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α -DEFENSINS EXPRESSION IN PERIPHERAL BLOOD MONONUCLEAR CELLS
FROM PATIENTS WITH HEPATITIS C VIRUS INFECTIONS
Dottorando: DANIELA FIOCCO
Dottorato di Ricerca in Biochimica- XVII Ciclo
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Università degli Studi di Roma “La Sapienza”
Coordinatore: Prof. PAOLO SARTI
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Università degli Studi di Roma “La Sapienza”
Docente guida: Prof. DONATELLA BARRA
Dipartimento di Scienze Biochimiche “A. Rossi Fanelli”
Università degli Studi di Roma “La Sapienza”
Docenti esaminatori:
Prof. MAURIZIO PACI - Dip. di Scienze e Tecnologie Chimiche
Università di Roma “Tor Vergata”
Prof. GIOVANNI ANTONINI - Dip. di Biologia
Università di Roma 3
Prof. NAZZARENO CAPITANIO - Dip. Scienze Biomediche
Università di Foggia
ABSTRACT
Hepatitis C virus (HCV) is the major causative agent of chronic liver diseases. Some reports
indicate that, besides hepatocytes, the virus can also infect lymphocytes. HCV core protein
(HCV C) was recently reported to activate IL-2 gene transcription through the NFAT
pathway. α-Defensins (HNPs) are antimicrobial peptides representing key components of the
innate immune system in humans. The presence of an NFAT putative binding site in the
HNPs promoter led us to investigate whether HCV infection could affect the expression of
HNPs. HNPs expression, in peripheral blood mononuclear cells (PBMC) from HCV-infected
patients, was studied and found significantly higher than that of healthy controls; a strong
correlation was observed between HNPs level and liver fibrosis. By monitoring for some
months patients undergoing INF-α- therapy, a drop in HNPs expression level was found. In
vitro incubation of PBMC with recombinant HCV C, triggered HNP expression, while no
induction was detected when stimulating with HBV core antigen; moreover, transcriptional
activation by HCV C was reduced by pre-incubation with an inhibitor of NFAT cascade. Our
data suggest that HCV might directly or indirectly affect HNPs expression, possibly through
the participation of NFAT; furthermore, our results support the hypothesis that HNPs might
be involved in HCV pathogenesis.
i
INDEX
1
1.1
1.1.1
1.5
1.6
1.6.1
1.6.2
1.6.3
1.6.4
INTRODUCTION……………………………………………………… 1
Antimicrobial peptides…………………………………………………… 1
Structural features and classification
of antimicrobial peptides………………………………………………….1
Mechanism of action of antimicrobial peptides………………………….. 3
Innate immunity………………………………………………………….. 5
The role of antimicrobial peptides in innate immunity…………………... 6
Human antimicrobial peptides: defensins and cathelicidins……………... 7
Defensins: structure and tissue distribution……………………………… 7
Defensin genes and regulation of their expression…………………….. 10
Activity of human defensins…………………………………………... 11
Cathelicidins…………………………………………………………... 12
Roles of defensins and cathelicidins
in the human immune system…………………………………………. 13
NFAT………………………………………………………………….. 13
Hepatitis C virus………………………………………………………. 16
HCV structure…………………………………………………………. 17
HCV diagnosis and therapy…………………………………………… 17
HCV pathogenesis…………………………………………………….. 18
HCV core protein……………………………………………………… 19
2
AIMS OF THE RESEARCH………………………………………... 21
3
3.1
3.1.1
MATERIAL AND METHODS……………………………………… 22
MATERIALS…………………………………………………………. 22
Patients. Study population: epidemiologic data,
biochemical and virological measurements…………………………... 22
Microorganisms……………………………………………………….. 24
Cells……………………………………………………………………. 24
Growth media …………………………………………………………. 24
Drugs and solutions…………………………………………………… 24
Oligonucleotides for RT-PCR…………………………………………. 26
METHODS …………………………………………………………… 27
PBMC preparation…………………………………………………….. 27
Granulocytes preparation ……………………………………………… 27
In vitro treatment of PBMC……………………………………………. 27
Peptide extraction……………………………………………………….28
Antibacterial activity…………………………………………………... 28
ELISA …………………………………………………………………. 28
Mass spectrometry……………………………………………………... 29
RNA preparation………………………………………………………. 29
Purification of DNA fragments from agarose gel……………………... 31
Quantitative real time RT-PCR ……………………………………….. 31
Statistical analysis…………………………………………………….. 35
1.1.2
1.2
1.3
1.4
1.4.1
1.4.2
1.4.3
1.4.4
1.4.5
3.1.2
3.1.3
3.1.4
3.1.5
3.1.6
3.2
3.2.1
3.2.2
3.2.3
3.2.4
3.2.5
3.2.6
3.2.7
3.2.8
3.2.9
3.2.10
3.2.11
4
4.1
RESULTS AND DISCUSSION …………………………………….. 36
Expression profiling of HNPs in HCV patients……………………….. 36
Quantitative real time reverse transcription PCR (qRT-PCR):
validation of the housekeeping genes ………………………………... 36
ii
Quantitative real time RT-PCR analysis of HNP genes ………………. 39
ELISA ………………………………………………………………….. 41
Antibacterial activity ………………………………………………….. 42
MALDI ToF analysis …………………………………………………. 43
4.2
4.3
4.8.
HNPs expression in other hepatic pathologies …………………………44
Expression profiles of other immune-related genes
in HCV-patients ……………………………………………………….. 46
HNPs expression in HIV patients ……………………………………... 48
HNPs expression in selected cell subsets ………………………………49
Correlation between HNP level and liver damage ……………………. 50
In vitro experiments on PBMC ………………………………………... 52
Effect of HCV C, cyclosporin A and the core protein
of hepatitis B virus …………………………………………………… 52
Effect of phorbol 12-myristate 13-acetate, ionomycin
and phytohemagglutinin ……………………………………………... 54
Selectivity of HCV C induction ……………………………………….. 56
HNP expression profile in HCV patients under antiviral therapy ……. 56
5
CONCLUSIONS …………………………………………………….. 58
6
ACKNOWLEDGMENTS …………………………………………... 60
7
REFERENCES ………………………………………………………. 61
8
PUBLICATIONS ……………………………………………………. 69
4.4
4.5
4.6
4.7
iii
1 INTRODUCTION
1.1 Antimicrobial peptides
For the survival of an organism, an efficient defence for protection against
microorganisms is primarily important. Antimicrobial peptides (AMPs) are essential effector
molecules of the natural defence system of most living organisms. AMPs are extraordinarily
widespread in nature, as they have been found in all forms of life, including bacteria, plants
and animals, both vertebrates and invertebrates. Therefore, they constitute one of the most
conserved theme in nature’s struggle to control pathogens.
Antimicrobial peptides are gene encoded molecules of 12-50 amino acid residues, that are
produced by the host organism and have the capacity to kill microbes. Although it represents
an ancient defence mechanism, the importance of the production of gene-encoded peptides in
the animal defence was recognised only recently. In fact, most known AMPs have been
identified and characterized during the past 20 years. Insect cecropins, mammalian defensins
and frog magainins were among the first antimicrobial peptides to be thoroughly
characterized and clearly related to bacteria-induced immunity in animals (Boman and
Hultmark, 1987; Selsted et al., 1985; Zasloff, 1987). Since then, a large number of
antimicrobial peptides have been identified in many different organisms, so that they are
currently counted in the hundreds.
By studying AMPs, insight has been gained into fundamental host defence systems and
into mechanisms of membrane-protein interaction. The interest in AMPs has been growing
even due to the exciting perspective to develop new antimicrobial agents, in face of the
declining efficacy of conventional antibiotics (Hancock and Leher, 1998).
