesting experiences, praiseworthy
accomplishments and other things well worth having. However, their defining
characteristic is that they are less important than the person who owns them.
The second type of possession is possession of people by things that are more
important than the people themselves. In our free society, such possession is
voluntary; people devote themselves to institutions and causes larger than
themselves because they believe that they should. Institutions and causes that
people may choose to serve include one’s country, one’s religion, and campaigns
against disease, hunger, political tyranny, or aging…
With both types of possession, it is important that people choose wisely.
The second type of possession is far more important than the first type of
possession to a person’s happiness and to the ultimate worth of a person’s life.
Although this truth is tragically underappreciated in our culture, there are still
many people committed to great causes that they believe in. Christianity, Islam,
environmentalism, scientific and technological progress, racial equality, feminism,
economic justice, civil liberties and so on all have dedicated servants. These causes
are very important to people.
One possible consequence of indefinitely long life spans is that people will be
more likely to outlive great causes that they believe in. Although Communism is
not entirely gone from Earth, it is no longer a major force, and its collapse broke
the hearts of many of its adherents—who had to find another cause for optimism
about the future, or go without. As people live longer, they may be more likely to
outlive the causes that they cherish.
On the other hand, political and social causes may themselves last longer if
their adherents refuse to give up.
According to the famous French proverb, “the more things change, the more
they remain the same.” In a society with universal access to Repnumi rejuvenation,
this truism will eventually become evident to most who follow politics. Certainly it
will be seen that political and social fashions—including mistakes—are cyclical.
The kinds of social causes most likely to benefit from Repnumi rejuvenation are
the non-controversial ones that require quiet dedication over long periods of time.
For many years, the human race has had amateur clubs devoted to any number of
pursuits: automobiles, bodybuilding, brazilian jiu-jitsu, chess, cooking, and so on.
However, in addition to this, there are many communities of people around the
world who participate in projects dedicated to some socially valuable purpose.
These are often collaborations between amateurs and professionals, and are
dedicated to such things as astronomy, ornithology, solving the prime number
problem, elucidating human population genetics, and searching visual data for
interstellar dust particles. There are many efforts to monitor the effects of the
changing environment on such things as plant blooming, cicada cycles, monarch
butterfly migration, and the appearance and disappearance of ice on water.
There is room for public participation in many other projects. Examples might
include recording contemporary history, monitoring the effects of diet and exercise
on health, measuring solar energy flux and local temperature, measuring local air
quality, measuring pollen counts, analyzing video-recorded bird overflights,
analyzing pathogenic microbes in the environment, and so on.
It will be interesting to see whether Repnumi rejuvenation changes people’s
attitudes toward long-term problems such as global warming. Will people be more
attentive to problems that they will someday personally face? Or are their attitudes
about global warming and other long-term problems determined by the same biases
that shape their political behavior in the present?
Repnumi, sin and redemption. Almost all people are subject to ethical
standards that they are expected to abide by. In this age before Repnumi, an
ordinary person can usually get through an normal lifespan without committing
any terrible crime or act of betrayal. However, as the average lifespan increases,
this may become harder.
The burden of extreme longevity may fall heaviest on people who must avoid
certain weaknesses without fail. For many marriages, even one instance of
infidelity by either partner would be enough to end the marriage.
In many professions certain transgressions will cause a person to be expelled
from that profession in disgrace, and render that person's career a failure. For
scientists and journalists, falsification of results and plagiarism are two such
transgressions. For soldiers, physical cowardice and deliberate breaches of security
are unforgiveable. For public officials, bribe-taking is unforgiveable. For medical
professionals, sexual misconduct involving patients is unforgiveable.
Many of these sins, such as cowardice, infidelity and greed are constant
temptations for normal people. Heretofore, a person could avoid these sins by
dying on schedule. Now, however, people will not die on schedule.
Repnumi and the limits of human mental capacity. Advances in information
technology are making it steadily easier to store and access both knowledge and
personal memories. Moreover, if the predictions made in Chapter 21 come to pass,
there may be an enormous increase in human mental capacity, as stored
information becomes accessible by thought.
However, our minds cannot expand forever, even with technical help. It may
eventually develop that very old yoppies will have to read personal diaries and
view old videos of themselves to remember their lives when they were young.
Although the saving of memory is a necessary condition for people remaining
familiar with their pasts, it is not enough. A person could accumulate hundreds of
years of experience, but review those memories only very intermittently. Under
such circumstances, it will be almost as though the experiences had happened to
someone else. For people to have ready access to their own pasts via thought, it
will be critically necessary that their memories be organized well.
Good organization of a person’s memories will probably require that access to
memories be directed by key words and summaries of memory. The summaries
themselves may be accessed by key words and shorter summaries. Developing and
improving such a system seems like a problem that could keep a great many clever
information specialists busy for a very long time.
If Repnumi rejuvenation becomes common, many people will desire this sort of
mental enhancement while many others probably will not. Many people will be
content to live and enjoy their lives in the normal fashion, forgetfulness included.
One can also imagine instances where people decide that they want to go on
living, but do not want the burden of decades or centuries of memories, and clear
out huge blocks of memory at a time. They may consign these to a static archive,
or simply let them vanish.
If Repnumi rejuvenation allows people to remain youthful for centuries, I
suspect that many people will undertake self-improvement projects only to
remember that they did much the same thing 150 years earlier. Moreover, older
people with excellent memories will probably experience frequent moments of
déjà vu.
Repnumi and meritocracy. All of modern society is animated by the belief
that people must earn the right to take themselves seriously and to be taken
seriously by others. This belief is the mainspring of most human productivity and
progress, and also of a great deal of evil, when people compete in anti-social ways
(such as by participating in organized crime).
Unfortunately, the competition inspired by this belief produces losers as well as
winners. There are many people, particularly among adolescents, who conclude
that they will never measure up, and despair as a result. These feelings of
inadequacy may contribute to a great deal of self-destructive behavior, including
smoking, binge drinking, illicit drug use, and reckless promiscuity.
In addition, in a meritocracy, some people acquire more money and power than
do others. They become more able than average to arrange society to suit
themselves, even if the arrangements hurt other people. American politics is full of
accusations that people have done this, with conservatives pillorying government
bureaucrats and leftists denouncing big corporations.
Xenobiotherapy, facilitated telepathy, and Repnumi rejuvenation will affect our
meritocracy in many ways. Knowledge, skill, experience, and personal connections
confer power upon those who have them. These things take time to acquire; hence,
if all else is equal, xenobiotherapy, facilitated telepathy and Repnumi should favor
older people over younger ones.
If society has only so much room for famous entertainers and athletes, it may
become even harder than it already is for youngsters to succeed in those endeavors.
On the other hand, the public might tire of seeing the same faces in sports and
entertainment, decade after decade, and demand new celebrities for that reason.
There may be a strong public desire to rehabilitate or preserve some sentimental
favorites. If Repnumi develops slowly, aged entertainers are likely to be
rejuvenated before aged athletes are, because the skin will probably be an earlier
target for Repnumi rejuvenation than will be the heart, skeletal muscles, or nervous
system.
Nearly all people will have time in their lives to learn more and experience
more than people currently do. Those who take special measures not to forget what
they have learned will likely rise higher than those who do not.
The combination of rejuvenation, facilitated telepathy, and enormous capacities
for information storage may raise many people’s intelligence to near genius levels.
Some will argue that there is no point to this—but others will see it as marvelous.
Adverse consequences of Repnumi rejuvenation. Although Repnumi
rejuvenation and xenobiotherapy are likely to be mostly beneficial, there will
probably be costs as well.
First, of course, since Repnumi rejuvenation will greatly decrease the death
rate, it may increase the human population of our already overcrowded planet.
Whether this actually happens, and how bad the effect will be, will depend on
whether the birth rate declines along with the death rate—as it has in the past.
Second, a reduction in the death rate might promote intellectual rigidity among
scientists and other members of the intelligentsia. As Max Planck famously said,
“A new scientific truth does not triumph by convincing its opponents and making
them see the light, but rather because its opponents eventually die, and a new
generation grows up that is familiar with it.” and (more bluntly) “Science advances
one funeral at a time.”
Thirdly, some members of the human race are evil geniuses, hugely destructive
people who are too clever to be foiled by the law. Perhaps society’s only real
defense against them is that they grow old and die like everyone else. If so,
Repnumi rejuvenation will greatly weaken this defense.
A fourth unfortunate consequence of xenobiotherapy and Repnumi may be that
with an increased sense of health security, people may resume old bad habits such
as smoking. They may feel that they can afford it.
Repnumi and people’s view of history. If Repnumi rejuvenation succeeds, it
will change human society enough that people will begin to think of Repnumi as
defining the modern world. Everything before Repnumi will eventually come to
seem prehistoric, the struggles of semi-human apes.
Repnumi and the human sense of purpose. Life’s questions fall into two
great categories: How? and Why? For most of humanity, most of the time,
concerns with “How” have predominated. How do we earn enough to stay
comfortable and healthy? How do we seduce a mate, protect our children, rise in
social status, cope with aging, leave a material and perhaps a spiritual legacy? How
do we protect our country from hostile foreigners? How do we advance political
ideas that we believe in? How do we speed scientific progress, protect the
environment, protect and improve our social institutions, and dispense justice?
People who are preoccupied with the Hows of life are inclined to accept the on
faith the worthiness of their goals. Those who are religious have God's reassurance
that life is worth living, that existence is worthwhile. But when the questions of
How have been solved, the questions of Why assert themselves. This is a problem,
because the evolutionary process that created our minds drives us away from death
and misfortune, rather than pulls us toward some overwhelming reason to go on
living.
Our instincts tell us that the ability to remain healthy and youthful indefinitely
is very valuable. But when people finally acquire this ability, they will have to ask
themselves why it is worthwhile.
It is very hard for humans to live without a sense of purpose, because the need
for purpose is in our DNA. A sense of purpose promotes survival; there are many
stories of people who have prospered because they were motivated to prosper or
have failed to prosper because of they were not.
Life itself is based on the principle that some outcomes are better than others.
Even organisms that are incapable of behavior must still organize the material
within themselves to within certain tolerances.
Even an amoeba that enters and then backs out of a region of excess alkalinity
has “chosen” between good and bad, although there is no reason to think that
amoebas are in any way sentient. The need to make wise choices preceded thinking
and probably spawned the ability to think.
The importance of purpose, and of distinguishing between good and bad,
increases as animals become more complex. It is well known that the lion who
predicts where the antelope will be and acts accordingly—and the antelope who
predicts where the lions will be and acts accordingly—will be more likely to
survive and leave offspring.
We have 4 billion years of evolution telling us that things must be done the
right way. Hence, when our view of the world tells us that the concepts of “good”
and “bad” have no meaning, and that all paths through existence are of equal
worth, it is no wonder we feel that something is missing. However Repnumi
rejuvenation and the other medical advances discussed in this book may help
create a society where purpose is no longer shaped by basic biological needs. We
will have to fill the gap that this leaves.
A thousand years is time for about 12 ordinary lifespans, arranged end-to-end.
Is there any real difference between allowing this natural process to continue and
instead keeping a single person alive and youthful for 1000 years? Will not that
aging person lose all mental connection with his or her childhood and young
adulthood? Indeed, will not that aging person lose all connection with his or her
past not just once, but many times in succession? It is more pleasant for the
original person not to have to wither and die; but will he or she in fact have died
slowly and imperceptibly many times during the interval? And, if so, is there
anything we can do to prevent that slow death?
Answering such questions may be as difficult, and require as much effort on the
part of as many people, as did the scientific journey that may produce Repnumi
rejuvenation. It will take wisdom to handle our new abilities, but the need for
wisdom is nothing new. There has never been a society where people could thrive
and be happy without wisdom.
Advanced uses of Repnumi. Some people undergoing Repnumi rejuvenation
will want more than replacement of aging nuclear and mitochondrial genomes with
youthful copies of the same genomes. They will want the replacements to be better
than what they have.
They may want their rejuvenated cells to be less prone to aging than are the
cells they were born with.
They may want their neural nuclei to be replaced by those of someone smarter,
or perhaps by nuclei that have been modified with many supposedly desirable
genes. They may want, and doctors may recommend, different nuclei for different
parts of their brains.
People may want the muscles of a leopard, a mule, a gorilla, or a chimpanzee.
People may want the retinas of an owl or eagle. Although whole nuclei from other
species (except perhaps chimps and gorillas) are unlikely to function normally in
human tissues, human nuclei modified to contain genes from foreign species might
well function normally.
We cannot yet induce the human immune system to accept the introduction of
foreign proteins and polysaccharides, but we probably will gain this ability within
the next decade or two. When that happens, no biological barriers to possibilities
such as the above will remain except the technical problems that accompany
Repnumi itself.
Society will have to decide what it will tolerate.
Xenobiotherapy and Repnumi rejuvenation over the long term. Eventually,
doctors will probably become able to monitor every cell and every structure of
their patients’ bodies. Patients will have ample warning of heart attacks, cancer,
aneurisms, osteoporosis and other afflictions. Many harmful processes, such as
cancer will be stopped automatically. Others, such as rheumatoid arthritis,
osteoarthritis and Alzheimer’s disease will be stopped as soon as they are
noticed—which will be soon after they begin.
Such monitoring and intervention will require the stationing of xenobiorgs
within all or most cells of a patient’s body, and perhaps the stationing of
extracellular xenobiorgs within the extracellular matrix of tissues. Upon first
consideration, the idea of engineered microbes being present in so many places in
the human body is very jarring. There is psychological comfort in knowing that
most of the human body is still mysterious.
