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The future role of molecular and cell biology
in medical practice in the tropical countries
David Weatherall
Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK
Molecular and cell biology have a great deal to offer tropical medicine in the
future. As well as helping to understand the population genetics and dynamics
of both infectious and non-infectious diseases, they promise to provide a new
generation of diagnostic and therapeutic agents, and to play a major role in the
development of new vaccines and other approaches to the control of disease in
tropical communities.
Correspondence to:
Professor Sir David
Weatherall, Institute of
Molecular Medicine.
University of Oxford,
John Radcliffe Hospital,
Oxford OX2 9DS, UK
Over the last 20 years there has been a gradual shift in the emphasis of
basic biomedical research from the study of disease in patients and their
organs to its definition at the level of molecules and cells. This new trend
has been underpinned by a remarkable new technology which has made
it possible to isolate and sequence genes, study their function and
transfer them across the species barrier. In the short time during which
this field has evolved, a great deal has been discovered about human
pathology at the molecular level. Many monogenic diseases have been
characterised, much has been learnt about the molecular and cell
biology of cancer, and a start has been made in defining the different
genes that comprise the complex interactions between nature and
nurture that underlie many of the major killers of Western society.
Enough is known already to suggest that this knowledge will have major
implications for the development of more precise diagnostic and
therapeutic agents in the future.
There is little doubt that tropical medicine will benefit from this new
technology, particularly as the demography of disease changes in
emerging countries. The World Health Organization have predicted that
by the year 2020 there will be a major shift in the pattern of disease1. As
social conditions and standards of hygiene and nutrition improve, there
will be a gradual decline in infant and childhood mortality due to
infectious disease. On the other hand, it is predicted that there will be a
steady increase in diseases of 'Westernisation', including heart disease,
diabetes, other forms of vascular disease, and the major psychoses. It is
already apparent from the epidemic of insulin-resistant diabetes that is
affecting many of these countries, that populations respond in quite
British Medical Bulletin 1998;54 (No. 2): 489-501
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Tropical medicine: achievements and prospects
different ways to the introduction of high energy diets2. Though it is likely
that both environmental and genetic factors are involved, there is
increasing evidence that inheritance may play an important role in these
different responses.
It is important, therefore, that medical practice in the tropics prepares
itself for the remarkable possibilities that the rapidly moving sciences of
molecular and cell biology will have to offer it in the future. This has
particular implications for medical education; specialists in the field will
have to be able to communicate with those working in the basic sciences
so that their technology can be adapted most effectively for the benefit
of the health of communities in the tropical world.
Technical advances
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490
The application of molecular and cell biology to the study of human
disease, or molecular medicine as it is rather optimistically called, has
developed on the back of the technical advances of molecular biology3'4.
One of the first was the discovery of how to isolate DNA and to cut it
up into pieces of different sizes using restriction endonucleases, that is
enzymes isolated from various bacteria that will slice DNA at
predictable sequences of nucleotide bases. An early and quite seminal
advance in the application of this approach to human pathology was
called Southern blotting after the name of its inventor, Edwin Southern.
In this technique restriction enzyme digests of DNA are separated into
different sized fragments by electrophoresis in gels and then simply
blotted onto nitrocellulose filters on which they can be immobilised. By
constructing radioactive probes to hunt for particular genes on these
filters it was possible to analyse potential disease loci for major deletions
or re-arrangements of the particular genes involved.
It soon became possible to take mixtures of restricted DNA and to
insert the different fragments into bacterial plasmids or other 'foreign'
DNA vectors. This was the beginning of the era of recombinant DNA
technology. The inserted DNA could be grown in bacteria and, hence, it
became possible to construct libraries containing most of the human
genome from which it was possible to isolate any gene of interest.
Methods were soon developed for rapid sequencing of DNA and hence
it became feasible to define the precise mutations in many single gene
disorders. And by carrying out genetic linkage studies using highly
variable regions of DNA as markers to pinpoint genes for diseases of
unknown cause, and to deduce the function of their products from their
sequence, a technique called 'reverse genetics', it became possible to
characterise the molecular pathology of many common monogenic
disorders of unknown cause.