1.1.1 Structural features and classification of antimicrobial peptides
While classical antibiotics are produced by microorganisms through a series of reactions
catalysed by different enzymes, AMPs are gene-encoded and hence ribosomally synthesised
from an RNA template. AMPs are usually synthesized as inactive, larger, precursor
molecules, which are then processed by specific proteases to release the mature, active
peptide. Most of the precursors consist of a signal sequence, for targeting to the endoplasmic
reticulum, and an anionic pro-region masking the activity of the peptide until it is secreted
(Hancock, 1997).
AMPs are typically composed of 12 to 50 amino acids and have molecular masses around
5 kDa. Although the AMPs vary considerably in length, sequence and secondary structure,
some common structural features can be recognized:
- they are cationic at physiological pH values, since they possess amino acids, such as
arginine and lysine, which are positively charged at neutral pH;
- they can fold in such a way as to have both a hydrophobic and a hydrophilic face of polar
and positively charged residues. This amphipatic nature is presumably the key to their
biological activity, enabling them to associate with the negatively charged phospholipids
of the bacterial membranes and disrupt normal membrane functions.
According to their chemical characteristics and 3D structure, AMPs can be classified into
three major groups (Boman, 1995):
1) linear peptides without cysteines, forming amphipatic helices;
2) linear peptides with a high proportion of one or more residues (often proline, arginine
or tryptophan);
3) peptides with an even number of cysteines intralinked by disulfide bonds; this class
can be further divided into a sub-group of peptides with one disulfide bond only and
1
α-helix loop structure, and a larger sub-group characterized by two or more disulfide
bonds and a predominant β–sheet structure.
Certain AMPs undergo post-translational modifications such as C-terminal amidation
(insect cecropins, amphibian temporins or bombinins), glycosylation (drosocin), amino acid
isomerization (D amino acids-containing peptides from amphibia), etc. These modifications
are likely to influence the biological properties of the mature peptide.
Table 1.1 outlines structure, biological source and activity of some common antimicrobial
peptides.
Table 1.1.
Structural class, source and activity of representative antimicrobial peptides
STRUCTURE
Linear,
cysteine-free
α-helix
PEPTIDE
cecropins
dermaseptins
magainins
temporins
bombinins
LL37
Linear with
high content
of certain
residues
1 disulfide
bond, cyclic
apidaecins
drosocin
PR-39
indolicidin
Bac -5, Bac-7
brevinins
esculentins
2 or more
α -defensins
disulfides
with
predominant
β-defensins
β–sheet
structure
defensins
θ-defensins
SOURCE
insect hemolymph
REFERENCE
Boman and Hultmark,
1987
frog skin
antibacterial and Mor A, et al., 1991
antifungal
Xenopus laevis skin antibacterial
Zasloff, 1987
Moore et al., 1991
and stomach
Rana species skin
antibacterial
Simmaco et al., 1996
Bombina species skin antibacterial
Simmaco et al., 1991
Human leukocytes
antibacterial,
Agerberth et al., 1995
chemotactic
and skin
bee hemolymph
antibacterial
Casteels et al., 1989
Drosophila
antibacterial
Bulet et al., 1993
hemolymph
Pig intestine
antibacterial
Agerberth et al., 1991
Bovine neutrophils
antibacterial
Selsted et al., 1992
Mammalian
antibacterial
Frank et al., 1990
leukocytes
Rana species skin
antibacterial,
Morikawa et al., 1992
hemolytic
Rana species skin
antibacterial,
Simmaco et al., 1994
antifungal
Mammalian
antibacterial,
Selsted et al., 1983; 1985
phagocytes, mouse
antifungal,
and human intestinal antiviral,
Paneth cells
cytotoxic
Mammalian epithelia, antibacterial,
Selsted et al., 1993
bovine neutrophils,
chemotactic
chicken leukocytes
Insect hemolymph,
antibacterial
Hoffmann and Hetru,
plant tissue
1992, Brokaert et al.,
1995
Monkey leukocytes
antiviral
Tang et al., 1999
2
ACTIVITY
antibacterial
1.1.2 Mechanism of action of antimicrobial peptides
AMPs usually have broad activity spectra and may kill both Gram-negative and Grampositive bacteria, as well as fungi. Some AMPs have also antiviral activity and cytotoxic
effects on eukaryotic cells. Susceptible microorganisms are killed in vitro by AMPs at
minimum inhibitory concentration (MIC) in the micromolar range (0.5-8 µg/mL). The killing
rate is usually much faster than that observed with conventional antibiotics; moreover, AMPs
do not induce resistant mutants so frequently as the conventional antibiotics do. Both the
above characteristics may be ascribed to their physical mechanism of action: most of the
AMPs appear to function as membrane-active agents disturbing its permeability and causing
its disgregation (Hancock et al., 1995)
Different approaches in in vitro studies have helped in elucidating the mode of action of
the cationic antimicrobial peptides, including the well studied peptides melittin, magainin,
cecropin and defensin (Christensen et al. 1988; Agawa et al., 1991; Kagan et al., 1990;
Matsuzaki et al., 1989). The use of convenient assay bacteria or common animal pathogens
have defined the spectra of action, while experiments on artificial membranes, lipid vesicles
and different spectroscopic methods, as well as the use of synthetic peptides, have been
applied to understand the lytic mechanism of the antibacterial peptides. These studies support
the formation of channels in some cases, and a general collapse of the membrane in other. By
contrast to the detailed and numerous in vitro studies, there are only a few in vivo experiments
to test the biological function of AMPs. Therefore, it is likely that the gap of complexity
between an artificial membrane and the envelope of a bacterium is too large to provide an
exhaustive, definitive conclusion about the mechanism of lysis occurring in vivo.
As a result of the extensive in vitro studies, a general mechanism of action can be outlined
(Hancock, 1997; Shai, 2002). The positively charged AMPs interact first with the negatively
charged sites on lipopolysaccharides (LPS), on the surface of Gram-negative bacteria, and are
subsequently taken up through a process called “self-promoted uptake”: the affected
membrane develops transient “cracks” allowing passage of a variety of molecules, including
the perturbing peptide itself. The killing event for both Gram-positive and Gram-negative
bacteria is then the chemical attack on the cytoplasmic (inner) membrane (Fig. 1.1): the
peptides interact with the negatively charged membranes and, as a consequence of this
interaction and of the high electrical potential of the membrane, they re-orient and undergo a
transition from an unstructured to a structured form. Thus, they aggregate into clusters,
directing their hydrophobic faces towards the membrane core, while pointing their
hydrophilic faces either inwards, to form channels, or towards the phospholipid headgroups of
the membrane. In both cases, either by the formation of transmembrane pores (barrel-stave
model) or by a general, progressive destabilization of the membrane structure (carpet model),
membrane collapses, and the free ions flux dissipates its potential causing cell lysis and death.
3
CARPET
MODEL
BARRELSTAVE
MODEL
Fig. 1.1. Schematic mechanism of interaction between the bacterial membrane and
antimicrobial peptides; hydrophilic faces are in red.
The selectivity for microbial cells compared to host cells is favoured by: i) the unique high
content of surface anionic lipids, ii) the large transmembrane potentials and iii) the lack of
cholesterol, that are all peculiar characteristics of the bacterial membrane.
This mechanism of action explains some of the clinically desirable properties of AMPs.
First of all, the difficult selection of mutant resistant strains: unlike classical antibiotics, which
have a specific single target (e.g. a key enzyme for the metabolism of the pathogen), AMPs
are directed against a fundamental, conserved structure as complex as the cytoplasmic
membrane is, and, therefore, only simultaneous, multiple genes mutations in the bacterial
genome could prevent their destructive action. Moreover, while most of the traditional
antibiotics promote endotoxinemia by releasing LPS (endotoxins) during cell killing, some
AMPs can bind to LPS, hence neutralizing their toxic effects for the host organism (Gough et
al., 1996). The protective mechanism appears to be the binding of LPS in such a way that it
fails to induce tumour necrosis factor. This protective role confers to these peptides a great
advantage over other antimicrobial agents. Another intriguing property of the AMPs is that
they can enhance the activity of other antibiotics, facilitating their penetration into the target
cell in a synergic effect.