Much of what is becoming possible will horrify many. However, people should
remember two things. First, what happens to us now is even more horrifying. We
are young at 20, mildly impaired at 40, seriously impaired at 60, decrepit at 80, and
extinct by 100—and that’s if we’re lucky. Repnumi rejuvenation could lift this
ancient curse. Second, we can pick and choose; if society wants Repnumi
rejuvenation but does not want facilitated telepathy, for example, we can allow one
and forbid the other.
Uses of Xenobiorgs Outside of Medicine (A Brief Preview of
Chapter 26)
Outdoor use and reliable containment. Engineered microbes may also find
uses outside of medicine. If engineered microbes are used in the open environment,
very convincing demonstrations that they cannot replicate will probably be
required for them to gain public acceptance.
Plant “immune systems.” Engineered microbes might be used to give corn and
peanut plants an “immune system” which could reduce the harm to consumers
caused by Aspergillus flavus and Aspergillus parasiticus infections.
Xenobiorgs and invertebrates. Engineered microbes might also be used to
direct the development of engineered invertebrates such as insects and spiders for
use as biodegradable pesticides and herbicides. Consider this scenario:
A scenario: xenobiorgs against Chagas disease. Chagas disease, caused by
Trypanosoma cruzi, is one of the world’s most horrifying infectious diseases. In
addition to the heart disease that it causes, DNA from the trypanosomes integrates
into human cells, including germ line cells. This DNA can be passed to subsequent
generations of humans.
Chagas disease is spread by “kissing bugs” of the subfamily Triatominae.
Imagine that an insect-killing spider is engineered that attacks and kills kissing
bugs without damaging any other organism. Such a spider might prevent enormous
amounts of sickness and death.
However, environmentalists would object—perhaps rightly—to the release of
any self-replicating genetically engineered animals into the environment. What
could be done to allow the spider to be used, while eliminating any chance that the
spider could reproduce in the wild?
Genetic engineers might prevent the spiders from replicating sexually by
removing the genes necessary for the spiders to develop sex organs. This would
prevent the spiders from replicating in the wild; but then, how could the spiders be
replicated under laboratory or factory conditions?
It might be possible to replicate the spiders directly from cultured cells, perhaps
in small artificial structures that would mimic female reproductive organs.
However, both the cells that destined to develop into future spiders, as well as any
accessory cells, would have to be directed somehow to follow the appropriate
developmental paths. Intracellular xenobiorgs might orchestrate this.
Arthropods as pest control agents. As pest control agents, genetically
engineered arthropods would have many advantages. They are would be
biodegradable and their behavior would be predictable. They could be engineered
to attack only the intended target. Since their replication requires a germ line and
reproductive organs, genetic ablation of these could prevent their reproduction.
Uses for invertebrate GMOs. Invertebrates do an enormous amount of work
on this planet, albeit it is simple work such as killing things. With the use of
genetic engineering, a portion of that work could be harnessed for human benefit.
One can imagine snails that only eat a specific weed, nematodes that attack the
roots of only certain weeds, dragonflies that gorge on malaria mosquitos, ants that
kill aphids instead of protecting them, pollen-eating insects that have been adapted
to eat spores of aflatoxin-producing fungi, and any number of other useful
examples.
However, public acceptance of any of such examples of genetic engineering
will require reliable containment. Intracellular xenobiorgs might contribute to such
containment.
A Guide to Resources Useful for Xenobiotherapy and
Repnumi Research (A Preview of Chapter 27)
Chapter 27 will present a list of techniques and resources useful in research
related to xenobiotherapy and Repnumi rejuvenation. I will try to keep this list upto-date.
The techniques listed will be specialized techniques that are not widely known
among molecular biologists. Possible examples include the techniques of
optogenetics (see Chapter 21), functional magnetic resonance imaging (see
Chapter 21), measurement of migration of human cells (see Chapter 11), special
techniques to measure the activity of antimicrobial peptides, special techniques to
measure replication of invasive bacteria within human cells, and so on. The
intention is to alert interested investigators to useful techiques that they otherwise
might not learn about.
The resources listed will include laboratories working on subjects related to
xenobiotherapy and Repnumi rejuvenation, relevant monoclonal antibodies,
specialized plasmid and viral genetic constructs, strains of bacteria that have been
engineered to operate in the human body, and so on. Again, the intention is to alert
interested investigators to resources that they otherwise might not discover.
Appendix 1 – Quick Internet Access Format
Although I cite very few references in this introductory chapter (Chapter 1),
most information presented in the remaining chapters is accompanied by cited
references. Almost all of the references cited are accessible via the Internet at least
in abstract form.
I use an inline citation method that I call Quick Internet Access (QIA) format.
In the text, cited references have this appearance: {
}
The reference information is shrunk to the smallest possible size and converted
to a tiny font. The font I use is Parchment, the smallest font available in Word 365.
When expanded to regular-sized text and to the Times New Roman font, the
above QIA references looks like this:
Krashes_MJ Front Behav Neurosci. 2014 Feb 28;8:57 Optogenetic and chemogenetic insights into the food addiction hypothesis, SR, YAO, M: hunger and hedonic pleasure, nr1
{Krashes_MJ Front Behav Neurosci. 2014 Feb 28;8:57 Optogenetic and
chemogenetic insights into the food addiction hypothesis, SR, YAO, M: hunger
and hedonic pleasure, nr1}
If the above citation makes intuitive sense, readers can skip the rest of this
section. Otherwise, please note the following.
1. The reference information is flanked by braces “{” and “}” which are not
shrunk.
2. Only the first author of the article or book is given, and the author’s surname
and initials are joined by an underscore.
3. The journal and page information is given in the form that I found it in. Usually,
this is the format that PubMed uses (see below for more about PubMed), but
sometimes it is in a different format used by a scientific journal.
4. The title of the article or book is given next.
5. I indicate whether the reference cited is a “hard reference” (i.e. original
reference) or a “soft reference” (i.e. a derivative reference). The abbreviations
that I use are “HR” and “SR”, respectively.
6. I indicate whether the entire article is available online. “YAO” means “Yes,
available online.” “NAO” means “Not available online.” Even if the entire
article is not available online, the abstract is usually available at PubMed’s
website.
7. If the full cited article is available online, I guide the reader to the cited
information using a marker text string present only once in the article. The text
string used to guide the reader is colored red.
8. I include a note to myself at the end of the citation. For those people who (like
me) just HAVE to know what abbreviations mean, “nr” stands for “noted
references”, “fn” stands for “free notes”, and “nin” stands for “not in notes.”
Some references in QIA format differ slightly from the above. If the cited
article or book were not available online except as an abstract, M: hunger and
hedonic pleasure would be replaced by M: abst.
If the cited reference is a web page, some of the information may instead by
replaced by a web address. The following is an example:
{Optogenetics, http://en.wikipedia.org/wiki/Optogenetics, M: temporal precision is
central, SR, YAO, nr4}
Pubmed. Usually, the best way to access an online article is to visit the
PubMed search page of the National Center for Biotechnology Information web
site {http://www.ncbi.nlm.nih.gov/pubmed}, and to enter into the search engine
only the title of the desired article. In most cases the title alone will lead the reader
to the correct article.
Appendix 2 - New Terminology
I have tried to avoid introducing new jargon in this e-book, but I use several
new terms:
Delinquent cells are somatic cells of the human body that because of mutation
or other disorder no longer perform their proper functions and may injure the rest
of the body. Cancer cells are delinquent cells, and other delinquent cell types can
be imagined, such as a brain cell that begins exporting gastric digestive enzymes.
Facilitated telepathy is the reading of information from and the writing of
information to human brain cells by bypassing the organs of sensation and
communication. Unlike the telepathy of science fiction, facilitated telepathy uses
ordinary physical phenomena such as light, magnetic fields, and sound.
Individualized nuclei are cell nuclei of the brain or other nervous system tissue
that have undergone individualized and unpredictable changes that are important to
a person’s identity. Repnumi replacement of such nuclei with other nuclei of the
same type, but not individualized, might weaken or erase a person’s memories or
other aspects of a person’s unique personality. It is still not known whether
individualized nuclei exist.
Repnumi rejuvenation is the replacement of aged nuclei and mitochondria by
more youthful counterparts. The ideal acronym for this process would be
“Renumi” (Renew-me); however, Renumi is a proprietary name for a form of
acupuncture. Hence, to avoid confusion, I use the term Repnumi.
Xenobiotherapy is the use of genetically engineered microbes as medical
agents.
Xenobiorgs are genetically engineered microbes used as xenobiotherapeutic
agents. Extracellular xenobiorgs operate outside of human cells, either in the blood
or within tissues. Intracellular xenobiorgs operate within human cells.
A leukocyte that contains an intracellular xenobiorg is called a
leukocyte/xenobiorg, a cardiocyte that contains a xenobiorg is called a
cardiocyte/xenobiorg, and so on.
In addition, instead of the cumbersome phrase “undergo apoptosis”, I use the
verb apoptose, although this is nonstandard.
Finally, in discussing biological function, I use words that imply intention in a
new way.
The human body came into existence according to Darwinian principles and
was not designed. And yet, the human body is extremely complex and must
accomplish many complex processes in order to survive and thrive—and most of
the English language’s words to describe complex objects and complex behavior
also imply thought.
If we say that the cell nucleus has a complex architecture, we might imply that
it was devised by an architect. If we say that a cell has a strategy to defeat
invaders, we might imply that the cell’s behavior was devised by a strategist. If we
say that a the body’s immune system must satisfy several distinct considerations,
we might imply that consideration is involved. Many other words useful in
describing the human body or its actions such as plan, design, purpose, goal,
tactic, solve, correct, proper, decision, demand, value, benefit, penalty,
function, reason (i.e. cause of action), detect, assure, disguise, and accident,
could be taken to imply judgment.
Rather than abandon most words used to describe complexity, I prefer to adopt
a new convention. This book presupposes Darwinism, and considers the “intent” of
any feature or process of the body to be the satisfaction of Darwinian demands.
I might say, for example, that the oxygen-carrying property of hemoglobin is
one of its purposes, but that the bright red color of hemoglobin is an accidental
characteristic. By this, I would mean that the ability to carry oxygen brought
hemoglobin into existence and maintains hemoglobin within the human
population, while the red color of hemoglobin did and does neither.
Appendix 3 – How Readers Can Help
Molecular biologists can have a very positive effect on this effort by doing
several things. First, they can publish their work in open-access journals and/or
describe their research at their website(s). Second, they can call my attention to any
mistakes I have made and any areas where the writing is unclear. Third, they can
bring to my attention any new research results that seem useful.
Members of the general public can also help in several ways. First, they can let
other people know about the project. Second, they can let me know about any
places in this e-book where the writing could be clearer to non-scientists. Third,
they can call my attention to new research and to mistakes in the text. Finally, they
can popularize the term Baboola Sumo.
Baboola Sumo is slang for “Baby Boomers Lacking Sufficient Money.” Baby
Boomers are Americans born between 1946 and 1965 during the post-World War
II “Baby Boom.” Boomers are at or approaching retirement age, but all too often
have saved far too little for a comfortable retirement. The aging of the Boomers
and the strain that this will put on Social Security and Medicare will soon become
America’s greatest social and economic problem.
Unfortunately for us Boomers, Repnumi rejuvenation will come to late to save
us. There is not much that I or anyone else can do for the Baboola Sumos.
However, if the slang term becomes popular, I will sell the URL
baboolasumo.com and use the proceeds to fund this work.
Appendix 4 – List of Medical Projects Discussed in this
Chapter and this E-Book
Below is a list of 96 medical projects discussed in this e-book, grouped by
subject. The shrunken text between braces {} is in Parchment font, font size 1.
Expand the text for more information about each individual project.
The information between the braces includes (i) a longer description of the
project, (ii) the Chapter in this book that the project is discussed in, (iii) the page in
Chapter 1 (this chapter) where there is a short summary of the project, and (iv)
(after the string “M:” and in red) a unique text string that readers can use to find
the discussion immediately. For example:
{(i) Use of intracellular xenobiorgs to deplete and then destroy adipose tissue.