British Medical Bulletin 1998,54 (No. 2)
Future role of molecular and cell biology
The next important step was to take isolated human genes and
persuade them to function, either in cultured cells or in laboratory
animals. This made it possible to learn about the major regulatory
regions that are involved in ensuring that genes are expressed in the
correct tissues at the right stages of development and at an appropriate
level. Furthermore, as methods for sequencing genes became more
efficient and were automated it became clear that it would be possible
to determine the complete sequence of the genome of any organism.
Currently, this has already been achieved in the case of some bacteria
and varieties of yeast, and the Human Genome Project, that is the
determination of the complete sequence of the DNA of a human being,
is well on course for completion early in the next millennium.
Although the full benefits of this field for the improvement of health
may not be evident for many years, and particularly until new
developments in biomathematics and computer technology help us to
understand how all our genes are orchestrated to subserve the complex
metabolic functions of intact cells, organs, and whole organisms, there
is no doubt that along the way there will be a steady accumulation of
information that will make a major impact on tropical medicine. It is
beyond the scope of this brief review to describe all these possibilities
and hence I shall simply summarise a few that are already well advanced
and try to predict some of the more important possibilities in the future.
Molecular genetics in the tropics
Single gene disorders
Although many diseases are inherited in a simple Mendelian pattern,
and are seen in every part of the world, most of them occur at a very low
frequency which probably reflects the mutation rate. However, there are
a few groups of genetic disorders which occur much more commonly
and which will pose an important public health problem in the future.
There is increasing evidence that they have reached their high frequency
in many tropical countries by natural selection, reflecting heterozygote
advantage against different forms of malaria. It is probably through this
mechanism that the inherited disorders of haemoglobin have become the
commonest human monogenic diseases5
Human adult haemoglobin consists of two pairs of a chains and two
pairs of P chains (<x,P2). The a and P globin chains are controlled by a
and P globin gene families which reside on chromosomes 16 and 11,
respectively. There are two classes of mutations at these gene loci. First
there are the structural haemoglobin variants, which result from single
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Tropical medicine: achievements and prospects
amino acid substitutions or other structural alterations in the a or P
globin chains. The second and more common disorders are those due to
a reduced rate of synthesis of the a or P globin chains, the a and P
thalassaemias. Studies at the molecular level have led to a broad
understanding of the structure and regulation of the globin genes and of
the molecular pathology of both the structural variants and the
thalassaemias5.
Although several hundred structural haemoglobin variants have been
described only three, haemoglobins S, C and E, reach very high
frequencies in some tropical countries. The homozygous state for the
sickle cell gene, sickle cell anaemia, is a major cause of childhood
morbidity and mortality in sub-Saharan Africa, the Mediterranean
region, the Middle East and in parts of India5. It also occurs frequently
in countries with large African immigrant populations. Although much
remains to be learnt about the mechanisms of sickling and the reasons
for the remarkable clinical heterogeneity of sickle cell anaemia,
considerable progress has been made towards its better management
and prevention from research at the molecular and cellular level6.
Haemoglobin E reaches very high frequencies throughout Bangladesh,
Burma, and in many countries in southeast Asia. Although its
homozygous state is characterised by a mild hypochromic anaemia,
because it is synthesised at a reduced rate, when inherited together with
P thalassaemia it often produces a crippling thalassaemic disorder. This
condition, haemoglobin E/p thalassaemia, is, globally, the commonest
serious inherited disorder of haemoglobin7.
The P thalassaemias, which result from over 150 different mutations
of the P globin gene, occur commonly throughout the Mediterranean
region, in parts of Africa, throughout the Middle East and the Indian
sub-continent, and in many regions of southeast Asia5. Together with
haemoglobin E/p thalassaemia, they constitute an increasing public
health problem now that childhood mortality rates due to malnutrition
and infection are falling and affected children are living long enough to
require treatment7. The a thalassaemias are also very heterogeneous at
the molecular level, and the severe forms, or a° thalassaemias, which
occur most frequently in the Mediterranean region and southeast Asia,
are associated with death in utero. The milder forms, the a+
thalassaemias, which occur frequently in many parts of sub-Saharan
Africa, throughout the Mediterranean region, the Middle East and
southeast Asia, are associated with mild hypochromic anaemias.