4
1.2 Innate immunity
Metazoans have evolved two basic host defence mechanisms against invading
microorganisms: the innate and adaptive immune systems. Innate, non adaptive immunity
represents a first line of defence and involves both a cellular response by specialized blood
cells and the rapid synthesis of proteins with a wide range of activities (opsonisation of
microbes, inhibition of proteases, intercellular signalling or direct antimicrobial action)
(Medzihtof and Janeway, 1997).
Whereas the innate immune response is present in all classes of the animal kingdom,
adaptive immunity is restricted to gnatostome vertebrates. Distinguishing features of each
type of immunity are given in Table 1.2. Summarizing, the innate immune system relies on a
limited number of receptors, able to recognize the vast variety of the molecular structures
associated with pathogens, and on defensive mechanisms that can counteract the broadest
spectrum of potential pathogens. Thereby, the innate immunity provides a selective but not
highly specific recognition system and a rapid initial response. By contrast, the adaptive
immunity lacks both rapidity and broader activity, while gaining extraordinary efficiency and
specificity.
From an evolutionary point of view, the innate immune system has probably predated the
adaptive immune response: first, innate host defences are found in all multicellular organisms,
while adaptive immunity is found only in higher vertebrates; second, the innate recognition
systems distinguish self from non-self perfectly; third, the innate immunity uses receptors and
signalling pathways which are strikingly conserved throughout the animal kingdom and seem
rather ancient in their lineage.
Table 1.2. Main characteristics of innate and adaptive immunity
Type of
response
Mechanisms
of
recognition
Distribution
INNATE IMMUNITY
Antigen independent
Not antigen specific
Immediate and maximal
Exposure results in no
immunologic memory
Pathogen recognized by receptors
encoded in the germ line
Receptors have broad specificity:
recognize many related molecular
structures called PAMPs
(pathogen-associated molecular
patterns)
PAMPs are essential
polysaccharides and
polynucleotides that differ little
from one pathogen to another, but
are not found in the host
Receptors are PRR (pattern
recognition receptor), such as the
Toll-like receptor family
In all metazoan
5
ADAPTIVE IMMNUNITY
Antigen-dependent
Antigen specific
Slow (because clones of responding cells
need to develop)
Exposure results in immunologic memory
Pathogen recognized by receptors generated
randomly by somatic rearrangement of
genes
Receptors have narrow specificity;
recognize a particular epytope
Most epytopes are derived from
polypeptides and reflect the individuality of
the pathogen
Receptors are B-cell (BCR) and T-cell
(TCR) receptors for antigen
In jawed vertebrates only
The innate immune response is based on all the pre-existing (pre-encoded) anti-infection tools
of the host. These include anatomical and physiological barriers, chemical factors and cellular
components, as summarised in Table 1.3.
Table 1.3. Defence tools of the innate immunity
Anatomical barriers
Physiological
barriers
Chemical factors
Cellular
components
skin, saliva, tears, intestinal movements, oscillation of
broncho-pulmonary cilia, etc.
body temperature and pH values which may inhibit microbial
growth
lysozyme; cytokines (interferons and TNF); complement;
transferrin and lactoferrin; peptide antibiotics, surfactants
proteins A and D, fibronectin, lectins, etc.
neutrophils, macrophages, monocytes, NK cells
The chemical factors, mostly secretory molecules, play a fundamental protective role:
lysozyme, in serum and tears, breaks down the bacterial cell walls; cytokines such as
interferons are secreted by virus-infected cells and prevent further viral replication; tumour
necrosis factor alpha (TNF-α) suppresses viral replication and triggers phagocytes; activated
complement is a group of serum proteins that participate in an enzymatic cascade, whose
ultimate goal is damaging the membrane of the pathogen; transferrin and lactoferrin deprive
invading organism of iron; antimicrobial compounds, such as peptide antibiotics, directly
damage microbes membrane, and surfactants proteins A and D enhance phagocytosis; lectins
can bind sugar molecules on the cell surface making them sticky and causing them to clump.
Also cellular components greatly contribute to host protection: neutrophils and macrophages
do so in virtue of their chemotactic and phagocytic capacity.
Innate immunity has long been considered as a separate entity from the adaptive immune
response and has been regarded to be of secondary importance in the hierarchy of immune
functions. In the past years, however, interest in innate immunity has grown considerably.
Recent studies demonstrate that the two systems are intimately correlated: the innate system
controls the initiation of the adaptive immune response and instructs it to develop a particular
effector response (Fearon and Locksley, 1996). The function of lymphocytes bearing clonally
rearranged receptors is dependent on signals provided by the innate recognition system
(Romagnani, 1992). Thus, innate and adaptive immune responses are integrated as a single
immune system, with the innate response preceding, and being necessary for, the adaptive
immune response.
1.3 The role of antimicrobial peptides in innate immunity
In animals, AMPs have been identified in several physiological contexts: secreted onto
mucosal surfaces and skin (e.g. human epithelial defensins and amphibian skin bioactive
peptides) or into internal body fluids (e.g. cecropins in the haemolymph of insects);
accumulated inside circulating fagocytic cells (e.g. the human neutrophil defensins). Both
their powerful antimicrobial properties and their “strategic” location in specialised immune
cells, or at the interface, between the organism and its environment, provide an indirect but
strong evidence that AMPs participate in resistance to pathogen attack. Moreover, the
induction of their expression in response to bacterial infection and tissue injury, highlights
their central role in what is generally indicated as the innate immune system.
Because host defence mechanisms are multiple and complex, it is difficult to demonstrate
the contribution of any single peptide or mechanism. Nevertheless, a compelling experimental
6
cue of the physiological importance of AMPs has come from studies on cystic fibrosis: the
salt inactivation of the airway epithelial AMPs (β-defensins) determines colonization and,
sometimes, fatal infections, by Pseudomonas aeruginosa, which are characteristic in patients
affected by this genetic disease (Goldman et al., 1997). More recently, a strong association
was shown between oral diseases and lack of measurable AMPs (LL37) in the saliva of
patients with Kostmann syndrome, a severe congenital neutropenia (Putsep et al., 2002).
According to Hans G. Boman (1996, 2003), AMPs represent an ideal first line of defence
and can be considered key effector molecules in innate immunity, particularly in mounting an
early and immediate response against invading micro-organisms. AMPs production is much
faster than that of immunoglobulins and this feature is not negligible when considering that
the growth rate of most microorganisms enormously overcomes that of a typical animal
lymphocyte. The adaptive immune system alone would not be able to efficiently counteract a
microbial infection. A peptide-based defence is not only necessary, it is easy to control and is
more energy- and information-saving, when compared to a clonally-based defence.
All these considerations underline the great survival value of peptide antibiotics in the
immune system of animals.
Innate immunity and, specifically, AMPs probably play a major role even in controlling
the natural symbiotic flora. In animals, including humans, normal flora of bacteria exists on
the skin, in the mouth, in the gastro-intestinal tract and in the airways, as well as in part of the
reproductive organs. New bacteria continuously colonize these organs, yet microbial
populations are kept in a quasi-steady state. It is likely that the control of the natural flora is
due to the continuative action of peptide antibiotics, rather than to the secretion of
immunoglobulins.
1.4 Human antimicrobial peptides: defensins and cathelicidins
In humans and other mammals, the two main antimicrobial peptide families are defensins
and cathelicidins. Although structurally and evolutionarily distinct, these two families are
similar for distribution, abundance and function, both classes being implied in antimicrobial
activity of phagocytes, inflammatory body fluids and epithelial secretions.