(ii) Discussed in Chapter 7. (iii) In this chapter, see p.41. (iv) M: xenobiorgs within
adipocytes}
Adipose tissue: Destruction, enhancement, and manipulation of adipose tissue
Depletion of “fat” cells – {
}
Destruction of depleted “fat cells” – {
}
Non-traditional breast augmentation – {
}
Marking of adipose stem cells for isolation – {
}
Taming of macrophages within adipose tissue – {
}
Taming of visceral adipose tissue – {
}
Using “fat cells” to express hormones – {
}
Removal of a hunger hormone from the blood – {
}
Use of intracellular xenobiorgs to deplete and then destroy adipose tissue. Discussed inChapter 7. In this chapter, see p.41. M: xenobiorgs withinadipocytes
Use of intracellular xenobiorgs to destroy adipocytes depleted by gastric bypass surgery. Discussed in Chapter 7. In this cha pter, see p.42. M: In formerly obese people
Use of adipocyte/xenobiorgs inbreast augmentation. Dis cussed in Chapter 7. In this chapter, see p.42. M: Breast augmentation sometimes
Use of xenobiorgs within adipose tissue to mark stem cells for isola tion. Discussed in Chapter 7. In this chapter, see p.42. M: Adipose tissue includes cell types
Use of xenobiorgs to control macrophages withinadipose tissue. Discussed in Chapter 7. In this chapter, see p.42. M: enter macrophages and control
Use ofxenobiorgs to reduce the danger of vis ceral adipose tissue. Dis cussed in Chapter 7. In this chapter, see p.42. M: the adipose tissue that surrounds organs
Use of intracellular xenobiorgs to alter the expression of adipocyte hormones, including anorectic peptide YY3-31, anti- obesity peptide enterostatin, anti-orectic cholecystokinins, and adiponectin. Discussed in Chapter 7. In this chapter, see p.43. M: shows promiseas ananorectic drug
Use of adipocyte/xenobiorgs to take up and destroy the hunger hormone ghrelin in Prader-Willi patients and other people. Discussed in Chapter 7. In this chapter, see p.43. M: Ghrelin is a peptide hormone
Apoptosis: Control of apoptosis-mediated tissue injury
Rescue from ischemia-reperfusion injury – {
}
Rescue from scleroderma, cutaneous lupus, and toxic necrolysis – {
Treating Crohn’s Disease and other gastrointestinal ailments – {
Treating cystic fibrosis – {
}
Treating chronic pulmonary obstructive disease – {
}
Preventing diabetes – {
}
Treating stroke and other brain disorders – {
}
Protecting kidneys during medical imaging – {
}
Use of intracellular xenobiorgs to reduce apoptosis-mediated is chemia-reperfusion injury in heart, brain, kidneys, liver, and intestines, and transplanted organs. Discussed in Chapter 7. In this chapter, see p.37. M: inju ry which affects the heart
Use of intracellular xenobiorgs to protect skin against exacerbation by apoptosis of scleroderma, cutaneous lupus, and toxic necrolysis. Discussed in Chapter 7. In this chapter, see p.37. M: be protected from scleroderma
}
Use of intracellular xenobiorgs to protect the gastrointestinal tract from exacerbation by apoptosis ofCrohn’s disease, ulcerative colitis, necrotizing enterocolitis, and infection with Helicobacter pylori. Discussed in Chapter 7. In this chapter, see p.38. M: protect the gastrointestinal tract
Use of intracellular xenobiorgs to ameliorate or reverse cystic fib rosis by blocking apopto sis.Discussed inChapter 7. In this chapter, see p.38. M: apoptosis that occurs in cystic fibrosis
Use of intracellular xenobiorgs to prevent apoptosis from exacerbating chronic obstructive pulmonary disease. Discussed inChapter 7. In this chapter, see p.38. M: in chronic obstructive pulmonary disease
Use of intracellular xenobiorgs to block apoptosis that exacerbates diabetes. Discussed in Chapter 7. In this chapter, see p.38. M: prevent or ameliorate diabetes
Use of intracellular xenobiorgs to prevent apoptosis from exacerbating ischemic stroke, Lou Gehrig’s disease, Huntington’s disease, Parkinson’s disease, and spinal cord injury. Dis cussed in Chapter 7. In this chapter, see p.38. M: apoptosis contributes importantly
Use of intracellular xenobiorgs to protect kidneys from apopto sis during medical imaging, kid ney transplantation, or physical obstructio n ofa ureter. Discussed in Chapter 7. In this chapter, see p.40. M: Kidneys can be injured by
}
Treating arthritis – {
}
Treating osteoporosis – {
}
Protecting the liver – {
}
Treatment of dilated cardiomyopathy – {
}
Treatment of muscular dystrophy – {
}
Prevention of eclampsia and pre-eclampsia – {
}
Treatment of arterial pulmonary hypertension – {
}
Protecting bystander cells from cancer treatments – {
Use of intracellular xenobiorgs to prevent apoptosis from exacerbating osteoarthritis and rheumatoid arthritis. Discussed in Chapter 7. In this chapter, see p.39. M: Arthri tis is a painfuland debilitating
Use of intracellular xenobiorgs against exacerbation of osteoporosis byapopto sis.Discussed inChapter 7. In this chapter, see p.39. M: Osteoporosis is skeletal fragility
Using intracellular xenobiorgs to protect liver from apoptosis in alcoholic fatty liver disease, non-alcoholic fatty liver disease, cholestasis (the obstruction of bile flow), acetaminophen overdose, hepatitis B and C, and liv er transplantation. Dis cussed in Chapter 7. In this chapter, see p.39. M: Apoptosis ofliver cells helps
Use of intracellular xenobiorgs to protect hearts from apoptosis in dilated myocardio pathy. Dis cussed in Chapter 7. In this chapter, see p.40. M: contributes to dilated cardiomyopathy
Use of intracellular xenobiorgs to block apoptosis inoculopharyngeal muscular dystrophy, congenital muscular dystrophytype 1A, Ullrich congenital muscular dystrophyand Bethlem myopathy inskeletal muscles. Discussed inChapter 7. In this chapter, see p.40. M: onset triplet expansion disease
Use of intracellular xenobiorgs to block apoptosis inpre-eclampsia and eclampsia. Dis cussed in Chapter 7. In this chapter, see p.40. M: serious complications ofpregnancy
Use of intracellular xenobiorgs to promote apoptosis that ameliorates arterial pulmonary hypertension. Discussed in Chapter 7. In this chapter, see p.41. M: The use of drugs to curb
Use ofintracellular xenobiorgs to block apopto sis inhealthy cells during anticancer treatments. Discussed in Chapter 7. In this chapter, see p.38. M: treatments used in cancer chemotherapy
Atherosclerosis
Detection and treatment of atherosclerosis – {
Use of leukocyte/xenobiorgs to detect and treat atherosclerosis. Discussed in Chapter 7. In this chapter, see p.32. M: leading cause of morbidity and death
}
}
Blood cleansing: Removal of harmful chemicals, hormones from the blood
Removal of harmful chemicals from the blood – {
}
Destruction of excess tumor necrosis factor – {
}
Lowering of blood sugar – {
}
Useof leukocyte/xenobiorgs to remove harmful chemicals, e.g. homocysteine, from the blood. Dis cussed in Chapter 7. In this chapter, see p.31. M: might be used to modify leukocytes
Use of adipocyte/xenobiorgs to take upand destroy excess tumor necrosis factor. Discussed in Chapter 7. In this chapter, see p.43. M: as its name im plies
Use of adipocyte/xenobiorgs to lo wer blood sugar. Discussed inChapter 7. In this chapter, see p.43. M: Removalof blood glucose
Cancer: Analysis of experimental and clinical cancers
Tracking fusions between cancer and non-cancer cells – {
Molecular documentation of cancer cell migration – {
Provoking immunity to cancer subpopulations – {
}
Use of intracellular xenobiorgs to monitor fusions between cancer and non-cancer cells. Discussed in Chapter 15. In this chapter, see p.103. M: fusions between cancer and
}
}
Use of intracellular xenobiorgs to monito r and record the types of tissuevisited by cancer cells. Discussed in Chapter 15. In this chapter, see p.104. M: record many aspects ofa cancer
Useof intracellular xenobiorgs to provoke an immune response to a subset ofcancer cells. Discussed in Chapter 15. In this chapter, see p.104. M: limited to specific cancer subtypes
Cancer: Attacking cancer using cytotoxic polypeptides
Cytotoxic polypeptides vs. cancer – {
}
Use of cytotoxic polypeptides (and perhaps the natural acidityof tumors) to destroy cancers. Discussed in Chapter 7. In this chapter, see p.20. M: One feature of solid cancers
Cancer: Detection of cancer based on surface antigens
Early detection of pancreatic cancer – {
}
Early detection of cervical cancer – {
}
Early detection of other cancers – {
}
Early detection of pancreatic cancer using leukocyte/xenobiorgs. Discussed in Chapter 7. In this chapter, see p.26. M: Pancreatic cancer is the fourth
Early detection of cervical cancer using leukocyte/xenobiorgs. Discussed inChapter 7. In this chapter, see p.27. M: Cervical cancer is the third
Use of leukocyte/xenobiorgs to probe for specific antigens, including cancer antigens, in the human body. Discussed inChapter 7. In this chapter, see p.28. M: Some humancells can nibble
Cancer: Detection or killing of cancers based on internal conditions
Detection of cancer by spatial changes – {
}
Stopping breast and prostate cancer very early – {
}
Using hypoxia to destroy cancers – {
}
Stopping melanoma very early – {
}
Intracellular xenobiorgs might detect very early stage cancers based on changes inthe spatialorganizationof cancer cells Discussed in Chapter 7. In this chapter, see p.47. M: Precancerous cells and cells
Use of intracellular xenobiorgs to kill breast, prostate and other cancers as soon as they begin to develop. Discussed in Chapter 7. In this chapter, see p.35. M: The human female breast
Use of cancer hypoxia to destroy cancers Discussed in Chapter 5. In this chapter, see p.15 & p.21. M: especially solid tumors & M: Bacteria of the genus Clostridium
Use of intracellular xenobiorgs to remove pigmented nevi (skin moles) and incipient melanoma. Discussed in Chapter 7. In this chapter, see p.35. M: because skin is easily
Cancer: Disruption of cancer signaling
Stopping cancer self-promotion – {
}
Stopping cancer from duping the body’s defenses – {
Stopping cancer’s digestion of tissues – {
}
Interruption of the signals bywhich cancers self-promote. Discussed in Chapter 5. In this chapter, see p. 22. M: xenobiorgs might also tame cancers
Cancer masquerades as wounded tissue, but xenobiorgs mig ht destroyor counteract the signals that cancer uses. Discussed in C hapter 5. In this chapter, see p. 15. M: The body has ways of
}
Disabling the proteases that cancers use to dig est tissue. Discussed in Chapter 5. In this chapter, see p.23. M: Mat rix metalloproteinases are
Cancer: Preventing metastasis through blood and lymphatic vessels
Stopping cancers from creating blood vessels – {
}
Stopping cancers from creating lymphatic vessels – {
}
Tracing lymphatic vessels to stop cancer spread – {
}
Waylaying of cancer cells within lymph nodes – { }
Destruction of cancers by prevention of new blood vessel growth.Discussed inChapter 5. In this chapter, see p.19. M: cancers requi re a generous blood
Use of xenobiorgs to prevent the formation of new lymphatic vessels by cancer cells. Discussed in Chapter 9. In this chapter, see p.68. M: Cancers induce the formation