Through an understanding of the molecular pathology of these
conditions, better forms of treatment have been developed and it has
been possible to establish prenatal diagnosis programmes which have
led to a marked reduction in their frequency in many parts of the
Mediterranean region8. However, their control by carrier detection and
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Future role of molecular and cell biology
prenatal diagnosis is not yet established in many countries, and in some
may not be acceptable for religious and other ethical reasons. Thus the
better management of these diseases offers a major challenge to the field
of molecular and cell biology, and for medical practice in the tropics, for
the next millennium.
The haemoglobin disorders and malaria
Our new-found ability to identify different forms of thalassaemia at the
molecular level has recently re-opened up the question of whether these
diseases have reached their high frequencies because of heterozygote
protection against severe forms of malaria. While this was known to be
the case for sickle cell disease9, until very recently there was little
evidence that the same mechanism underlies the particularly high
frequency of the thalassaemias. Recent work has shown, however, that
the milder forms of a thalassaemia offer a highly significant level of
protection against Plasmodium falciparum malaria, though, remarkably,
affected babies during the few years of life seem to be more prone to
infection, by both P. vivax and P. falciparum10'11. This quite unexpected
observation, which highlights the value of direct DNA analysis for
genotyping large populations, offers a novel conceptual framework for
understanding how a* thalassaemia might protect against malaria11. It
appears that homozygotes are more susceptible to malaria, but only at a
time of development when the disease rarely kills. Thus, it is possible
that this may provide them with an immunising dose of malaria which
offers later protection. Interestingly, in areas in which both types of
malaria occur, the earliest infections in life are usually due to P. vivax.
Furthermore, there is some evidence that there may be crossimmunisation between the two common forms of malaria12. Thus the
fact that young babies with a thalassaemia are more susceptible to P.
vivax early in life may explain their later resistance to P. falciparum.
Other genetic polymorphisms and malaria
It is now clear that many other red cell disorders and polymorphisms
have been shaped by malaria. Again, it is research at the molecular level
which has enabled these issues to be clarified. Glucose-6-phosphate
dehydrogenase (G6PD) deficiency results from many different mutations
at the G6PD locus. Recent studies suggest that both female
heterozygotes and male hemizygotes have a reduced risk of malaria, in
the range of 50%13. Protection is also mediated by different red cell
surface antigens; the molecular basis for the absence of the Duffy
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Tropical medicine: achievements and prospects
antigen, observed many years ago to be associated with resistance to P.
vivax malaria, has now been identified as a substitution in the binding
site for the GATA1 erythroid transcription factor at position -156 to the
promoter14. By gene mapping analyses, the Melanesian form of
ovalocytosis, which is quite difficult to identify with certainty, has been
shown to provide complete protection against cerebral malaria (S.J.
Allen, personal communication).
Genetic variability due to selection by malaria is not confined to genes
that are expressed in red cells15. Certain polymorphisms of the HLA-DR
system are associated with protertion against both cerebral malaria and
severe malarial anaemia16. While earlier studies of associations of this type
were bedevilled by the possibility that the histocompatibility antigens
were simply acting as informative markers for other genes that might be
genuine immune response determinants, analyses at the molecular level
has shown quite clearly that these genes and their products are, in
themselves, immune response agents through determinant selection. For
example, studies of the HLA-B53 association with malaria have shown
how this might be mediated through the identification of HLA-B53
cytotoxic lymphocytes that identify a particular parasite epitope17.
Another important malaria-related polymorphism that has been
discovered recently involves the gene encoding tumour necrosis factor-a
(TNFa). Gambian children who are homozygous for a single base change
at position -308 of the promoter of the TNFa gene have a significantly
increased likelihood of dying from cerebral malaria18. In vitro reporter
gene studies suggest that this polymorphism can increase levels of TNF
expression, an observation that is compatible with evidence that excessive
TNF production is a major factor in the pathogenesis of cerebral malaria.
Another potentially important functional polymorphism related to
malarial infection which has been unearthed recently involves a member
of the immunoglobulin supergene family called ICAM-1. This protein
has a wide range of immunological functions and it has also been found
to have an important role in the adherence of P. falciparum-iniected red
cells in cerebral malaria. It has been found that, in parts of Africa, there
is a high prevalence of a polymorphism of this protein which appears to
be a predisposing factor for cerebral malaria19. It is possible that this
may have been selected for the role that it may play in other forms of
infection but that it is disadvantageous in the case of those due to P.
falciparutn.