1.4.1 Defensins: structure and tissue distribution
Among all the AMPs, defensins are particularly prominent in humans, as evidenced by the
large number of expressed genes, the various forms present in human tissues and their
occurrence in inflamed or infected tissues (Lehrer, 2004). After their original isolation from
mammalian leukocytes (Lehrer et al., 1983; Ganz et al., 1985), defensins were also found to
be produced by various epithelial cells (Ouelette and Selsted, 1996; Diamond et al., 1991).
Besides mammals, distantly related defensins have been found also in insects and plants
(Hoffmann and Hetru , 1992; Brokaert et al., 1995)
Defensins are arginine-rich cationic peptides characterized by a β-sheet fold and a
framework of six disulfide-linked cysteines. The two main defensin subfamilies are α and βdefensins, differing in the spacing and connectivity of their cysteine residues, which are
typically conserved among members of each subfamily. α-Defensins comprise the group of
human neutrophil peptides (HNP-1 to 4) and human defensins 5 and 6 (HD-5 and HD-6). βDefensins include the group of human β-defensins (HBD-1 to 4). Their sequences are
reported in Fig. 1.2.
7
α-defensins
HNP-1
HNP-2
HNP-3
HD-5
HD-6
HNP-4
ACYCRIPACIAGERRYGTCIYQGRLWAFCC
CYCRIPACIAGERRYGTCIYQGRLWAFCC
DCYCRIPACIAGERRYGTCIYQGRLWAFCC
ARATCYCRTGRCATRESLSGVCEISGRLYRLCCR
RAFTCHCRR-SCYSTEYSYGTCTVMGN-HRFCCL
VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRV
* **
*
*
* *
*
**
30
29
30
34
32
33
β-defensins
HBD-1
DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK
HBD-2 GGIG--DPVTCLKSGAICKPVFCPRRYKQIGTCGLPGTKCCKKP
HBD-3 GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK
HBD-4 RSEFELDRICGIGTARCR-KKCRSQEYRIGRCPN-TYACCLRKPWDESLLNRTK
*
. *
*
* *
**
36
42
45
52
Fig. 1.2. Sequence alignment and cysteine bonding of human α- and β-defensins. Conserved residues
in each subgroups are indicated by asterisks. Underlined amino acids may or may not be
part of mature peptides. Gaps (-) were inserted to maximize the identities. The number of
residues is indicated for each peptide.
8
α-Defensins are synthesized as 90-100 amino acids-long pre-pro-peptide precursors,
consisting of an N-terminal signal sequence and an anionic propiece followed by the mature
peptide at the C-terminus (~ 30 amino acids); in many cases, the charge of the propiece
counterbalances that of the mature peptide, and this might be relevant both for proper folding
and for preventing intracellular damage to cell membranes. β-Defensins are longer (~
40 aa), with a stronger cationic nature, and their precursor has a short or absent propiece.
As determined by X-ray-crystallography and 2D NMR (Hill et al., 1991), both α and βdefensins consist of a triple stranded β-sheet structure (Fig. 1.3).
Fig. 1.3. Structure of HNP-3 and HBD-3. The essential structure is a triple-stranded β-sheet.
Cysteine disulfides are in orange, basic and acidic regions are blue and red,
respectively.
In mammals, the pattern of defensin tissue distribution is species-dependent, and varies
considerably even when comparing closely related species.
The main sites of α-defensins expression in humans are neutrophils and intestinal Paneth
cells.
- The primary (azurophil) granules of neutrophils contain high amounts of four distinct αdefensins also referred to as human neutrophil peptides (HNP-1 to 4). The HNPs are
stored as fully processed mature peptides (~ 3 kDa). They constitute 30-50% of the total
proteins of these organelles, greatly contributing to the oxygen-independent killing of
invading pathogens. HNP-1, 2 and 3 are the most abundant, while HNP-4 concentration
is about 100-fold lower.
- The Paneth cells of the small intestinal crypts express the human α-defensins HD-5 and 6,
and store them in specialized secretory granules as pro-peptides. Homologous defensins
(cryptidins) are expressed also in the mouse intestine. The presence of AMPs in the
9
gastrointestinal tracts of insects, amphibians and mammals supports the hypothesis that
the expression of epithelial defensins serves to limit the proliferation of intraluminal
flora.
α-Defensins are expressed also in some epithelia, such as that of the female uro-genital
tract and of the nasal and bronchial airways; sensitive analytical techniques have
demonstrated the expression of HNP-1 and 3 also by monocytes, macrophages, B and NKcells (Agerberth et al., 2000).
Human β-defensins (HBD) have been isolated from many cell types, mainly epithelial,
confirming that, in addition to many other biological functions, these cells actively participate
in host defence, not simply constituting a barrier for this purpose. So far four β-defensins
(HBD-1 to 4) have been described, but genomic analyses have recently found additional βdefensin genes, whose corresponding peptide products have yet to be characterized. HBD-1 is
mainly expressed in the epidermis and in the epithelia of pancreas, kidney and urinary tract;
HBD-2 and 3 are found in the skin and in airway epithelia; HBD-4 is expressed in testis,
stomach, lung and neutrophils. HBD-2 is stored as mature peptide in the lamellar bodies of
the skin cells.
1.4.2 Defensin genes and regulation of their expression
Since polymorphisms in copy number has been observed for both HNP and HBD genes,
the number of human defensin genes cannot be exactly defined; the main cluster of α and βdefensin genes maps on chromosome 8p22-23, with other minor clusters, more recently
characterized, in other genomic regions (Liu et al., 1997; Schutte et al., 2003). The structural
organization of defensin genes and the close proximity of α and β-defensin genes in the main
cluster suggest a common evolutionary origin of the two defensin families. From a common
ancestral gene, events of genomic duplication end insertions have led to a multiplicity of
defensins, differing markedly in amino acid sequence, net charge and quaternary structure,
capable to cope successfully with different types of pathogens.
Despite defensin genes share a common ancestor, control mechanisms and pattern of their
expression differ markedly. As a general rule, α-defensins are expressed constitutively and
their synthesis is closely linked to cell differentiation processes, whereas most β-defensins are
inducible and their synthesis seems to be triggered by infectious signals.
α-Defensins
HNPs are synthesized constitutively by the bone marrow precursors, during specific
differentiation stages of neutrophil development: transcription of the genes is likely to be
massive and brief in the stage of granulocyte precursor, while little or no transcription is
observed at the stage of mature neutrophil. The promoter region of HNPs appears well
conserved and contains transcription factor binding sites, such as Ets-like elements, C/EPB
and c-Myb sites, which are also involved in the transcriptional regulation of many myeloidspecific genes. These sites resulted essential for transcription of HNP genes in the HL60
myeloid cell line (Ma et al., 1998; Tsutsumi-Ishii et al., 2000). Having completed their
maturation, and having accumulated in their granules high and definite levels of HNPs,
together with several other antimicrobial proteins (including lactoferrin, bactericidal
permeability ìncreasing protein, BPI, lysozyme, etc), neutrophils are released into the blood
and enter the tissues.
Plasma concentration of HNP-1 and 3 was found significantly higher during severe
bacterial infections (Ihi et al., 1997): this increased level might reflect an enhanced neutrophil
activation at sites of infection. It even raises the possibility that HNP biosynthesis might be
stimulated in response to infections, by means of signalling pathways as yet unknown,
10
therefore implying a mode of expression for HNP genes that is not only constitutive. Recent
findings support that peripheral leukocytes retain substantial ability to transcribe HNP1-3
genes, although the expression level was not affected by ex vivo treatment with bacterial
components such as LPS (Fang et al., 2003).