Use ofextracellula r xenobiorgs to trace lymphatic vessels to block cancer spread. Discussed inChapter 9. In this chapter, see p.67. M: Mapping of the lymphatic
Discussed in Chapter 9. In this chapter, see p.67. M: Current t echniques to prevent
Differentiation: Reading of cells’ differentiated states and related projects
Assessment of cells’s internal state from the outside – {
}
Assessment of cells’ internal state from the inside – {
}
Detection of hidden injury – {
}
Assessing the state of frozen and thawed cells – {
}
Isolation of rare and transient cell types – {
}
Prevention of graft-vs-host disease – {
}
Analysis of tissue differentiation – {
}
Protecting bystander cells from medicines – {
}
Mapping of solid tissues – {
}
Tracking of biological rhythms – {
}
Moving cell organelles – {
}
Sampling of cells’ internal state from the outside using reverse-orientation receptors. Discussed in Chapter 7. In this chapter, see p.28. M: addition to detecting surface antigens
Use of intracellular xenobiorgs to monitor the conditio ns withinhuman cells and report to doctors.Discussed inChapter 7. In this chapter, see p.52. M: monitor and report on
Use of intracellular xenobiorgs to detect hidden injury in apparently normal tissues byelucidating cell morphogies.Discussed inChapter 7. In this chapter, see p.49. M: Cells that have been damaged
Useof intracellular xenobiorgs to assess injury and death incells that have been frozen and thawed. Dis cussed in Chapter 7. In this chapter, see p.47. M: after some procedure such
Use of intracellular xenobiorgs to mark specific cell types for isolation by fluorescence-activated cell sorting and simila r techniques. These could include transient cell types of great interest. Discussed in Chapter 7. In this chapter, see p.46. M: manypurposes in research
Use of intracellular xenobiorgs to purge bone marrow of cells causing graft-vs-hostdisease. Discussed in Chapter 7. In this chapter, see p.46. M: selectively remove certain cell
Use of intracellular xenobiorgs to dissect the steps of tissue differentation by interfering with selected genes. Discussed in Chapter 7. In this chapter, see p.49. M: dissecting the regulatory networks
Use of intracellular xenobiorgs to limit the action ofmedicines to desired cell types. Discussed in Chapter 7. In this chapter, see p.46. M: action ofmedicines to desired
Use of intracellular xenobiorgs to mapsolid tissues cell by cell, including mapping over time. Discussed inChapter 7. In this chapter, see p.46.M: report the differentiated states
Use of intracellular xenobiorgs to track biological rhythms and their effects. Discussed in Chapter 7. In this chapter, see p.47. M: Many human tissue types
Use ofintracellular xenobiorgs to alter positions ofintracellular organelles and perhaps affect differentiated state. Discussed in Chapter 15. In this chapter, see p.115. M: its position within the body
Drugs: Delivery of drugs to specific sites & conversion of pro-drugs
Moving drugs to desired locations in the body – {
}
Creation of drugs at desired locations in the body – {
}
Use of circulating liposomes, micelles, xenobiorgs or other particles to carry drugs to chosen sites inthe body. Discussed in Chapter 2 & 11. In this chapter, see p.3 & p.90. M: in situnear a target tissue & M: The first such technology is
Conversion of circulating inactive pro-drugs to active drugs or creation of bio active agents ata chosen sitewithin the body. Discussed in Chapter 2. In this chapter, see p.3. M: convert an inactive blood
Gene manipulation: Gene therapy and gene conversion by xenobiorgs
Standardized cassettes for gene therapy – {
}
Directors of targeted gene conversion – {
}
Use of intracellular xenobiorgs as cassettes for gene therapy. Discussed in Chapter 7. In this chapter, see p.50. M: Intracellular microbial parasites
Use of intracellular xenobiorgs to direct targeted gene conversion. Discussed inChapter 7. In this chapter, see p.51. M: engineered to direct target ed gene
Immune system: Allergy-related, immune suppression
Local suppression of immunity – {
}
Local suppression of allergy – {
}
Use of xenobiorgs to suppress the immune response ina restricted location. Discussed inChapter 19. In this chapter, see p.112. M: Repnumi rejuvenation are likely
Use of extracellular or int racellular xenobiorgs to quench local allergy flare-ups via individ ual lymph nodes. Discussed in Chapter 9. In this chapter, see p.67. M: Current techniques to prevent spread
Infectious Disease: Suppression of protozoan, bacterial, and viral diseases
Suppression of tooth decay – {
}
Eradication of malaria – {
}
Using cytotoxic polypeptides to kill invading microbes – {
}
Shielding bystander cells from cytotoxic polypeptides – {
}
Preserving the body’s antimicrobial peptides – {
}
Preserving the body’s antibacterial traps made of DNA – {
}
Destroying the body’s antibacterial traps made of DNA – {
}
Preventing bacterial disruption of the human immune system – {
}
Enhancing defective neutrophils – {
}
Removing HTLV-1–infected lymphocytes – {
}
Protecting white blood cells against HIV – {
}
Protection against papillomavirus – {
}
Emergency protection against influenza virus – {
}
Protecting cells from invading bacteria – {
}
Analyzing infections of solid tissues – {
}
Use of extracellular xenobiorgs to kill or confuse the bacteria that cause toothdecay. Discussed inChapter 3. In this chapter, see p. 4.M: Microbes are seldom used
Use of intracellular xenobiorgs in a scheme to eradicate malaria. Discussed in Chapter 7. In this chapter, see p.36. M: Malaria is spread to human hosts
Use of cytotoxic polypeptides against microbial invaders. Discussed inChapter 4. In this chapter, see p.9. M: Cytotoxic polypeptides have been
Protection of healthy cells from cytotoxic polypeptides using cytoxic polypeptide mim ics. Discussed in Chapter 4. In this chapter, see p.7. M: some soluble polypeptides resemble
Preventing bacteria from evading or destroying antibacterial cytoxic polypeptides. Discussed in Chapter 7. In this chapter, s ee p.25. M: new ability might be the ability
Controlling the movement ofinvading bacteria by preserving the body’s antibacterial DNA traps. Discussed in Chapter 7. In this chapter, see p.25. M: 3 leukocyte types
Deliberate destruction of the body’s antibacterial DNA t raps in cases where bacteria exploit these for thei r own benefit. {Discussed in Chapter 7. In this chapter, see p.26. M: there is also one case
Neutralization of signals from bacteria that disrupt human immunity. Discussed in Chapter 7. In this chapter, see p.25. M: generat ed chemical rest raints
Enhancement of defective neutrophils in newborns and cystic fibrosis patients. Discussed inChapter 7. In this chapter, see p.26. M: especially premature infants
Selective destruction of lymphocytes infected by HTLV-1. Discussed in Chapter 7. In this chapter, see p.25. M: leuk emia and tropical spastic
Use of intracellular xenobiorgs to protect against human immunodeficiency virus. Discussed in Chapter 7. In this chapter, see p.33. M: Although Human Immunodeficiency
Use of intracellular xenobiorgs to protect against humanpapillomavirus. Discussed in Chapter 7. In this chapter, see p.34. M: is more dangerous to women
Use of intracellular xenobiorgs to protect lungs against influenza virus onan emergency basis. Discussed in Chapter 7. In this chapter, see p.34. M: protect cells against virus infection
Use of intracellular xenobiorgs to defend human cells againstsingle-cell pathogens.Discussed inChapter 7. In this chapter, see p.54. M: invasionby pathogenic microbes
Use of intracellular xenobiorgs to analyze infection ofsolid tissues bya dis ease agent. Dis cussed inChapter 15. In this chapter, see p.47. M: are likely to be affected unevenly
Injury detection: Detection of disease and hidden injury
Detection of disease by high polyamine levels – {
Detection of and action against conditions that produce highpolyamine levels: cancer, some infections, Parkinson’s disease, and stroke. Discussed in Chapter 7. In this chapter, see p. 30. M: Biologically sig nificant polyamines
}
Neurological Disease
Combatting Alzheimer’s toxic plaque – {
}
Stopping neuron suicide in Alzheimer’s Disease – {
Prevention of Huntington’s Disease – {
}
Action against Alzheimer’s Disease by co mbatting toxic plaque [Discussed in Chapters 6 & 7. In this chapter, see p25 & p.30. M: Xenobiorgs Against Alzheimer & M: It is incurable and fatal
Action againstAlzheimer’s Disease by combatting neural apoptosis.Discussed inChapter 7. In this chapter, see p.38. M: apoptosis contributes importantly to
}
Use of intracellular xenobiorgs to treat Huntington’s disease and other protein expansion/contraction diseases . Discussed inChapter 7. In this chapter, s ee p.36. M: delinquent cells that have undergone
Other: Hair color, baldness, tanning, telomeres, p53, collagen remodeling, etc.
Preservation of hair color – {
}
Reversal of baldness – {
}
Better artificial tanning – {
}
Protection of telomeres from destructive agents – {
}
Relaxation of anticancer controls to prolong life of selected cells – {
}
Remodeling of the extracellular matrix – {
}
Use of intracellular xenobiorgs to preserve hair color. Discussed in Chapter 7. In this chapter, see p.31. M: Graying of hair is the most
Use of intracellular xenobiorgs to reverse baldness. Discussed in Chapter 7. In this chapter, see p.31. M: Baldness increases with aging
Use of intracellular xenobiorgs to tan human skin. Discussed inChapter 7. In this chapter, see p.32. M: Although tanning of the skin
Use of intracellular xenobiorgs to restore the lengths of telomeres shortened by diseaseor anticancer treatments. Discussed in Chapter 7. In this chapter, see p.44. M: Telomerase is an enzyme
Use of intracellular xenobiorgs to lengthen selected tissue life by suspending the activities of proteins p53 and retinoblastoma protein. Dis cussed in Chapter 7. In this chapter, see p.48. M: proliferative ability to human tissues
Use of xenobiorgs to remodel collagen and other extracellular matrix components in skinand other organs. Discussed in Chapter 11. In this chapter, see p.91. M: This remodeling consists mainly
Repnumi: Repnumi and removal of intracellular garbage
Rejuvenation of human cells – {
}
Removal of intracellular garbage – {
}
Rejuvenationof human cells in situ byreplacement of aged nuclei and mito chondria withyouthful co unterparts (Repnumi rejuvenation). Discussed in Chapter 12. In this chapter, see p.91. M: Although the purpose of Repnumi
Use of intracellular xenobiorgs to remove oxidized proteins and other intracellula r garbage. Discussed in Chapter 7. In this chapter, see p.50. M: degrade surplus or damaged proteins
Stem cells: Stem cell-related
Stopping stem cells from forming teratomas – {
}
Creation and preservation of human stem cells – {
}
Creation and preservation of non-human stem cells – {
Use of intracellular xenobiorgs to prevent stem cells from producing teratomas. Dis cussed in Chapter 7. In this chapter, see p.35. M: Pluripotent stem cells may
Use of intracellular xenobiorgs to mark, create or preserv e human stem cells for research or medicine. Discussed in Chapter 7. In this chapter, see p.48. M: Humanstem cells can differentiate