Now that it is possible to study genetic disease and polymorphisms in
large populations at the DNA level, a remarkable picture of their pattern
of distribution is evolving. It is clear, for example, that in the case of a
and B thalassaemia, every population has its own particular mutations.
The sickle cell mutation occurs in Africa, the Mediterranean and India,
but not further east; haemoglobin E is found all over southeast Asia,
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Future role of molecular and cell biology
Burma and Bangladesh, but not further west. Melanesian ovalocytosis is
confined to Melanesia and adjacent regions. Different HLA-DR
polymorphisms appear to be protective in particular populations. It is
likely that these observations reflect the relatively recent appearance of
malaria as the principal agent that has maintained these polymorphisms,
a notion that is strengthened by the molecular analysis of the
relationship between globin gene polymorphisms and their associated
restriction fragment length polymorphism haplotypes9. Malaria must
have been a particularly powerful selective agent to have recruited so
many deleterious and other traits in such a short evolutionary time.
Genetic susceptibility to infections other than malaria
Although it has been suspected for a long time that individual responses
to infection may have a strong genetic basis, it is only with the advent of
the molecular era that it has been possible to investigate this important
possibility20*21. Until very recently studies in this field utilised the
'candidate gene' approach, that is research workers made an educated
guess about what type of gene might be involved in host-defence
mechanisms and then proceeded to test whether particular
polymorphisms are associated with different degrees of susceptibility.
One of the first candidates was the HLA-DR gene family; early studies
using serotyping have now been strengthened by an analysis of
polymorphisms of this system at the DNA level. Convincing HLA-DR
associations have been found with a variety of parasitic illnesses
including leishmaniasis, onchocerciasis and filariasis20. Strong
associations have also been demonstrated with either chronic carriage or
rates of viral clearance in patients infected with hepatitis B virus22-23.
The candidate gene approach has also been valuable for studying
genetic variability in susceptibility to bacterial infection. As well as
malaria, TNF polymorphisms have been found to modify the course of
meningococcal meningitis, lepromatous leprosy and trachoma24"26.
Increased susceptibility to bacterial infections has also been related to
several polymorphisms of the gene for the mannose-binding protein
(MBP)27. It has long been known that the ability to secrete the soluble
form of ABO blood group antigens into saliva and other body fluids,
that is the secretor status, may be associated with variable susceptibility
to bacterial infection. Recently, the molecular basis for the non-secretor
phenotype has been found to be a nonsense allele at the gene locus
involved, Sec218. Preliminary studies suggest that this polymorphism
occurs widely as the basis for the non-secretor phenotype and, hence, it
should now be relatively easy to relate secretor status to varying
susceptibility to infection by studies at the DNA level.
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Important progress is also being made towards a better understanding
of individual susceptibility to viral disease using the candidate gene
approach. The relationship between HLA-DR polymorphisms and the
rates of clearance of hepatitis B virus has already been mentioned. The
puzzling observation that some patients who are regularly exposed to
the human immunodeficiency virus (HIV) do not become infected has
recently been explained, at least in some cases, at the molecular level29.
It turns out that CD4-positive T-cells of such individuals are highly
resistant in vivo to the entry of primary macrophage-tropic virus but are
regularly infected with transformed T-cell adapted viruses. These
individuals are homozygous for a defect in the Ckr-5 gene, which
encodes for the co-receptor for primary HTV isolates. The molecular
defect in this case appears to be a defective Crk-5 allele containing an
internal 32 basepair deletion.
Quite recently, a different approach has been used to search for genes
that may modulate response to infection. In this case, linkage analyses
have been carried out using DNA polymorphisms to pinpoint genes that
govern the intensity of infection by Schistosoma mansoni. Hitherto such
'blind' genome searches have been extremely difficult, but, with the
availability of a linkage map of most of the human genome, this approach
offers an extremely valuable way of finding disease-susceptibility genes. In
this case, a gene that modifies the degree of infection by S. mansoni was
found to lie on chromosome 5q31-q33, a region that encodes IL4, IL5 and
several other immunological mediators30. There is no doubt that this
powerful approach will be extremely valuable for identifying other genes
that modify susceptibility to infection.