Paneth cell α-defensin genes, similarly to the hematopoietic defensins, have a constitutive
level of transcription that is part of a developmental program of the highly differentiated cells
in which they are expressed. Nonetheless, the presence of several nuclear factor interleukin-6
(NF-IL6) recognition sites, in the promoter of both HD-5 and 6, does not rule out that these
genes might be up-regulated in response to inflammatory stimuli (Mallow et al., 1996). Once
accumulated in specialized granules, the enteric defensins are secreted into the lumen in
response to bacteria by a process involving receptors and transduction pathways that have not
been characterized yet.
β-Defensins
HBD genes are characterized by both constitutive and inducible expression.
HBD-1 gene promoter contains NF-IL6 and γ-interferon consensus sites; HBD-2 gene has
several nuclear factor-κB (NF-κB) and NF-IL6 consensus sites. The presence of these
regulatory cis-elements is consistent with the inducibility of these genes, either directly by the
infection or by the production of inflammatory cytokines.
HBD-1 seems to be constitutively synthesized by many epithelial cells, whereas HBD-2
and 3 are expressed when epithelial tissues are stimulated with bacteria. HBD-2 gene
transcription in the skin is induced by bacterial contact through the intermediate synthesis of
interleukin-1 (IL-1) by myeloid cells (Liu et al., 2003). Transcriptional activation of HBD-2
gene in airway epithelia results upon treating with bacterial molecules such as lipoteichoic
acids (LTA) and is mediated by an epithelial expressed toll like receptor (TLR) that
recognizes components of the Gram-positive bacteria, thereby activating the NF-κB pathway
(Wang et al., 2003). NF-κB binding sites in the promoter region of HBD-2 gene, confer
responsiveness to both LTA and proinflammatory cytokines such as IL-1. Transcription of
HBD-1 and 2 is also strongly induced in LPS-treated peripheral leukocytes (Fang et al.,
2003).
HBD-3 and 4 genes are regulated by NF-κB–independent mechanisms, that remain to be
characterized.
In bovine tracheal epithelial cells, LPS exposure or bacterial contact induce transcription
of a β-defensin, the tracheal antimicrobial peptide (TAP), and transcription is accompanied by
NF-κB activation and binding to the TAP promoter sequence. Involvement of the well known
transcription factors of the Rel/NF-κB family seems quite a conserved theme in the activation
of genes which are infection- and inflammation-induced, and not only in mammals: these
proteins, in facts, have been implied in the activation of several inducible AMP genes, in quite
a broad range of phylogenetically distant organisms, including insects, amphibians and
mammals (Boman, 1998).
1.4.3 Activity of human defensins
Most defensins have antimicrobial activity against Gram-positive, Gram-negative bacteria
and fungi especially when tested under low concentration of salts and plasma proteins. In
these conditions, the activity is in the low µM range (1-10 µg/mL). Antiviral properties have
been ascribed to HNPs: in vitro incubation with these defensins inactivate several types of
enveloped viruses such as cytomegalovirus, herpes simplex (HSV) 1 and 2, vesicular
stomatitis and influenza A virus (Daher et al., 1986).
HNPs inhibit also human immunodeficiency virus 1 (HIV-1) replication (Nakashima et
al., 1993). It has been recently suggested that HNPs could be major components of the CD8+
11
T cell antiviral factor (CAF) and contribute to its anti-HIV activity (Zhang et al., 2002). CAF
is thought to play an important role in HIV-non-progressor patients, that is HIV-infected
individuals, whose infection remains stable for a long time without developing the distinctive
symptoms of AIDS. The involvement of α-defensins in CAF activity has been argued and
finally ruled out, since no mRNAs for HNPs could be detected in highly purified CD8+ T
cells. Yet the anti-HIV property of HNPs remains undisputed and represents an intriguing
subject to understand how the immune system can prevent HIV infection.
Interestingly, members of the most recently identified defensin subfamily, the θ-defensins,
were found to possess a potent anti-HIV activity. These cyclic octadecapeptides have been
isolated from leukocytes of the rhesus monkey and seem to arise from post-translational
splicing of two α-defensin-like nonapeptides (Tang et al., 1999). The θ-defensins apparently
evolved in all the primates, but were subsequently lost in humans, due to mutations in the
corresponding and still functional genes. Artificially created human θ-defensins (named
“retrocyclins”) exert substantial anti-HIV activity (Cole and Lehrer, 2002). Phylogenetic
evidence indicates that loss of retrocyclin production might date back to the hominid stage:
this evolutionary loss could account for the HIV-1 susceptibility in modern humans.
Many diverse biological functions, other than a direct antimicrobial effect, have been
ascribed to mammalian and human defensins; most of them somehow contribute to host
defence system. These activities can be summarized as follows.
-
-
Both α- and β-defensins chemoattract several types of immune cells (monocytes,
dendritic cells, lymphocytes and cytokine-activated neutrophils), influencing and
enhancing antimicrobial immunity (Yang et al., 1999; 2000).
Some defensin stimulate and/or modify cytokines production (Yang et al., 2002).
HNPs interact with complement (van den Berg et al., 1998).
Some defensins, such as rabbit NP-3A, act as corticostatin by binding the ACHT receptor
(Zhu et al., 1989)
HNPs participate in wound repair, by enhancing extracellular matrix deposition, and
promote cell growth (Murphy et al., 1993; Oono et al., 2002).
HNPs can have cytotoxic effects and contribute to lysis of tumour cells (Lichtestein et al.,
1986).
Many α- and θ-defensins are capable of binding membrane glycoproteins: this lectin-like
behaviour could be important for their antiviral properties as well as for their antibacterial
activity, since polysaccharides are integral components of fungal and bacterial cell walls
(Wang et al., 2003).
1.4.4 Cathelicidins
Cathelicidins constitute a unique mammalian gene family. They are structurally organized
as an N-terminal signal peptide, a highly conserved prosequence - the cathelin domain, after
which the family takes its name - and a variable cationic peptide at the C-terminus (Zanetti et
al., 1995; Bals and Wilson, 2003) The conservation of the cathelin domain is striking between
species and indicates that the diverse members of this family evolved from a common
ancestor gene.
Unlike most mammals, in humans the cathelicidin family is restricted to a single gene
product, the LL37 peptide (elsewhere indicated as hCAP18). Both halves of the human
cathelicidin are active: the cathelin domain is antimicrobial and acts as a protease inhibitor;
LL37 peptide, characterized by an α-helical conformation, is active against Gram-positive and
Gram-negative bacteria.
Like defensins, also LL37 is a multifunctional AMP. It participates in host defence not
only by direct antimicrobial activity, but also by: i) recruiting cellular defence (LL37 is
12
chemotactic for monocytes, neutrophils and T cells) and ii) promoting wound healing and
tissue repair (LL37 stimulates angiogenesis).
LL37 was originally identified in specific granules of neutrophils, where it is stored as
propeptide, being processed and released upon appropriate stimulation; subsequently, it has
been found in various epithelial tissues (skin, gastrointestinal tract, airways, etc.), in different
body fluids, as well as in macrophages, B cells, NK cells, and γδ T cells.
The promoter region of the gene encoding LL37 contains several putative binding sites for
many transcription factors, including NF-κB and NF-IL6, acute-phase response factor and
C/EPB. All these sites suggest that transcription of LL37 gene may be regulated by infection
and inflammatory cytokines. LL37 is induced in inflamed skin and its serum concentration
rises during infection, whereas, in intestinal cells, its synthesis seems rather differentiationdependent. To date, little is known about constitutive or regulated expression of LL37 gene.