Use of intracellular xenobiorgs to mark, create or preserv e non-human stem cells for research or commerce. Discussed in Chapter 7. In this chapter, see p.48. M: the germ lines of experimental animals
}
Telepathy: Facilitated telepathy-related
Facilitated telepathy – {
}
Use of intracellular xenobiorgs in facilitated telepathy. Discussed in Chapter 21. In this chapter, see p.126. M: The most amazing use
Toxins: Defense of cells against toxins
Defending cells against toxins – {
Use ofintracellular xenobiorgs to protect cells f rom environmental hazards, chemotherapy agents, P450 drug-inactivationproducts, aflatoxin, and acetaminophen.Discussed in Chapter 7. In this chapter, see p.49. M: might protect particular tissues
}
Non-medical: Non-medical use of xenobiorgs
Engineered insects and other arthopods - {
Useof intracellular xenobiorgs to replace sexual reproduction in engineered insects and other arthropods.Discussed inChapter 26. In this chapter, see p.148.M: development of engineered invertebrat es
}
Appendix 5 – Key Words
Below are key words and phrases in 2-point font. They can be used to search
the above chapter. Increase the font size to view the key words. Text enclosed by
parentheses is descriptive, and is not part of a search term. The numbers in
parentheses indicate the number of times that each key word or phrase appears in
the chapter text. Be sure to search with only the key words or phrases, and not with
any spaces that precede or follow them.
Numbers
$350 billion (1) /:/ 30-90 nm diameter (1) /:/
A
α-1-antichymotrypsin (1) /:/ β-amyloid (6) /:/ β-catenin (2) /:/ β-galactoside (1) /:/ α-helices (1) /:/ α-ketoglutarate (1) /:/ β-lactam (1) /:/ β-sheet (1) /:/ α4β1 (an integrin) (1) /:/ α4β7 (an integrin) (2) /:/ αeβ7 (an integrin) (1) /:/ αlβ2 (an integrin) (2) /:/ αMβ2 (an integrin) (2) /:/ αVβ3 (an integrin) (2) /:/ αVβ5 (an integrin) (1) /:/ A+T-rich (1) /:/ A2a (adenosine receptor) (2) /:/ A3 (adenosine receptor) (1) /:/ abbreviations (used herein) (2) /:/ abilities (11) /:/ ability (56) /:/ ablation (2) /:/ abnormal (20) /:/ abortion (2) /:/ absorb
excess fluid (1) /:/ absorb toxins (1) /:/ abstract (3) /:/ abuse (of Repnumi) (2) /:/ abuse (of xenobiotherapy) (2) /:/ academically brighter (1) /:/ accomplice (cells) (4) /:/ accusation (1) /:/ Acetabularia (1) /:/ acetaminophen (2) /:/ acetaminophen overdose (1) /:/ acetylcholine (1) /:/ acid-dependence (2) /:/ acid-dependent (2) /:/ actin (2) /:/ acupuncture (1) /:/ acute lymphoblastic leukemia (2) /:/ addict (4) /:/ adenosine (7) /:/ adenosine-mediated (1) /:/ adhesion molecule (9) /:/ adipocyte (2) /:/ adipocyte/xenobiorgs (6) /:/
adiponectin (8) /:/ adipose (42) /:/ adipose-derived (1) /:/ adipose-related (1) /:/ adrenal (3) /:/ adult T-cell leukemia (1) /:/ Aequorea victoria (jellyfish) (2) /:/ affinity maturation (2) /:/ aflatoxin (4) /:/ aflatoxin B1 (1) /:/ aflatoxin metabolites (1) /:/ aflatoxin-producing fungi (1) /:/ African eyeworm (1) /:/ African trypanosome (1) /:/ agricultural (1) /:/ AIDS (3) /:/ airway (3) /:/ albino (1) /:/ albumin (1) /:/ alcoholic fatty liver disease (1) /:/ alkaline phosphatase (1) /:/ allele (1) /:/ allergic (1) /:/ allergy (3) /:/ allergy flare-ups
(1) /:/ allograft (1) /:/ aluminum (1) /:/ alveolar (2) /:/ alveoli (1) /:/ Alzheimer (28) /:/ Alzheimer’s plaque (1) /:/ amastigotes (1) /:/ AMD3100 (1) /:/ ammonia (1) /:/ amoeba (9) /:/ Amoeba proteus (4) /:/ amphipathic (2) /:/ ampullae (1) /:/ amylin (2) /:/ amyloid (3) /:/ amyloid plaque (1) /:/ amyloid plaque polymerization (1) /:/ amyotrophic lateral sclerosis (2) /:/ anabolic drugs (1) /:/ anaerobic metabolism (1) /:/ analog (2) /:/ Anaplasma phagocytophilum (7) /:/ anaplasmosis (1) /:/ ancient (4) /:/ anemia (2) /:/
anesthetic (1) /:/ aneuploidy (1) /:/ aneurism (1) /:/ angiogenesis (18) /:/ angiogenic (4) /:/ angiogenic factor (2) /:/ angiogenin (1) /:/ angiopoietins (1) /:/ angiostatic (4) /:/ angiostatic factor (2) /:/ anion (1) /:/ anionic (lipids) (2) /:/ AnkA (protein) (1) /:/ ankylosing (1) /:/ ankylosing spondylitis (1) /:/ Anopheles (3) /:/ anorectic (1) /:/ anoxia (2) /:/ anserine (1) /:/ anti-adhesive /:/ anti-Alzheimer (1) /:/ anti-angiogenic (1) /:/ anti-apoptotic (1) /:/ anti-cancer (9) /:/ anti-CD44 (1) /:/ anti-fibronectin (1) /:/ anti-immune (1)
/:/ anti-inflammatory (2) /:/ anti-inflammatory factor (1) /:/ anti-obesity (1) /:/ anti-oxidant (1) /:/ anti-parallel (1) /:/ anti-social (1) /:/ antibacterial (3) /:/ antibiotic (4) /:/ antibodies (42) /:/ antibody (20) /:/ antibody-mediated (1) /:/ antibody-producing (2) /:/ anticancer (3) /:/ antigen (34) /:/ antigen signature (3) /:/ antigen-antibody (1) /:/ antigen-presenting (8) /:/ antigenic (16) /:/ antigenic variation (4) /:/ antimicrobial (13) /:/ antisense (1) /:/ antiviral (2) /:/ AP-1 (1) /:/ apelin (2) /:/ aphid (1) /:/ aplastic anemia (1) /:/
apolipoprotein(a) (1) /:/ apoptose (1) /:/ apoptosis (77) /:/ apoptotic (1) /:/ aptamer (1) /:/ arabidopsis (1) /:/ arachidonic (1) /:/ arginase (1) /:/ arginine (2) /:/ aromatase/estradiol (1) /:/ array (tandem DNA sequence array) (2) /:/ arrest (a step of leukocyte homing) (13) /:/ arterial (8) /:/ arteries (8) /:/ arteriosclerosis (2) /:/ artery (6) /:/ arthritis (10) /:/ arthropods (3) /:/ Aspergillus flavus (2) /:/ Aspergillus fumigatus (1) /:/ Aspergillus parasiticus (2) /:/ aspirational pneumonia (1) /:/ asymmetric dimethyl-L-arginine (1) /:/
asymmetric segregation of replicons (1) /:/ ataxia telangiectasia (1) /:/ atherosclerosis (12) /:/ atherosclerotic (12) /:/ atherosclerotic lesions (1) /:/ atherosclerotic plaque (9) /:/ athlete (4) /:/ ATP (1) /:/ attractant (5) /:/ autocrine (1) /:/ autoimmune (4) /:/ autoimmunity (3) /:/ autologous Repnumi (2) /:/ autologous transplantation (1) /:/ autoreactive T cells (1) /:/ auxotroph (2) /:/ axenic (1) /:/ axon (1) /:/
B-C
B cell (12) /:/ B lymphocyte (1) /:/ B-cell (1) /:/ B10 cells (1) /:/ B16 (melanoma) cells (1) /:/ Babesia (1) /:/ babies (1) /:/ baboolasumo.com (2) /:/ baby (3) /:/ Bacillus anthracis (1) /:/ Bacillus pertussis (1) /:/ bacteria (73) /:/ bacteriocin (2) /:/ bacteriophage (1) /:/ bacterium (16) /:/ baldness (5) /:/ band 5q31 (of chromosome 5) (1) /:/ barrel stave mechanism (1) /:/ basement membrane (3) /:/ Bcl-2 (1) /:/ Bdellovibrio (1) /:/ bees (venom) (1) /:/ Bethlem myopathy (2) /:/ bilayer (1) /:/ bile (1) /:/ bile flow (1) /:/ BIO-1211
(1) /:/ bio-electrospraying (1) /:/ biodegradable pesticides (1) /:/ biofilm (1) /:/ biological rhythm (4) /:/ blastocyst (1) /:/ blood circulation (6) /:/ blood flow (3) /:/ blood glucose (3) /:/ blood platelets (1) /:/ blood pressure (1) /:/ blood vessel (11) /:/ blood-borne (3) /:/ blood-brain barrier (3) /:/ blood-group (1) /:/ blood-oxygen-level (1) /:/ bloodstream (2) /:/ bodybuilder (1) /:/ bodybuilding (1) /:/ bone fractures (1) /:/ bone marrow (17) /:/ bone resorption (3) /:/ Bordetella bronchisepta (1) /:/ Bordetella filamentous
hemagglutinins (1) /:/ Bordetella pertussis (2) /:/ bradykinin (1) /:/ brain (95) /:/ breast (21) /:/ breast cancer (11) /:/ bronchial (2) /:/ Brucella abortus (1) /:/ Brugia malayi (8) /:/ Brugia pahangi (1) /:/ Brugia timori (1) /:/ Burkholderia cepacia (1) /:/ c-kit (1) /:/ c-reactive protein (1) /:/ c5a peptidase (1) /:/ Ca2+ (5) /:/ cachexia (1) /:/ cadaverine (2) /:/ cadherins (1) /:/ Caenorhabditis elegans (15) /:/ calcium (5) /:/ calcium phosphate (1) /:/ cancer (252) /:/ cancerous transformation (2) /:/ Candida (1) /:/ capillaries (26) /:/
carbohydrate (2) /:/ carbonic anhydrase (6) /:/ carcinogen (2) /:/ carcinoma (2) /:/ cardiac (2) /:/ cardiac stents (1) /:/ cardiocyte (2) /:/ cardiocyte/xenobiorg (1) /:/ cardiolipin (1) /:/ cardiomyopathy (2) /:/ cardioprotective (1) /:/ carnosine (1) /:/ carotenoid (2) /:/ carpet mechanism (1) /:/ carrion eaters (1) /:/ cartilage (3) /:/ catalase (3) /:/ catecholamine (1) /:/ cation (2) /:/ caveolin (1) /:/ CCL16 (1) /:/ CCL17 (2) /:/ CCL19 (1) /:/ CCL21 (6) /:/ CCL22 (1) /:/ CCL25 (1) /:/ CCL27 (2) /:/ CCR10 (1) /:/ CCR4 (1) /:/
CCR5 (2) /:/ CCR7 (1) /:/ CCR9 (2) /:/ CD155 (1) /:/ CD16 (1) /:/ CD28 (1) /:/ CD4 (7) /:/ CD4+ (5) /:/ CD4(+)CD25(+)FOXP3(+) (1) /:/ CD43 (1) /:/ CD44 (3) /:/ CD46 (1) /:/ cell lineages (1) /:/ cell suicide (apoptosis) (5) /:/ cell type-specificity of parasitization (1) /:/ cervical (13) /:/ cervixes (1) /:/ Chagas (6) /:/ chelator (1) /:/ chemerin (2) /:/ chemical beacon (1) /:/ chemical mutagenesis (1) /:/ chemoattractant (3) /:/ chemoattraction (2) /:/ chemokine (11) /:/ chemoreceptor (1) /:/ chemotaxis (7) /:/
chemotherapy (5) /:/ chicken (2) /:/ chilies (and aflatoxin) (1) /:/ chimp (3) /:/ chimpanzee (muscles of) (1) /:/ chirality (1) /:/ Chlamydia (4) /:/ Chlamydophila (2) /:/ Chlamydophila pneumoniae (1) /:/ cholecystokinin (5) /:/ cholestasis (1) /:/ cholesterol (5) /:/ chondrocyte (2) /:/ chromatin (9) /:/ chromosomal (1) /:/ chromosome (20) /:/ chronic obstructive pulmonary disease (2) /:/ cigarette smoke (1) /:/ circadian (11) /:/ circulatory (3) /:/ circumcision (2) /:/ CLA+ve (1) /:/ Clostridium (4) /:/ CMV (3) /:/ co-stimulatory
(1) /:/ coagulase (1) /:/ cocaine (2) /:/ cofactor (3) /:/ colipase (1) /:/ collagen (9) /:/ colostrum (5) /:/ complement (5) /:/ complement C5 (1) /:/ conformation (9) /:/ congenital (3) /:/ continuous-feed reservoirs (1) /:/ copper (2) /:/ “copy number variation” of chromosome segments (1) /:/ corn (and aflatoxin) (2) /:/ coronaviruses (1) /:/ cotton seed (and aflatoxin) (1) /:/ countercultural activists (1) /:/ cowpox (1) /:/ Coxiella burnetii (4) /:/ CpG dinucleotides (1) /:/ cranium (1) /:/ Crohn’s (5) /:/ cross-linking (of receptors) (2) /:/
cross-resistance (2) /:/ cryptic tissue heterogeneity (1) /:/ ctl (cytotoxic t lymphocyte) (1) /:/ Cu+ (1) /:/ cutaneous lupus (1) /:/ Cutaneous Lymphocyte Antigen (1) /:/ CX3C (1) /:/ CXC (1) /:/ CXC13 (1) /:/ CXCL1 (1) /:/ CXCL10 (1) /:/ CXCL12 (3) /:/ CXCL13 (1) /:/ CXCL4 (1) /:/ CXCL8 (1) /:/ CXCR2 (2) /:/ CXCR3 (2) /:/ CXCR4 (8) /:/ CXCR5 (2) /:/ CXCR5-CXCL13 (1) /:/ cyclosporine (1) /:/ cystic fibrosis (6) /:/ cystic fibrosis transmembrane conductance regulator protein (1) /:/ cytochrome (2) /:/ cytokine (2) /:/
cytomegalovirus (1) /:/ cytoskeleton (6) /:/ cytotoxic polypeptide aggregation (1) /:/
D-E
D-form (amino acids) (1) /:/ decidualised uterus (1) /:/ decoy components (2) /:/ decoy receptor (1) /:/ dedifferentiate (1) /:/ dedifferentiation (1) /:/ delayed-type hypersensitivity (1) /:/ delinquent cells (9) /:/ dementia (2) /:/ dendrites (1) /:/ dendritic (7) /:/ deoxyribonuclease (1) /:/ depolarizing (cell membrane) (1) /:/ destructive tissue remodeling (1) /:/ desynchronized (biological rhythms) (1) /:/ detoxify (3) /:/ diabetes (10) /:/ diabetic (5) /:/ diabetic foot (ulcers) (4) /:/ diapedesis (6) /:/ differentiated state (21) /:/ diffusible