Studies of the genomes of infectious organisms
The development of techniques for rapidly sequencing DNA, and thenautomation, are making it possible to sequence the entire genomes of
micro-organisms in a relatively short time. A full list of bacteria and
other agents that are being analysed in this way has been published
recently31.It is already clear that a knowledge of the primary DNA
sequence of an infectious organism can yield valuable information,
particularly the identification of different virulence genes. These studies
can be combined with assays for function. For example, it is possible to
define different gene transcripts at particular phases of infection. The
methodology for this type of analysis is developing rapidly; using
microchip technology it is feasible to study the output of literally
thousands of genes at different phases of infection. This provides a
valuable profile of which genes are likely to be of particular functional
importance with respect to virulence. This information has major
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Future role of molecular and cell biology
implications for strategies directed at the development of new antimicrobial, anti-viral and anti-parasitic agents.
Analysis of the genomes of pathogenic bacteria have other important
implications. These organisms face a particularly daunting task because
infections occur within a very short time during which they encounter a
wide variety of host polymorphisms, a range of immune mechanisms,
and environments which may already be modified by the administration
of anti-microbial drugs. It is now becoming clear that bacteria, and
probably other organisms, have a remarkable repertoire of different
genetic systems with which to tackle these problems32.
One mechanism of this kind, that is of considerable current interest, is
the generation of mutator alleles, genes or gene families which can lead
to an increase in mutation rate. Such alleles may predispose a wide
variety of different genes to potentially beneficial mutations, and may
hitchhike because the mutator allele and the selected alleles may be
linked, especially in asexual clonal populations. It is now clear that
viruses, bacteria and parasites may all have subsets of genes that are
excessively prone to mutation through a variety of molecular
mechanisms. These hypermutable genes encode surface molecules, such
as adhesins and invasins, which are intimately involved in interactions
with host molecules. Hence, populations of micro-organisms can use
these combinatorial systems rapidly to generate phenotypic variation
which can influence antigenicity, motility, chemotaxis, attachment, and
a variety of other qualities which may alter their virulence. The potential
molecular mechanisms for these remarkable adaptive changes, which are
not yet fully understood, have been summarised recently32. Clearly, these
genes offer another potential target for novel chemotherapeutic agents.
Diseases due to 'Westernisation'
As mentioned earlier, there is a remarkable variation between different
populations in their response to the diets and lifestyles of more affluent
Western societies. For example, there is currently an increasingly serious
world epidemic of type 2 diabetes, with or without associated obesity. In
some countries that have been exposed to high energy diets for the first
time, up to 70% of the population is affected. In the Pima Indians, this
form of diabetes is associated with gross obesity and an extremely high
frequency of gallstones, a constellation of disorders which has been
called 'the new world syndrome'. Although there is currently much
debate about the role of nature and nurture in the generation of the high
frequency of diabetes it seems very likely that genetic factors play an
important role; this form of diabetes shows an extremely high degree of
heritability in twin studies2.
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Tropical medicine: achievements and prospects
This remarkable susceptibility to diabetes as a response to high energy
diets may reflect what has been called a 'thrifty genotype'. The idea is
that, during the early dissemination of Homo sapiens, populations that
had to undergo long journeys and periods of dietetic deprivation may
have undergone intense selection for a variety of different genes involved
in more effective energy storage; it is just this genetic make-up that now
makes their distant progeny more likely to develop diabetes. Since there
are probably many ways in which this type of susceptibility might be
mediated, different genes may be involved in different populations. It
will be extremely productive, therefore, to try to identify them by DNAbased linkage studies. Because this form of diabetes is an important risk
factor for vascular disease, a better understanding of its pathogenesis is
of particular importance.
Diabetes is not the only common disease of Westernisation that is
being found at different frequencies in the emerging countries. There are
remarkable differences in the frequency of coronary artery disease and
hypertension which may also have a genetic basis. Similarly, it is
becoming clear that childhood respiratory disorders, particularly
asthma, also vary in frequency in a way which may reflect genetic as
well as environmental factors.