1.4.5 Roles of defensins and cathelicidins in the human immune system
The role of defensins and cathelicidins in innate immune system of mammals and humans
is being established by recent experiments on transgenic mice, but additional studies in these
and related models would be desirable to document the function of these AMPs in in vivo
contexts (Nizet et al.,2001; Morrison et al., 2002; Salzman et al., 2003). Nonetheless, there is
already an expanding body of experimental researches pointing to the relevance of defensins
and cathelicidins in human diseases: several clinical studies have begun to associate
alterations in AMP synthesis or function with human infectious diseases, inflammatory
syndromes, or immune deficiencies. AMP production increases in response to specific
infections and acute damage to the epithelial barrier (e.g. psoriasis, Helicobacter pylori
gastritis, etc.) in agreement with the expected protective role of these molecules. By contrast,
increased and dysregulated AMP synthesis is also associated with some chronic inflammatory
disorder (e.g. lung fibrosis) reflecting the dual function of AMPs in the immune system (Gallo
and Nizet, 2003).
1.5 NFAT
NFAT (nuclear factor of activated T cells) was originally described as a putative
transcription factor present in the nuclear extracts of activated Jurkat T cells that binds to
human interleukin-2 (IL-2) promoter (Shaw et al., 1988). The NFAT family contains at least
four major isoforms, named NFATC1-4, each of which exhibits a characteristic pattern of cell
type specificity.
Transcription factors of the NFAT family regulate the expression of several immunerelated genes, such as those encoding cytokines and their receptors, in response to antigenic
stimulation of cells of the immune system (Crabtree and Clipstone, 1994; Rao et al.,1997).
NFAT proteins are also involved in heart valve development, angiogenesis of peripheral
vessels, myogenesis and signalling mechanisms in the hippocampus (Horsley and Pavlath,
2002).
NFAT consists of two components: a pre-existing cytosolic component (NFATc), whose
activation is regulated by Ca2+ concentration and blocked by cyclosporin A (CsA), and a
nuclear component, NFATn, whose induction requires de novo protein synthesis upon T cell
stimulation. Both components are required for binding to the NFAT site, a purine-rich core
motif, (A/T)GGAAAA, with surrounding bases varying according to the specific nuclear
partner of NFATc.
NFATn is expressed under the control of the p21ras pathway (Woodrow et al., 1993);
members of the AP-1 (activator protein-1) family (such as Fra-1, Jun B, and c-Fos) have been
13
demonstrated to be able to functionally substitute for NFATn (Northrop et al., 1993). Recent
evidence indicates that several other proteins, such as the zinc finger proteins GATA4 and cMAF, can also cooperate with NFATc.
NFATc proteins share some cardinal functional and structural features:
- their cytoplasmic location;
- their induction via a peculiar calcium/calcineurin-activated pathway;
- their inhibition by clinically important immunosuppressive drugs CsA and FK506
(Mattila, et al. 1990);
- the presence of a conserved regulatory SP repeated motif and serine rich domain;
- the conserved Rel homology DNA-binding domain;
- a conserved calcineurin binding site.
The schematic structure of NFATc family members is depicted in Fig.1.4.
Rel-homology DNA-binding domain
NFAT homology region
SRR
Cn
NLS
Cn
NLS
SP-repeats motifs
Fig.1.4. Structural features of a typical NFATc protein. Cn, calcineurin binding site; SRR,
serine-rich region; NLS, nuclear localization signal.
The signalling pathway leading to NFAT activation has been mainly characterized in T
cells and is illustrated schematically in Fig. 1.5. A key step in NFAT activation is its nuclear
import: in resting T cells, NFAT is phosphorylated and resides in the cytoplasm; upon T cell
stimulation, which elicits a sustained rise of intracellular Ca2+ concentration, NFAT is
dephosphorylated by the Ca2+-activated Ser/Thr phosphatase calcineurin, which leads to its
nuclear import, likely as a result of the exposure of the nuclear localization signal (NLS).
Once accumulated in the nucleus, dephosphorylated NFATc recruits the nuclear partner,
NFATn, to form a complex on the ARRE-2 (antigen receptor response element) site of the IL2 promoter (Timmerman et al., 1996).
Thus, the Ca2+-dependent and p21ras-dependent pathways, that emanate in parallel from
the T cell receptor upon stimulation, are reintegrated through NFAT-mediated assembly of a
transcriptional complex on lymphokine gene promoters to initiate the immune response. The
activation of NFAT can be reversed by several constitutively active kinases which have been
implicated in the rephosphorylation of NFAT and its nuclear export (Beals et al., 1997). In
addition, phosphorylation of NFAT in vitro inhibits its ability to bind DNA.
Surprisingly, in the animal kingdom, relatively few biochemical pathways appear to
transmit all signals from receptors into the nucleus and virtually all of these pathways are
present in both vertebrates and invertebrates. Conversely, NFAT signalling seems to have
arisen in vertebrates only, since, so far, no invertebrate homolog of NFATc has ever been
found. This observation supports the suggestive hypothesis that NFATc arose just to suit
vertebrates’ specialized and distinctive needs, such as certain aspects of the nervous system
and a complex adaptive immune response. NFATc genes might have derived from a process
of exon shuffling between a Rel domain and a precursor gene with a calcineurin-sensitive
Ca2+ region. This process could have provided a new link between Ca 2+ signals and the
nucleus, useful for the control of genes primarily dedicated to cellular interactions.
14
Fig.1.5. Schematic illustration of intracellular signalling pathways resulting in activation of IL2 transcription. Antigen recognition by TCR initiates two pathways: one, p21rasdependent, stimulates de novo synthesis of NFATn; the other promotes an increase of
intracellular Ca 2+ levels. This activates calcineurin that dephosphorylates NFATc
allowing it to enter the nucleus, where it cooperates with NFATn to bind specific sites
on the promoter of various cytokine genes, thus activating transcription of the
corresponding genes. The process is reversed by kinases that re-phosphorylate NFAT,
inducing its nuclear export.
15
1.6 Hepatitis C virus
Hepatitis C virus (HCV) is the major aetiological agent of blood-borne non-A, non-B
hepatitis and a leading cause of liver cirrhosis and hepatocellular carcinoma (Kuo et al. 1989;
Purcell, 1994; Houghton et al., 1991).
HCV represents one of the most significant health problems since it affects an estimated
170 million people worldwide. The prevalence of HCV infections varies in different parts of
the world: for example, in Scandinavia, it is less than 0.5% of the population; whereas, in
Egypt, over 20% of the population is infected. In the USA and Western Europe, the
complications of HCV chronic hepatitis and cirrhosis are the most common reasons for liver
transplantation.
One of the major problems with HCV infections is that up to 85% of individuals initially
infected become chronically infected, usually for decades. The other 15-20% have an acute
infection that is resolved spontaneously in a few weeks or months. The propensity of HCV to
cause chronic, persistent infections is explained by its extraordinary ability to escape
destruction by the host immune system. Once established, chronic HCV infection causes an
inflammation of the liver called chronic hepatitis; this condition can further progress to
scarring of the liver, called fibrosis, or more advanced scarring, called cirrhosis. Some
patients with cirrhosis can develop liver failure and complications such as hepatocellular
carcinoma (HCC).
HCV is spread most efficiently trough the blood, thereby it is transmitted by infected
blood or blood products, transplantation of infected solid organs and the sharing of
contaminated needles among intravenous drug users.
In retrospect, HCV was the most common cause of transfusion-associated hepatitis in the
1980s: at that time, HCV had not been identified yet, and post-transfusion cases of hepatitis
were called non-A non-B hepatitis. In the mid 1980s, when the practice of using paid blood
donors was stopped and blood started to be screened for HIV, the risk of HCV transmission
fell to about 5%. Isolation and identification of the virus, and subsequent development of
specific serologic tests, have dramatically lowered the risk of acquiring HCV through blood
transfusion. At present, the most common cause of HCV transmission is by intravenous drug
abuse (Fig. 1.6).
Fig 1.6.
Risk factors for infection with hepatitis C virus
16
Despite the striking reduction in the number of new cases of hepatitis, the number of
deaths and the need for liver transplantation, due to complications of chronic infections, are
expected to increase within the next two decades: this is because of the large number of
individuals who became infected 10-20 years ago.