inhibitor (1) /:/ digestion of fats (1) /:/ dimethyl-L-arginine (1) /:/ diphtheria toxin (1) /:/ Discosoma (mushroom coral) (1) /:/ divalent (antibodies) (1) /:/ diversifying selection (1) /:/ DNA sequence (7) /:/ DNA trap (4) /:/ DNA-binding (2) /:/ DNA-recognition (1) /:/ docking structures (1) /:/ draining (lymph node) (2) /:/ Drosophila melanogaster (2) /:/ drug (16) /:/ drug-carrying (2) /:/ drug-resistant (1) /:/ DSCAM (down syndrome cell adhesion molecule) (3) /:/ DsRed (1) /:/ dummy receptors (2) /:/ dyskeratosis congenita (1) /:/
E-cadherin (4) /:/ E-selectin (9) /:/ early endosomal autoantigen 1 (EEA1) (1) /:/ eclampsia (3) /:/ economic (7) /:/ ectopic dermatitis (1) /:/ eczema (1) /:/ edema (1) /:/ EEA1 (early endosomal autoantigen 1 ) (1) /:/ EFF-1 (a nematode protein) (1) /:/ effector (5) /:/ efflux (of fatty acids) (1) /:/ egress (of lymphocytes from lymph nodes) (5) /:/ elastin (1) /:/ elderly (11) /:/ electric field (1) /:/ electrodes (in brain) (1) /:/ electromagnetic field (2) /:/ electromagnetic radiation (4) /:/ electromagnetism (1) /:/ electrostatic (binding of cytotoxic
polypeptides) (6) /:/ elephantiasis (2) /:/ embryogenesis (1) /:/ embryonic (2) /:/ embryos (3) /:/ encephalitis (1) /:/ end-tagging (of peptides) (1) /:/ endocrine (3) /:/ endocytosis (5) /:/ endogenous cytotoxic peptides (1) /:/ endometrial (1) /:/ endonuclease (site-specific) (1) /:/ endopeptidase (1) /:/ endosome (3) /:/ endospore (1) /:/ endostatin (1) /:/ endosymbionts (9) /:/ endothelia (3) /:/ endothelin (5) /:/ endothelin-1 (1) /:/ endothelium (16) /:/ energy (6) /:/ enforced (lymphatic flow) (2) /:/ entactin (2) /:/ enterostatin (7) /:/
entrapment (of engineered microfilariae) (1) /:/ enucleated (cells) (1) /:/ enveloped (viruses) (1) /:/ enzyme (8) /:/ eosinophil (1) /:/ epidermal cells (2) /:/ epidermis (2) /:/ epigenetic (6) /:/ episodic (homing) (1) /:/ epithelial (1) /:/ epithelial-mesenchymal (1) /:/ epithelial-mesenchymal transition (1) /:/ epithelium (1) /:/ epitope (7) /:/ erythrocyte (2) /:/ Escherichia coli (1) /:/ Escherichia coli k12 /:/ /:/ estradiol (2) /:/ etiology (of diabetes) (1) /:/ eukaryote (unicellular) (1) /:/ eukaryotic (unicellular) (2) /:/ evade (the immune
system) (21) /:/ evaluations (of donor nuclei) (1) /:/ evasion (of the immune system) (12) /:/ evolution (of cytotoxic polypeptides) (5) /:/ evolve (resistance to cytotoxic polypeptides) (9) /:/ exacerbate (damage) (1) /:/ exacerbation (of arthritis) (1) /:/ exogenous cytotoxic polypeptides (1) /:/ exonucleases (1) /:/ exosomes (5) /:/ expansion (protein expansion diseases) (6) /:/ expel (expulsion of nuclei and mitochondria) (1) /:/ expend (energy in entering a host cell) (1) /:/ expense (involved in repnumi) (6) /:/ export (proteins, mRNA, etc) (8) /:/
extinction of costs (1) /:/ extracellular adenosine (2) /:/ extracellular domain (protein domain) (2) /:/ extracellular fluid (1) /:/ extracellular matrix (34) /:/ extracellular pathogen (1) /:/ extracellular space (2) /:/ extracellular xenobiorg (1) /:/ extraneous hormones (1) /:/ extravasate (2) /:/ extravasating (1) /:/ extravasation (20) /:/ extravillous trophoblasts (1) /:/ extreme longevity (1) /:/ extruded DNA sequences (1) /:/ extruded nuclei (1) /:/
F-G-H
facial skin (1) /:/ facilitated telepathy (42) /:/ facultatively intracellular (1) /:/ Fanconi anemia (1) /:/ fat-graft (1) /:/ fatty acid (5) /:/ faux-germinal center (1) /:/ Fe3O4 (magnetite) (1) /:/ feathers (from cultured cells) (1) /:/ feces (xenobiorg exit via the feces) (2) /:/ feeder cells (1) /:/ feet (of diabetics) (1) /:/ female mosquitos (2) /:/ ferrimagnetic iron oxide (1) /:/ fertilization (molecular machinery) (2) /:/ fetus (and immunotolerance) (3) /:/ fever (1) /:/ fiber optics (1) /:/ fibrate drugs (1) /:/ fibrinogen (1) /:/ fibroblast (3) /:/ fibroblast
growth factor (3) /:/ fibrocyte (1) /:/ fibrocytes locomote (1) /:/ fibronectin (7) /:/ fibrous connective tissue (and atherosclerosis) (1) /:/ filarial nematode (5) /:/ financial (11) /:/ fingernail (1) /:/ fingolimod (3) /:/ fish (cytotoxic polypeptides) (1) /:/ flexible molecular stalk (1) /:/ flocculate (of cytotoxic polypeptides) (1) /:/ flow (of lymph) (36) /:/ flow cytometry (1) /:/ fluidity (of cell membranes) (1) /:/ fluorescence-activated cell sorting (2) /:/ fluorescent protein (derivatives of gfp) (9) /:/ fluorescing protein (1) /:/ fluorescing xenobiorg (1)
/:/ fMRI (functional magnetic resonance imaging) (12) /:/ fn (free notes) (1) /:/ foam cells (3) /:/ font (8) /:/ food intake (1) /:/ forced expression (1) /:/ foreign antigen (2) /:/ foreign DNA (1) /:/ foreign peptide (1) /:/ foreign protein (2) /:/ foreign species (proteins from) (1) /:/ foreskin (1) /:/ fosmid (1) /:/ fouling the kidneys (1) /:/ four-chain antibodies (1) /:/ FOXP3 (3) /:/ fragile bone (1) /:/ fragments (of proteins) (11) /:/ Francisella tularensis (1) /:/ free radicals (1) /:/ free-living nematode (1) /:/ freezing/thawing (1) /:/ frog
(cytotoxic polypeptides in skin) (1) /:/ fruit flies (1) /:/ fruit fly (2) /:/ functional magnetic resonance imaging (fMRI) (4) /:/ fuse (cancer cells with non-cancer cells) (9) /:/ fusion (cell-cell) (18) /:/ fusion proteins (from viruses etc) (1) /:/ G-protein (1) /:/ gadolinium (1) /:/ galectin (6) /:/ gastric bypass (2) /:/ gastric bypass surgery (2) /:/ gastric cancer (1) /:/ gastric parasite (1) /:/ gastrointestinal parasite (1) /:/ “gateway” tissues (1) /:/ gene conversion (12) /:/ genetic disease (1) /:/ genetic palindromes (1) /:/ geomagnetic field sensing (2)
/:/ germ line (6) /:/ germ plasm (1) /:/ germinal center (5) /:/ ghrelin (7) /:/ gingival diseases (1) /:/ glia (1) /:/ glioblastoma (1) /:/ glioma (1) /:/ glomerulonephritis (1) /:/ glucose-induced apoptosis (1) /:/ glutathione (2) /:/ glycolysis (2) /:/ glycoprotein (3) /:/ glycosaminoglycans (2) /:/ glycosidases (1) /:/ glycosylation (1) /:/ glycosyltransferase (1) /:/ GMO (genetically modified organism) (3) /:/ gorilla (muscles of) (1) /:/ gp130 (4) /:/ gp150 (1) /:/ gp63 (1) /:/ gradient (chemical) (5) /:/ graft-versus-host (2) /:/ gramnegative (3) /:/ gram-positive (3) /:/ granulocyte-macrophage colony-stimulating factor (1) /:/ granulocytic anaplasmosis (1) /:/ graying (of hair) (5) /:/ green fluorescent protein (GFP) (6) /:/ greigite (1) /:/ H2O2 (9) /:/ H5N1 (1) /:/ habitability (of lymphatic vessels) (1) /:/ Haemophilus influenza (1) /:/ hair color (3) /:/ hair follicles (9) /:/ heart (29) /:/ heartbeat (1) /:/ heat (7) /:/ heat shock proteins (2) /:/ Helicobacter pylori (4) /:/ helminth (2) /:/ helper T cells (1) /:/ hemagglutinin (1) /:/ hematopoiesis (1) /:/ hematopoietic (2) /:/
hemoglobin (4) /:/ heparan (7) /:/ heparan sulfate (7) /:/ heparin (2) /:/ hepatic (3) /:/ hepatic stellate cells (1) /:/ hepatitis (2) /:/ hepatocytes (2) /:/ HER2 (1) /:/ herbicides (biodegradable) (1) /:/ Hermes-1 (1) /:/ herpes simplex (1) /:/ heterochronic (1) /:/ heterodimer (1) /:/ heterogeneity (12) /:/ heterogeneous (4) /:/ heterologous (9) /:/ heteroplasmy (7) /:/ hexosamine (1) /:/ high density lipoprotein (1) /:/ high-affinity (3) /:/ high-protein plant nectar (1) /:/ high-throughput sequencing (of dna) (1) /:/ histamine (1) /:/ histatins
(1) /:/ histidine (1) /:/ histidine-rich cytotoxic polypeptide (1) /:/ histocompatibility (2) /:/ HIV (Human Immunodeficiency Virus) (13) /:/ HIV-1 (5) /:/ HIV-2 (2) /:/ HLA (human leukocyte antigen) (15) /:/ homeostasis (1) /:/ homing (100) /:/ homing pigeons (navigation) (1) /:/ homocysteine (5) /:/ homotypic adhesion (1) /:/ hormonal (3) /:/ hormone (7) /:/ host-derived membrane (1) /:/ host-specified (entry) (2) /:/ HTERT (human telomerase reverse transcriptase) (1) /:/ htlv-1 (2) /:/ HTLV-I (human T-lymphotrophic virus I) (2) /:/
HTLV-I-infected (1) /:/ human circulatory system (1) /:/ human cytomegalovirus (1) /:/ human endogenous cytotoxic peptides (1) /:/ human genome (3) /:/ human growth hormone (1) /:/ human heart tissue (1) /:/ human immune system (16) /:/ human innate immunity (4) /:/ human life span (1) /:/ human lymphatic vessels (1) /:/ human mitochondria (5) /:/ human papilloma virus (1) /:/ human protocadherin genes (1) /:/ human saliva (1) /:/ human salivary cytotoxic polypeptides (1) /:/ human stem cells (6) /:/ human surgeons (and repnumi
rejuvenation) (3) /:/ human-animal (thought exchanges) (1) /:/ human-machine (thought exchanges) (1) /:/ human-machine-human (thought exchanges) (1) /:/ human-to-human (2) /:/ human-to-machine (1) /:/ humoral (immunity) (6) /:/ hunger-inducing (hormone) (1) /:/ huntingtin (1) /:/ Huntington’s (7) /:/ hyaluronan (1) /:/ hyaluronate (1) /:/ hybrid receptors (1) /:/ hydrogen peroxide (4) /:/ hydrolases (2) /:/ hydrophobic (2) /:/ hydrophobicity (4) /:/ hyenas (1) /:/ hypermethylation (1) /:/ hyperplasia (1) /:/ hypertension (5) /:/
hypertensive (1) /:/ hyperthermia (5) /:/ hypochlorite (1) /:/ hypomethylation (1) /:/ hypoxia (11) /:/ hypoxic (2) /:/
I-J-K-L
ICAM-1 (8) /:/ ICAM-2 (1) /:/ Ig-superfamily (1) /:/ IgA (1) /:/ IGF-1 (insulin-like growth factor 1) (3) /:/ IgG (1) /:/ IL-10 (1) /:/ IL-6 (4) /:/ immature immune systems (1) /:/ immortalized (human cells) (3) /:/ immune surveillance (1) /:/ immune-deficient (1) /:/ immunity-blocking (2) /:/ immunocompromised (1) /:/ immunodominance (3) /:/ immunodominant (2) /:/ immunogenic (4) /:/ immunoglobulin (2) /:/ immunologic (1) /:/ immunomodulator (3) /:/ immunosuppressant (1) /:/ immunosuppression (8) /:/
immunosuppressive (1) /:/ impetigo (1) /:/ infant (6) /:/ infarction (1) /:/ infiltrate (1) /:/ infiltration (6) /:/ inflamed (4) /:/ inflammation (19) /:/ inflammatory (6) /:/ inflammatory bowel disease (1) /:/ infliximab (3) /:/ influenza virus (4) /:/ ingested bacteria (ingested by human cells) (1) /:/ injure (1) /:/ innate immunity (9) /:/ inositol (1) /:/ inositol triphosphate (1) /:/ insulin (3) /:/ insulin-like growth factor 1 (igf-1) (1) /:/ integrin (10) /:/ intellectual rigidity (1) /:/ inter-cellular synchrony (1) /:/ interleukin (2) /:/ interleukin-10
(1) /:/ interleukin-12 (1) /:/ interleukin-1α (1) /:/ interleukin-1β (2) /:/ interleukin-2 (1) /:/ interleukin-3 (1) /:/ interleukin-6 (2) /:/ internalin (1) /:/ interstitial fluid (of tumors) (1) /:/ intracellular garbage (4) /:/ intraepithelial lymphocytes (2) /:/ intragenic triplet tandem arrays (1) /:/ intraluminal valves (of lymphatic channels) (2) /:/ invasin (1) /:/ invasive front (of a tumor) (1) /:/ invertebrate (1) /:/ ion channel (1) /:/ iron (3) /:/ Isaria sinclairii (1) /:/ ischemia (3) /:/ ischemia-reperfusion (2) /:/ ischemia-reperfusion injury (2) /:/
ischemic (9) /:/ ischemic hind limbs (1) /:/ ivory (1) /:/ jellyfish Aequorea victoria (2) /:/ karyotypic rearrangements (1) /:/ kidney (6) /:/ kidney dialysis (1) /:/ kidney stone (1) /:/ killing spectrum (1) /:/ L-arginine (2) /:/ L-carnitine (1) /:/ L-form amino acid (1) /:/ L-NG-nitroarginine methyl ester (1) /:/ L-selectin (5) /:/ lactate (3) /:/ lactic acid fermentation (Warburg effect) (1) /:/ lactose (1) /:/ laminin (3) /:/ laminin-111 (1) /:/ laminin-binding (1) /:/ leather from cultured cells (1) /:/ lectin (70) /:/ Legionella pneumophila (2) /:/