These common non-infectious diseases will play an increasingly
important role in determining the pattern of disease in the tropical
world. Since susceptibility to them may reflect the action of completely
different genes to those that are involved in Western societies, this aspect
of medicine in the tropics will be increasingly important in the future.
The tools of molecular genetics, particularly linkage analysis using DNA
polymorphisms to find the particular genes involved, will provide an
extremely valuable approach to this important problem (see also
Forrester, Cooper and Weatherall in this issue).
Diagnosis and therapy
The recombinant DNA era offers a wide variety of new possibilities for
the more accurate diagnosis and definitive treatment of many of the
current diseases of tropical climates as well as for the non-infectious
disorders that may gradually take their place.
Diagnostics
DNA-based technology offers a wide variety of new diagnostic
approaches to infectious disease3-4. The development of the polymerase
chain reaction (PCR), which makes it possible rapidly to amplify any
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Future role of molecular and cell biology
chosen piece of DNA from the genome of a micro-organism, has
provided a valuable new approach to the diagnosis of bacterial, viral
and parasitic illnesses. As the genes that are involved in the development
of resistance to antibiotics and other therapeutic agents are defined, this
approach is also providing valuable information about the emergence of
resistant strains of organisms in populations. PCR-based technology is
also being used for both carrier detection and prenatal diagnosis of
important genetic diseases in the tropics, particularly the
haemoglobinopathies. DNA can be obtained by chorion villus sampling
during the first trimester of pregnancy; the increasing use of this
approach in prenatal diagnosis programmes has already resulted in a
marked reduction in the number of cases of fJ thalassaemia in many
Mediterranean populations8. Currently, plans are being developed to
apply this technology more widely in the rural populations of southeast
Asia. For example, in Thailand it is hoped that it will be possible to
reduce the number of births of children with serious forms of
thalassaemia by approximately 30% over the next 20 years. Similarly, as
more is learnt about the genes involved in susceptibility to diseases, such
as type 2 diabetes, it should be possible to define individuals at
particularly high risk for developing these conditions in response to
changes in diet and lifestyle.
Prevention and treatment of tropical diseases
Recombinant DNA technology offers many different new approaches to
the development of vaccines33. A variety of methods are being explored.
One of the earliest successes was the development of a vaccine against
hepatitis B. In this case, a gene for an appropriate antigen was cloned in
yeast to produce a safe and effective vaccine. The advantage of this
technique is that the genetically engineered yeast cell contains
information for only a particular viral gene, and hence there is no
possibility that a complete virus or any other infectious agent could arise
during the production of the vaccine. In other cases, live recombinant
vectors are being used. More recently there has been considerable
interest in DNA immunisation. In this case, inoculation with plasmid
DNA vectors that encode immunogenic proteins appears to induce both
humoral and cell-mediated immune responses, which quite often seem
to provide protective immunity. Although this approach is still in its
infancy, and its overall efficacy and safety remain to be determined, in
the long-term it may offer a safer and cheaper alternative to more
conventional vaccines.
As more is learnt about the virulence genes of microorganisms, and
about_the__gattern of gene expression during infections, it should be
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Tropical medicine: achievements and prospects
possible to define specific targets for the development of new forms of
chemotherapy. In effect, therapeutics will move from traditional
medicinal chemistry to the era of designer drugs. There is also much
current interest in defining the different molecular mechanisms for drug
resistance. A variety of members of a ubiquitous family of membrane
transporter proteins have been identified as being major players in the
development of resistance to a wide variety of chemotherapeutic agents.
As more is learnt about how drug resistance is mediated, through
changes in the genes that encode for these proteins, it should be possible
to design drugs to by-pass these resistance pathways.
Postscript
It has only been possible to outline a few of the applications of
molecular and cell biology to medicine in the tropics. But enough has
been achieved already to make it clear that this field has enormous
possibilities for improving the health of the populations of the emerging
countries. Currently, this is a complex research field, the fruits of which
are expensive. International health agencies and the governments and
pharmaceutical industries of richer countries will have to recognise its
importance for world health in the future, and ensure that its
applications for the diseases of the emerging countries are not neglected.
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