1.6.1 HCV structure
The identification of HCV is relatively recent (Choo et al, 1989). HCV, unrelated to the
other common hepatitis viruses (A, B, D and E), is an enveloped, single-stranded positivesense RNA virus belonging to the Hepacivirus genus of the Flaviviridae family (Fig. 1.7). Its
9,6-kb genome consists of a single RNA molecule with conserved terminal 5’ and 3’ noncoding elements, flanking an open reading frame encoding a single large polyprotein, which is
cleaved by host and viral proteases into three structural (core, E1, E2) and at least six nonstructural (NS) proteins (Grakoui et al., 1993) (see Fig. 1.8). Structural proteins contribute to
build up the envelop, while non structural proteins are involved in RNA replication. HCV
infects mainly hepatocytes; its genome does not integrate into that of the infected cell, for it is
directly translated and replicated by the host machinery.
Based on sequence diversity, HCV has been categorized into 6 major genotypes and many
more subtypes. Different genotypes show a distinct geographic distribution. The influence of
genotype on the long-term prognosis of HCV is still unclear, however, it seems that patients
infected with genotype 1 (particularly genotype 1b) are more likely to develop chronic
infection and do not respond well to pharmacological therapy.
core
Fig. 1.7.
Morphology of hepatitis C virus.
1.6.2 HCV diagnosis and therapy
Viral hepatitis may develop without clinical signs, or nonspecific symptoms may appear
for a short time, with or without jaundice. These symptoms may vary from non-specific flulike symptoms to liver failure. Diagnosis of hepatitis often depends on accumulation of
findings considered together.
HCV infection can be diagnosed and monitored by evaluating biochemical and
histological parameters. Abnormally high serum levels of hepatic enzymes (ALT and AST)
can be symptomatic of liver injury and are detected by routine blood analysis. Immunoblot
and ELISA tests are specifically used to detect anti-HCV antibodies in the blood and indicate
whether an individual has been exposed to the virus. Detection of viral RNA, through
sensitive molecular tests (such as RT-PCR), can confirm that a positive anti-HCV result
17
reflects an active infection and, although viral load does not correlate with the severity of
disease, may be helpful to monitor patient response to therapy. Liver biopsy assesses the
extent of fibrosis and the level of inflammation; though invasive, this type of analysis
provides important information about the severity, and thus the prognosis, of the disease.
The pharmacological antiviral therapy to treat hepatitis C is currently based on pegylated
IFN-α (PEG INF-α), alone or in combination with ribavirin (Fried et al., 2002). These drugs
enhance immune reaction to viral infection: PEG IFN-α, like other naturally occurring
interferons, has antiviral, antiproliferative and immunomodulating functions; ribavirin, a
nucleoside analogue, interferes with the RNA metabolism of the viral genome.
Three types of responses to the antiviral therapy have been described:
- sustained virological response is the optimal response: viral RNA becomes undetectable 6
months after therapy is stopped; most of sustained responders will remain in remission (no
signs of disease) indefinitely;
- relapse: relapser patients initially respond, but virus becomes again detectable within 3-6
months after stopping therapy;
- non-response: virus RNA remains or becomes detectable during therapy.
No vaccine is currently available for preventing HCV infection.
1.6.3 HCV pathogenesis
A deep knowledge of HCV pathogenesis is not yet clear. Since there is no definite
experimental evidence for a direct cytopatic effect of HCV, the liver damage in chronic
infection is probably caused by the interplay between the virus and the host immune reaction.
Since HCV infects only humans and chimpanzees, and replicates inefficiently in cell cultures,
studies aimed at assessing abilities of the virus to modulate host response have been difficult
to perform.
Cell-mediated specific response seems pivotal both for a successful viral clearance and in
mediating hepatic injury. Individuals who experience complete virological recovery, show
vigorous, multispecific CD4+ T helper 1 responses, while poorer CD4+ T helper 1 responses
are typical of chronic hepatitis (Cerny and Chisari, 1999). Induction of virus-specific CTLs
(cytotoxic T lymphocytes) is a well established mechanism for virus elimination during
infection; however, in the case of HCV, the lymphocytes may not be sufficient to eliminate
the virus completely. Conversely, cytotoxic response may cause extensive necrosis of both
infected cells and adjacent healthy cells, contributing to tissue damage.
The role of the humoral response is still controversial, but mostly considered of minor
relevance; indeed, it is documented that chronicization is associated to an unbalanced
cytokine production which enhances the humoral rather than the cell-mediated response
(Cooper et al., 1999).
The reasons why the host immune system often fails to eradicate the virus are not known.
HCV has probably evolved mechanisms to elude the host response. One proposed mechanism
of immune evasion is the generation, during the infection, of viral variants that could
circumvent antibody and CTL recognition. This is consistent with the high error rate of the
viral polymerase, which generates multiple virus quasi-species within the same host (Mc
Michael and Phillips, 1997).
Alternatively, as noted for several other viruses, HCV may encode products that act to
inhibit viral clearance by the host and can lead to progressive, persistent infection. Besides
hepatocytes, HCV infects lymphocytes and dendritic cells: by affecting these immune cells
the virus might compromise host immune functions (Pavio, 2003).
The viral product that is more likely to participate in this immune-interference is the core
protein.
18
1.6.4 HCV core protein
HCV core protein (HCV C) derives from the N-terminal 191 amino acids of the large
precursor polyprotein and is highly conserved among all isolates, with a sequence identity in
the range of 85-100% (Fig. 1.8). HCV C is highly basic, consistently with its RNA-binding
activity; it has two potential nuclear localization signals and a conserved putative DNAbinding motif (DBD), which support the hypothesis that HCV C also functions as a gene
regulatory protein; a predominantly hydrophobic domain is present in the C-terminal region
and is probably involved in protein-protein interactions during assembly of the viral
nucleocapsid (Bukh et al., 1994).
HCV C has a packaging function, as it is the major component of the viral nucleocapsid
(Fig 1.7); however, emerging evidence suggests that this protein is multifunctional and
several of its biological properties might have implications in HCV distinctive pathogenesis.
HCV C localizes both in the cytoplasm, where it has been found to associate with the
endoplasmic reticulum, lymphotoxin-β receptor and TNF receptor 1, and in the nucleus,
where, especially its C-terminal-truncated forms, seems to participate in transcriptional
regulation.
3’
C E1
E2
p7
5’
NS2
NS3
NS4A/B
NS5A/B
MSTnPKPQRkTkRNTnrRPqDvKFPGGGQIVGGVYILPRRGPR
IGVRatRKtSERQPRGRRQPIPkaRrpeGrsWaqPGyPWPIYg
nEGcgWAGWLLSPrGSrPsWGptDPRrrSRNlGkVIDTlTCgf
ADLMGYiPlVGaPlGGvArALAHGVRvlEDGvNyATGNlPGCs
FSIFlLALlSCltvPasa
Fig.1.8. Diagram of the HCV genome and consensus amino acid sequence of the core protein.
The lines indicate the 5’ and 3’ non-coding regions, while the boxes stand for the
reading frame. Regions encoding structural proteins are coloured. NS, non structural
proteins. In the sequence, capital letters indicate conserved amino acids. Three basic
regions, probably involved in RNA interaction, are marked in yellow; two putative
nuclear localization signals are underlined; a potential DNA-binding domain is dotunderlined.
HCV C has been implicated in cellular proliferation and there are several experimental
studies ascribing to this protein an oncogenic potential (Ray et al., 1996; Tsuchinhara et al.,
1999); the tumorigenic properties of HCV C has been associated to its capability to bind and
inactivate the endogenous LZIP factor, which functions as a cellular tumour suppressor (Jin et
al., 2000). The oncogenic nature of HCV C is noteworthy, since it could directly contribute to
the onset of HCC.