Leishmania donovani (6) /:/ lentivirus (2) /:/ leopard (muscles of) (1) /:/ leprosy (2) /:/ leptin (3) /:/ Leu-8 (homing receptor) (1) /:/ leucine-rich repeat region (1) /:/ leukocyte/xenobiorg (42) /:/ LFA-1 (4) /:/ ligand (17) /:/ light-activated protein (1) /:/ light-emitting diode (1) /:/ light-sensitive protein (3) /:/ Lincoln (Abraham) (2) /:/ LINE-1 element (1) /:/ lipid bilayer (1) /:/ lipid rafts (1) /:/ lipid-depleted adipocytes (1) /:/ lipophosphoglycan (1) /:/ lipopolysaccharide (3) /:/ lipopolysaccharides (1) /:/ lipoprotein (4) /:/ liposome
(delivery of drugs) (1) /:/ liposuction (1) /:/ lipoteichoic (1) /:/ Listeria innocua (1) /:/ Listeria ivanovii (1) /:/ Listeria monocytogenes (3) /:/ listeriosis (1) /:/ lithium (1) /:/ liver cancer (2) /:/ liver inflammation (1) /:/ liver metastasis (1) /:/ liver resection (1) /:/ liver toxicity (1) /:/ liver toxin (1) /:/ Loa loa (african eyeworm) (1) /:/ local immunodeficiency (1) /:/ Lorentz effect (1) /:/ Lou Gehrig’s disease (3) /:/ low density lipoprotein (3) /:/ low-affinity (2) /:/ low-energy (1) /:/ low-pressure (1) /:/ lung (13) /:/ lung cancer (1) /:/
lung compartments (1) /:/ lung function (1) /:/ lung mucosa (1) /:/ lung vasculature (2) /:/ lung-homing (1) /:/ lupus erythematosus (1) /:/ LY294002 (1) /:/ lymph (62) /:/ lymph channel (3) /:/ lymph node (7) /:/ lymph-traveling (1) /:/ lymphatic (124) /:/ lymphatic channel (3) /:/ lymphatic filariasis (2) /:/ lymphedema (1) /:/ lymphoblastic (2) /:/ lymphocyte (10) /:/ lymphoid (5) /:/ lymphoid follicles (1) /:/ lyse (7) /:/ lysis (11) /:/ lysosome (4) /:/ lysosome fusion (2) /:/ lysosome-phagosome (1) /:/ lysyl moieties (1) /:/ lytic
antibodies (1) /:/ lytic polypeptides (4) /:/
M
Mac-1 (integrin) (2) /:/ macrophage (5) /:/ macrophage/xenobiorg (2) /:/ MAdCAM-1 (2) /:/ magnetic field (4) /:/ magnetic resonance imaging (7) /:/ magnetism (1) /:/ magnetite (18) /:/ magnetite (Fe3O4) (18) /:/ magnetotactic bacteria (1) /:/ malaria (14) /:/ malaria-free (mosquitos) (1) /:/ malarial Pacific island (1) /:/ malignant (12) /:/ malignant pleural mesothelioma (1) /:/ mammal (1) /:/ mammary (2) /:/ mammary gland (1) /:/ mannose (1) /:/ Mansonella (1) /:/ mapping (of information from one brain to another) (8) /:/
mapping (of solid tissues) (8) /:/ marine organism (cytotoxic polypeptides) (1) /:/ mariner-class transposon (1) /:/ marrow (bone marrow) (23) /:/ masquerade (cancer) (1) /:/ mast cell (3) /:/ mast cell degranulation (1) /:/ maternal (immunosuppression) (2) /:/ maternal antipaternal cell-mediated immunity (1) /:/ maturation (of dendritic cells) (5) /:/ maturation (of erythrocytes) (5) /:/ mature phagolysosomes (3) /:/ measles (fusion protein) (1) /:/ meat (from cultured cells) (3) /:/ mechanical guides (to control homing) (1) /:/ melanin (4) /:/
melanocyte (4) /:/ melanoma (11) /:/ membrane (44) /:/ membrane-bound (2) /:/ membrane-crossing (1) /:/ memories (21) /:/ memory (26) /:/ mental (12) /:/ mercury (1) /:/ meritocracy (3) /:/ mesenchymal (5) /:/ mesenchyme (1) /:/ mesothelioma (1) /:/ messenger RNA (1) /:/ metalloproteinase (5) /:/ metastases (2) /:/ metastasis (21) /:/ metastasize (3) /:/ metastasizing (4) /:/ metastatic (6) /:/ methylation (4) /:/ Mg2+ (2) /:/ MGAT5 (1) /:/ MHC (2) /:/ MHC-I (3) /:/ MHC-II (1) /:/ mice (25) /:/ micelle (delivery of
drugs) (1) /:/ micro-inflammation (1) /:/ microbe (22) /:/ microbial (22) /:/ microfilament (1) /:/ microfilaria (20) /:/ microorganism (1) /:/ microparticle (2) /:/ microparticle bombardment (2) /:/ microRNA (1) /:/ microsporidian (1) /:/ microsporidian polar tube (1) /:/ microtube (1) /:/ microvasculature (1) /:/ microvilli (3) /:/ middle ear (infection) (1) /:/ milk (from cultured cells) (3) /:/ millet (and aflatoxin) (1) /:/ minicells (1) /:/ mitochondria (93) /:/ mitochondrion (2) /:/ Mn2+ (2) /:/ moles (pigmented nevi) (1) /:/ monkey (1)
/:/ monoclonal (antibodies) (12) /:/ monocyte (3) /:/ monomeric human actin (1) /:/ monovalent (antibodies) (2) /:/ morpholinos (1) /:/ mosquito (7) /:/ mother (immunorestraint) (1) /:/ motile (7) /:/ motility (2) /:/ motor (neuron or neurone) (2) /:/ mouse (6) /:/ mRNA (6) /:/ MSCRAMMS (2) /:/ MSH3 (1) /:/ Msp2 (2) /:/ MT1-MMP (2) /:/ MUC16 (2) /:/ MUC5 (6) /:/ mucin (4) /:/ mucin AgC10 (4) /:/ mucosa (5) /:/ mule (muscles of) (1) /:/ multi-antigen (1) /:/ multi-factorial (1) /:/ multifunctional (2) /:/ multigene
family (1) /:/ multiplicity of infection (3) /:/ multispecies biofilm (1) /:/ mumps (1) /:/ muscle (19) /:/ muscle fiber (2) /:/ muscular (5) /:/ muscular dystrophy (5) /:/ mushroom coral (1) /:/ mutant (2) /:/ mutate (1) /:/ mutation (5) /:/ Mycobacterium (10) /:/ Mycobacterium leprae (1) /:/ Mycobacterium lepraemurium (1) /:/ Mycobacterium tuberculosis (4) /:/ mycophenolic (3) /:/ myeloid (1) /:/ myeloma (2) /:/ myoblasts (3) /:/ myocytes (1) /:/ myopathy (1) /:/ myxoma (1) /:/
N-O
NaCl (inactivation of cytotoxic polypeptides) (2) /:/ Nanog (1) /:/ nanotech robots (4) /:/ nasal mucosa (1) /:/ naïve T cells (1) /:/ nebulized spray (1) /:/ necrolysis (1) /:/ necrotizing enterocolitis (1) /:/ nectar (and mosquitos) (6) /:/ nectin (protein family) (1) /:/ Nef (protein) (4) /:/ Neisseria gonorrhea (1) /:/ Nematoda (1) /:/ nematode (25) /:/ nematode anatomy (2) /:/ nematode behavior (4) /:/ nematode locomotion (1) /:/ neointima (1) /:/ neonate (1) /:/ nerve (5) /:/ nervous (tissue or system) (8) /:/ network (lymphatic) (3) /:/
network (regulatory) (3) /:/ neural (28) /:/ neural plasticity (1) /:/ neuraminidase (3) /:/ neurobiological (1) /:/ neurobiology (2) /:/ neuroblasts (1) /:/ neurodegenerative (2) /:/ neurological (3) /:/ neuron (10) /:/ neurone (1) /:/ neuropeptide (1) /:/ neuroses (1) /:/ neutrophil (14) /:/ neutrophil peripheral lymph node homing receptor (1) /:/ nevi (pigmented) (2) /:/ newborn (2) /:/ NF-κB (4) /:/ nibble (nibbling of antigens) (1) /:/ nidogen (1) /:/ Nijmegen breakage syndrome (1) /:/ nitric oxide (5) /:/ Nod-Like Receptors (1) /:/ nonalcoholic fatty liver disease (1) /:/ non-biting (mosquitos) (1) /:/ non-coding RNA (1) /:/ non-obese diabetic mice (1) /:/ non-shivering thermogenesis (1) /:/ non-solid tissues (1) /:/ non-surgical breast augmentation (1) /:/ non-target (cells) (3) /:/ non-toxic binding polypeptides (1) /:/ normoxic (1) /:/ nourish (xenobiorgs within cells) (4) /:/ nuclear (24) /:/ nuclear localization (1) /:/ nucleases (5) /:/ nucleus (29) /:/ nucleus accumbens (1) /:/ nursing home facilities (1) /:/ obese (people) (6) /:/ obesity (3) /:/ obligate anaerobe (1) /:/
obligately (intracellular) (2) /:/ obligatory (endosymbionts) (5) /:/ obstruction (of flow from kidney or liver) (3) /:/ Oct4 (1) /:/ octanoylated (1) /:/ oculopharyngeal (2) /:/ oligomers (4) /:/ Onchocerca lienalis (1) /:/ Onchocerca volvulus (1) /:/ oncogene HER2 (1) /:/ oncolysis (1) /:/ oncolytic (1) /:/ Oncorhynchus mykiss (rainbow trout) (1) /:/ online (information facilities) (3) /:/ oogenesis (2) /:/ operons (1) /:/ opsin proteins (1) /:/ optical fiber (1) /:/ optical tweezers (1) /:/ optogenetic (15) /:/ orectic (factors) (1) /:/ organ (29) /:/
organ-specific (3) /:/ organelle (1) /:/ ornithine (1) /:/ orthotopic (cancer transplantation) (2) /:/ osteoarthritis (5) /:/ osteoblasts (4) /:/ osteoclasts (2) /:/ osteocytes (3) /:/ osteogenesis (1) /:/ osteogenesis imperfecta (1) /:/ osteoporosis (6) /:/ ovarian (3) /:/ ovum (2) /:/ owl (retinas of) (1) /:/ oxidation (2) /:/ oxidative (stress) (1) /:/ oxidized (lipoprotein) (8) /:/ oxidized (proteins) (8) /:/ oxidized lipids (1) /:/ oxidizing agents (1) /:/ oxygen (diffusion through tissue) (13) /:/ oxygen (partial pressure) (13) /:/ oxygen-carrying
(hemoglobin) (1) /:/
P-Q
P-selectin (11) /:/ P-selectin glycoprotein ligand-1 (PSGL-1) (1) /:/ p16 (tumor supressor) (1) /:/ p450 (2) /:/ p53 (3) /:/ PABPN1 (2) /:/ palindrome (1) /:/ pancreas (6) /:/ pancreatic (16) /:/ pancreatic lipase (1) /:/ pancreatic peptide amylin (IAPP) (1) /:/ pandemic (1) /:/ papillary (1) /:/ papilloma (1) /:/ papillomavirus (6) /:/ parabiosis (7) /:/ parabiotic (4) /:/ paracytophagy (1) /:/ paraendothelial (4) /:/ parainfluenza (1) /:/ paralogs (3) /:/ Paramecium (1) /:/ parasite (27) /:/ parasite-encoded (1) /:/ parasite-specified (3) /:/
parasite-synthesized (1) /:/ parasitic (15) /:/ parasitization (1) /:/ parasitize (3) /:/ parathyroid (1) /:/ parathyroid hypertensive factor (1) /:/ Parchment (font) (2) /:/ Parkinson’s (9) /:/ pathogen (18) /:/ pathogen-derived (1) /:/ pathogen-infected (3) /:/ pathogenicity island-1 (1) /:/ pathogenicity island-2 (1) /:/ peanut (1) /:/ (Penicillium) echinulatum (1) /:/ Penicillium stoloniferum (1) /:/ penile (3) /:/ penis (1) /:/ pentapeptide (enterostatin) (1) /:/ (peptide) lipidation (1) /:/ peptide-mediated (cell fusion) (1) /:/ peptide-membrane binding
(1) /:/ peptide-presentation (1) /:/ pesticides (living) (1) /:/ Peyer's (2) /:/ pH (low pH of cancers) (10) /:/ pH-dependent cytotoxic polypeptides (1) /:/ phage display libraries (2) /:/ phagolysosome (1) /:/ phagolysosomes (6) /:/ phagosome (6) /:/ phagosome-lysosome (3) /:/ phosphatase (1) /:/ phosphatidylinositol (1) /:/ phosphatidylserine (2) /:/ phosphoinositide (2) /:/ phosphoinositide 3-kinase (2) /:/ phospholipases (1) /:/ phospholipids (1) /:/ phosphorothioate (1) /:/ phosphorothioate ester (1) /:/ pigment (antioxidant) (2) /:/ pili
(1) /:/ pilin (1) /:/ placenta (3) /:/ Planck (Max) (1) /:/ plant (5) /:/ plasminogen (2) /:/ plasminogen activator inhibitor-1 (1) /:/ Plasmodium falciparum (7) /:/ Platelet-Derived Growth Factor (PGDF) (2) /:/ pluripotency (3) /:/ pluripotent (17) /:/ pockets of immune deficiency (1) /:/ polarity (and cancer) (3) /:/ poliovirus (1) /:/ polyalanine (1) /:/ polyamine (2) /:/ polyglutamine (1) /:/ polymerase chain reaction (1) /:/ polystyrene beads (2) /:/ pore formation (3) /:/ post-capillary venules (1) /:/ post-ischemic inflammation (1) /:/ postmenopausal (1) /:/ post-natal (1) /:/ post-nodal lymphatic vessels (1) /:/ post-translational (protein modifications) (1) /:/ PP2 529573 (a Src protein inhibitor) (1) /:/ Prader-Willi (3) /:/ pre-collecting vessels (lymphatic) (4) /:/ pre-eclampsia (4) /:/ pre-implantation embryos (1) /:/ pre-nodal collecting vessels (lymphatic) (4) /:/ precancerous (3) /:/ premature (aging) (5) /:/ premature (infant) (5) /:/ prenatally (1) /:/ preprocholecystokinin (1) /:/ presenilin (1) /:/ primate lentiviruses (1) /:/ probiotics (1) /:/ prodrug (6) /:/ “professional”
phagocytes (1) /:/ progesterone (4) /:/ prokaryotic endosymbionts (2) /:/ proline (1) /:/ promastigotes (2) /:/ prostaglandin (1) /:/ prostaglandin E2 (1) /:/ prostate (5) /:/ prostatic (1) /:/ prosthetic (2) /:/ protease (4) /:/ protein Ki-67 (1) /:/ protein-protein (1) /:/ proteobacteria (1) /:/ proteoglycans (1) /:/ proteolysis (2) /:/ protocadherin (6) /:/ protozoa (1) /:/ pseudogenes (1) /:/ Pseudomonas aeruginosa (2) /:/ pseudopod (2) /:/ PSGL-1 (2) /:/ psoriasis (2) /:/ psoriatic arthritis (1) /:/ pterins (1) /:/ Pubmed (6) /:/ purine (2) /:/
putrescine (3) /:/ putrid (1) /:/ QIA format (1) /:/ quadriplegia (1) /:/ quench (of light signal in optogenetics) (2) /:/ Quick Internet Access (QIA format) (1) /:/ quorum (1) /:/ quorum-sensing (1) /:/
R
rabbit pox (1) /:/ radiation-resistant (1) /:/ radioactive (tracers or contrast agents) (2) /:/ radioactive colloid (1) /:/ rainbow trout (1) /:/ rapidly-degrading (2) /:/ rats (8) /:/ reactive nitrogen species (1) /:/ reactive oxygen intermediates (2) /:/ reactive oxygen species (2) /:/ receptor (37) /:/ receptor-ligand (3) /:/ recirculation (of lymphocytes) (2) /:/ recognition between seeker cells and endothelial cells (1) /:/ recombineering (2) /:/ recruitment of stem cells (1) /:/ redifferentiation (of cells) (4) /:/ redirection (of immune system cells) (1) /:/
reduced lipids (1) /:/ regenerate (7) /:/ regeneration (7) /:/ regulate (4) /:/ regulation (5) /:/ regulator (1) /:/ regulatory cascade (1) /:/ regulons (1) /:/ rejection (of tissues and organs) (6) /:/ rejuvenable (2) /:/ rejuvenate (7) /:/ rejuvenating (1) /:/ rejuvenation (127) /:/ relay (of light signals) (5) /:/ remodeled (lymphatic networks) (1) /:/ remodeling (of the extracellular matrix) (15) /:/ renal (2) /:/ reoxygenation (of tumors) (2) /:/ repellant (3) /:/ repelling (of cytotoxic polypeptides) (2) /:/ replicons (1) /:/ Repnumi (284) /:/ repositioned
(organelles) (2) /:/ reprogram (cells) (2) /:/ resistin (1) /:/ respiration (2) /:/ respiring (1) /:/ retina (1) /:/ retinoblastoma (2) /:/ retinoic (2) /:/ retinoic acid-inducible (1) /:/ retinoic acid-inducible gene-i-like receptors (1) /:/ retinol-binding protein 4 (2) /:/ retrocyclins (1) /:/ reversal of baldness (1) /:/ reverse-orientation receptor (1) /:/ rheumatoid (8) /:/ rhythmic pumping (of lymphatic vessels) (1) /:/ rice (1) /:/ Rickettsia (4) /:/ Rickettsiae (2) /:/ right lymphatic duct (3) /:/ right of perpetual veto (1) /:/ RNA interference (21) /:/
RNA-induced transcriptional silencing (1) /:/ ruffling (of cell membrane) (1) /:/
S
Saccharomyces castellii (1) /:/ safeguards (2) /:/ safely (4) /:/ safety (6) /:/ saliva (2) /:/ salivary cytotoxic polypeptides (1) /:/ Salmonella (14) /:/ Salmonella enterica (4) /:/ Salmonella leucine-arginine auxotrophs (1) /:/ Salmonella typhimurium (3) /:/ Sarcina lutea (1) /:/ satellite cells (muscle stem cells) (1) /:/ satiety hormone (leptin) (1) /:/ saturation (tissue invasion scheme) (4) /:/ scalp (induction of hair follices in) (2) /:/ scavenge (expelled nuclei and mitochondria) (2) /:/ scavenging (of expelled nuclei and mitochondria) (3) /:/
scleroderma (2) /:/ sclerosis (3) /:/ sclerotized (4) /:/ scorpion (venom) (1) /:/ SDF-1α (Stromal-Derived Factor 1α) (3) /:/ secretoneurin (2) /:/ selectin (1) /:/ self-avoidance (neural) (2) /:/ self-organization (of rejuvenated cells) (2) /:/ self-promoting (cancers) (1) /:/ self-protection (against cytotoxic polypeptides) (1) /:/ self-reacting T cells (1) /:/ self-renew (cancer stem cells) (1) /:/ self-tolerance (immunological) (1) /:/ sell (1) /:/ semen (1) /:/ semi-human (1) /:/ send (2) /:/ sender (2) /:/ senders (2) /:/ sender’s (1) /:/ sending (4) /:/
senesce (1) /:/ senesced (6) /:/ senescence (4) /:/ senescence-acceleration (1) /:/ senescesced (1) /:/ seniority (1) /:/ sensation (5) /:/ sensations (3) /:/ sensitization by acid (of cancers) (1) /:/ sentinel node (1) /:/ separable (functions of macromolecules) (1) /:/ sepsis (neonatal) (1) /:/ sequential (receptor-ligand recognition) (2) /:/ serine protease inhibitor (serpin) (1) /:/ serpin (serum protease inhibitor) (1) /:/ serum killing of bacteria (1) /:/ Severe Acute Respiratory Syndrome (SARS) (1) /:/ shear flow (or shear stress) (1) /:/ shear force (of
blood flow) (1) /:/ shed (shedding of antigens, receptors, etc) (4) /:/ shedding of l-selectin (1) /:/ shift (of antigenic profile) (4) /:/ shigella (2) /:/ shock wave therapy (1) /:/ shrimp (cytotoxic polypeptides) (1) /:/ shuffling (of protein domains) (1) /:/ sialic acid (2) /:/ sialoglycoprotein (1) /:/ sialyated mucins (1) /:/ sialyl-LewisX (2) /:/ signaling (23) /:/ silver bullet (against cancer) (1) /:/ single-celled (8) /:/ single-chain (15) /:/ single-chain antibody mimics (2) /:/ singlet oxygen (3) /:/ sinusoidal endothelium (2) /:/ site-specific (nuclease
or endonuclease) (3) /:/ Sjogren’s syndrome (2) /:/ skeletal fragility (1) /:/ skeletal muscle (4) /:/ skin (38) /:/ skull (6) /:/ slither (microfilariae) (1) /:/ smooth muscle (4) /:/ Snail (cohesion protein) (1) /:/ snake (venom) (1) /:/ SODCI (superoxide dismutase) (1) /:/ soft plaque (2) /:/ solid (tissues) (33) /:/ solid (tumors) (33) /:/ soluble β-amyloid monomers (1) /:/ soluble decoy receptors (1) /:/ somatic cell (4) /:/ somatic mutation (3) /:/ soporifics (1) /:/ sorghum (and aflatoxin) (1) /:/ spatial organization (of cells) (2) /:/ spatial
restriction (within the body) (3) /:/ species (30) /:/ sperm (2) /:/ spermidine (2) /:/ spermine (2) /:/ sphingosine (1) /:/ sphingosine-1-phosphate (1) /:/ SpiC (1) /:/ spices (and aflatoxin) (1) /:/ spider venom (1) /:/ spinal cord (3) /:/ spinal injury (1) /:/ spleen (3) /:/ splicing isoforms (1) /:/ spores (of aflatoxin-producing fungi) (4) /:/ squamous cell (cancers) (3) /:/ Src protein (1) /:/ stage-specific (RNA interference) (1) /:/ staging areas (2) /:/ staining (of lymphatic vessels) (2) /:/ Staphylococcus (6) /:/ Staphylococcus aureus (6) /:/
statin (drugs) (1) /:/ statistical techniques (1) /:/ stem cells (117) /:/ step-by-step development (1) /:/ stereotyped actions (of nematodes) (1) /:/ sterility (and telomerase) (1) /:/ stochastic theory (of cancer) (1) /:/ stomach (2) /:/ streptococcus (5) /:/ Streptococcus agalactiae (2) /:/ Streptococcus mutans (1) /:/ stress (12) /:/ string (text string) (2) /:/ stroke (neurological) (8) /:/ stromal-derived factor 1α (SDF-1α) (2) /:/ subcutaneous (injection) (1) /:/ subsonic vibration (1) /:/ sugar (5) /:/ sugars (2) /:/ sulfatases (1) /:/ sun-protective (1) /:/
sunflower seeds (and aflatoxin) (1) /:/ superable (1) /:/ superantigen-like (3) /:/ superfamily (1) /:/ superfluous xenobiorgs (1) /:/ superinfection (1) /:/ superoxide (9) /:/ superoxide anion generators (1) /:/ superoxide dismutase (4) /:/ supracellular (senescence) (3) /:/ surfactant (1) /:/ survivin (1) /:/ synapse (4) /:/ synergy (between cytotoxic polypeptides) (1) /:/ syngeneic mice (1) /:/
T
T-cell (5) /:/ T-lymphocytes (10) /:/ T-lymphotrophic (1) /:/ tandem peptide repeats (1) /:/ tandemly repeated peptide sequences (1) /:/ tanning (4) /:/ tarantula (venom) (1) /:/ tarantula venom (1) /:/ Tat (1) /:/ Tat-mediated (1) /:/ tax (1) /:/ taxis (in nematodes) (3) /:/ teeth (1) /:/ teichoic (2) /:/ telangiectasia (1) /:/ telepathic (facilitated telepathy) (1) /:/ telepathy (facilitated) (45) /:/ telomerase (23) /:/ telomerase-deficient (1) /:/ telomere (2) /:/ telomere-protective (1) /:/ teratoma (1) /:/ Th1-type (1) /:/ Th2 (2) /:/ Th2-inducing
(1) /:/ Th2-type (1) /:/ thermogenesis (1) /:/ thoracic duct (3) /:/ three-layered walls (of lymphatic vessels) (1) /:/ thrombospondin (1) /:/ thrombospondin-related (1) /:/ thromboxane (1) /:/ thymus (6) /:/ thymus gland (3) /:/ TIMP (1) /:/ tissue plasminogen inhibitor (1) /:/ TLR3 (1) /:/ TLR7 (1) /:/ TLR9 (1) /:/ toenails (protection from chemotherapy) (1) /:/ toll-like (6) /:/ toroidal pore mechanism (of cell lysis) (1) /:/ tortuous course (of lymphatic vessels) (2) /:/ totipotent (1) /:/ toxic (12) /:/ toxicity (2) /:/ toxin (7) /:/
toxoplasma (2) /:/ Toxoplasma gondii (2) /:/ trachomatis (Chlamydia) (1) /:/ trans-endothelial (13) /:/ trans-sialidase (1) /:/ trans-splicing (3) /:/ transcellular diapedesis (1) /:/ transcellular transmigration (1) /:/ transcription factor (1) /:/ transduce (1) /:/ transduction (2) /:/ transendothelial (2) /:/ transfected (1) /:/ transferability (of xenobiorgs between species) (1) /:/ transient (cell types) (1) /:/ transmembrane (5) /:/ transmigration (7) /:/ transmigratory (1) /:/ transplant (1) /:/ transplantation (10) /:/ transporter (protein) (1) /:/
transposition (1) /:/ transposon (2) /:/ transposon-mediated (1) /:/ trap-forming leukocytes (DNA traps) (1) /:/ trapped (engineered microfilariae) (2) /:/ treatment-resistant (cancer) (1) /:/ tree nuts (and aflaxtoxin) (1) /:/ tree-like (lymphatic vessels) (1) /:/ Treg (2) /:/ triacylglycerides (1) /:/ Triatominae (kissing bugs) (1) /:/ Trichinella spiralis (1) /:/ triglyceride synthesis (1) /:/ trimethylamine-n-oxide (1) /:/ triplet expansion disease (1) /:/ trophoblasts (2) /:/ tropical spastic paraparesis (1) /:/ Trypanosoma (13) /:/ Trypanosoma brucei (6)
/:/ Trypanosoma brucei gambiense (1) /:/ Trypanosoma cruzi (7) /:/ trypanosome (3) /:/ trypanosomes (6) /:/ trypomastigotes (1) /:/ trypsin (1) /:/ tubules (abnormal) (1) /:/ tumor (59) /:/ tumor necrosis factor (TNF) (13) /:/ tumor suppressor protein (1) /:/ tumor-associated (1) /:/ tumor-carried (1) /:/ tumor-specific (1) /:/ tumorigenic (1) /:/ two-component immunosuppressant (2) /:/ type-1-polarized effector T cells (1) /:/
U-V-W-X-Y-Z
ubiquitin (1) /:/ ubiquitin ligase (1) /:/ ubiquitin-proteasome (1) /:/ ulcer-causing bacterium (1) /:/ ulcerative (2) /:/ ulcerative colitis (2) /:/ ulcers (2) /:/ Ullrich congenital muscular dystrophy (1) /:/ ultrafast laser surgery (1) /:/ uncircumcised men (and xenobiorgs) (1) /:/ undifferentiation signature (2) /:/ unicellular eukaryotes (1) /:/ unidirectional flow (in lymphatic channels) (1) /:/ unmethylated CpG dinucleotides (1) /:/ urea (and atherosclerosis) (1) /:/ ureter (obstruction of) (1) /:/ urogenital tract (1) /:/ urogenital tract (cytotoxic
polypeptides) (1) /:/ uterus (and eclampsia) (1) /:/ vaccination (3) /:/ vaccine (2) /:/ vaccinia (cowpox) (1) /:/ vacuole (cytoplasmic) (13) /:/ vaginas (1) /:/ valve (lymphatic) (4) /:/ Variable Surface Glycoproteins (2) /:/ Vascular Endothelial Growth Factor (VEGF) (4) /:/ vasculature (16) /:/ vasculogenesis (7) /:/ vasculogenic (1) /:/ vasculogenic mimicry (1) /:/ VCAM-1 (Vascular Cell Adhesion Molecule 1) (6) /:/ VE-cadherin (3) /:/ vein (blood) (2) /:/ venereal disease (1) /:/ venules (3) /:/ vernal keratoconjunctivitis (1) /:/ Very Late
Antigen-4 (1) /:/ vesicle (1) /:/ vessel wall (4) /:/ vinculin (1) /:/ viral (14) /:/ viral single-stranded RNA (1) /:/ viremia (1) /:/ virological (1) /:/ virulence (3) /:/ virulence factor (1) /:/ virulent (3) /:/ virus (14) /:/ visceral adipose tissue (4) /:/ visfatin (2) /:/ vitronectin (1) /:/ VLA-4 (2) /:/ Warburg (4) /:/ wasp (venom) (1) /:/ web address (1) /:/ Werner syndrome (1) /:/ wheat (and aflatoxin) (1) /:/ white adipose tissue (1) /:/ white blood cells (8) /:/ white matter (brain) (1) /:/ whooping cough (Bordetella pertussis) (1) /:/
Wikipedia (5) /:/ Wnt (1) /:/ Wnt1 (1) /:/ Wolbachia (7) /:/ Word 365 (1) /:/ worms (2) /:/ wound (injury) (8) /:/ wound chamber (1) /:/ wound healing (3) /:/ wrist (2) /:/ Wuchereria bancrofti (5) /:/ xenobiorg (113) /:/ xenobiorg-host (1) /:/ xenobiorg-synthesized (1) /:/ xenobiotherapeutic (1) /:/ xenobiotherapy (48) /:/ yeast (1) /:/ Yersinia pestis (2) /:/ YGRKKRRQRRR (1) /:/ yippies (1) /:/ yoppies (14) /:/ yuppies (1) /:/ YY3-31 (2) /:/ zeta potential of colloids (1) /:/ zinc finger (nuclease) (2) /:/ zinc-dependent (1) /:/
zipper (entry mechanism) (1) /:/ zirconia (window into brain) (1) /:/ Zn2+ (1) /:/ zwitterionic lipids (1) /:/ zygote (2) /:/
Version August 18, 2014, 4:34 PM