Another relevant biological function of HCV C is its well documented, anti-apoptotic
action: this effect might be advantageous for the virus, since it would allow the infected
hepatocytes to avoid the apoptosis, induced by the host immune system as a protective
mechanism, thereby allowing persisting and spreading infection (Marusawa et al., 1999).
HCV C has the potential to affect host immune gene expression through the activation of
the NFAT pathway: in fact, a recombinant core protein was found to activate NFAT-mediated
transcription from IL2-promoter in Jurkat cells (Bergqvist and Rice, 2001). The potential of
19
this effect in vivo is even more intriguing when considering that several other cytokines and
many immune-related genes are NFAT-regulated. Dysregulation of cytokine expression could
be part of the strategy used by the virus to interfere with the host immune system, thereby
hampering an effective defence response.
Studies on animal models (mice infected with a panel of recombinant vaccinia, expressing
the various HCV proteins) confirm that HCV core is sufficient to induce immune suppression
(Large et al., 1999).
In addition to cytoplasmic and nuclear localization, HCV C is secreted from stably
transfected cell lines, and likely from HCV-infected hepatocytes in vivo; indeed, circulating
core protein is detectable in the blood stream of infected patients, where it may provide the
virus with an indirect mechanism of immune dysregulation, influencing even host cells not
directly infected (Kanto et al., 1995; Sabile et al., 1999). Consistently with this immunomodulatory action, exogenous addition of HCV C was shown to affect the expression of
various cytokines implicated in CTLs activation, such as IL12 and INF-γ (Eisen-Vandervelde
et al., 2004).
20
2 AIMS OF THE RESEARCH
The aim of this research was to investigate the immuno-modulatory effect of HCV,
studying the expression of some immune genes in peripheral blood mononuclear cells
(PBMC) from HCV-infected patients. In this regard, we focussed our attention particularly on
the expression of HNPs. In fact, taking into account that: i) the expression of a recombinant
HCV core protein was found to induce the NFAT pathway (Bergqvist and Rice, 2001), and ii)
a putative NFAT core binding site is present in the promoter of HNP-1 and 3 genes (Fig 2.1),
we intended to find out whether, during the viral infection, the expression of these
antimicrobial peptides could be affected.
The study was carried out analyzing the profile of expression in cells directly extracted
from healthy and diseased individuals; another part of the study concerned with a set of in
vitro experiments to assess the potential effect of HCV core protein.
The choice to use PBMC rather than hepatic tissue, that is actually the main target of
HCV, is based on the following motivations: i) PBMC comprise relevant elements of the
innate and adaptive immune systems; ii) they are part of a “moving tissue”, the blood, which
can be informative about the status of the whole organism, as it reaches every district within
the body; iii) PBMC can be easily collected from a blood sample, without any need for
invasive and/or risky biopsy.
ets
-90
-80
-70
-60
-50
-40
TTTAATGGACCCAACAGAAAGTAACCCCGGAAATTAGGACACCTCATCCCA
HNP promoter
NFAT
Fig. 2.1.
Nucleotide sequence of the HNP promoter in the region -90/-40. The shaded sequence
indicates the Ets binding site, while the putative NFAT recognition site is underlined.
21
3 MATERIALS AND METHODS
3.1 MATERIALS
3.1.1 Patients. Study population: epidemiologic data, biochemical and virological
measurements
We investigated a total of consecutive 117 patients with chronic HCV infection (64 men,
53 women, mean age 47.6+11.9, range 30-72 years). Patients had serum antibodies to HCV,
detected by enzyme-linked immunosorbent assay and confirmed by recombinant immunoblot
assay. All patients had high aminotransferase (: aspartate and alanine aminotransferases, AST
and ALT) levels for at least 6 months. The epidemiologic characteristics, and the biochemical,
virological and histological data of these patients are shown in Table 3.1.
None of the patients had received or was receiving antiviral therapy at the time of the liver
biopsy or at the time of blood tests, with the exception of those included in the specific study
(see results). Exclusion criteria were coinfection with HIV, hepatitis B virus, other causes of
liver disease diagnosed by standard clinical, serological and biochemical criteria. Patients
with alcohol intake >80 g/day in men and >60 g/day in women or chronic drug intake,
dyslipidemia, hyperglycemia and severe steatosis on liver biopsy were also excluded.
As control group, we studied 22 sex- and age-matched normal healthy volunteers. Patients
with acute C, A and B hepatitis, chronic and cirrhotic B hepatitis, hepatocellular carcinoma
(HCV-), HIV infection, and various types of bacterial infections were also included in this
study.
The study was approved by the institutional review board of the Department of Infectious
Diseases, Università La Sapienza, Roma.
All patients and controls gave their written informed consent before inclusion.
Evaluation of α-defensin levels and determination of histological score were done in a
blind manner.
22
No.
%
18
21
78
66 ± 48
15
18
67
HCV transmission route
Intravenous drug use
Transfusion
Other
AST level (mean ± SD)
ALT level (mean ± SD)
HCV-RNA level (mean ± SD)
95 ± 67
2567000 ± 4159000
HCV genotype
Genotype 1
Genotype non-1
68
49
58
42
15
102
13
87
56
46
55
45
10
36
21
16
19
9.8
35.2
20.7
15.6
18.7
HISTOLOGY
Hepatocellular carcinoma
Chronic hepatitis
HAI
1-6
>7
Fibrosis
0
1
2
3
4
Table 3.1. Epidemiologic characteristics and biochemical, virological, and histological
data of 117 patients with HCV chronic infection. HAI, histological activity
index. ALT, alanine aminotransferase. AST, aspartate aminotransferase.
SD, standard deviation.
Serum HCV-RNA detection, quantification and genotyping
Serum HCV-RNA was detected by reverse transcriptase polymerase chain reaction
(Amplicor Roche) and quantified by branched-DNA assay (b-DNA assay, Quantiplex TM,
Chiron Co., Emmeryville, CA, USA). The detection limit of the test is 600 IU/ml. HCV
genotype was done using the reverse hybridization line probe assay (INNO-LIPA TM HCV
assay, Innogenetics NV, Ghent, Belgium), according to the manufacturer’s instructions.
Histological evaluation
The histological activity index (HAI) was quantified according to Knodell score. The
index represents the sum of periportal + bridging necrosis (0-10), intralobular degeneration
(0-4) and portal inflammation (0-4).
Fibrosis is classified as follows: 0 = none; 1 = enlarged, fibrotic portal tracts; 2 =
periportal or portal-portal septa but intact architecture; 3 = fibrosis with architectural
distortion but no obvious cirrhosis; 4 = cirrhosis
23
3.1.2 Microorganisms
Bacillus megaterium Bm11, Gram-positive, streptomycin resistant, was used in the
antibacterial activity assays.
3.1.3 Cells
CD4+ and CD8+ T cells, from peripheral blood of healthy donors, were a gift from
IRCCS, Santa Lucia, Rome. Cells were obtained by magnetic cell sorting, with a level of
purity >95%, as evaluated by flow cytometry.
Peripheral blood mononuclear cells (PBMC) and granulocytes were purified by density
gradient purification (see methods).
3.1.4 Growth media
For PBMC, the medium used was RPMI 1640, supplemented with freshly added 10 %
fetal bovine serum (all from Sigma). RPMI is stored at 4 °C; fetal bovine serum aliquots are
stored at -80 °C.
For bacteria Luria Bertani (LB) broth was prepared:
Bacto-tryptone
10g/l
Yeast extract.
5g/l
NaCl
5g/l
Solid LB is prepared by adding 15 g/l agar
Soft agarose LB is prepared by adding 10 g/l agarose.
Sterilize the media by autoclaving at 120 °C for 120 min.
3.1.5 Drugs and solutions
Cyclosporin A (CsA) stock solution is prepared by dissolving the powder in ethanol at a
concentration of 1mg/ml. Store aliquots at -20 °C.
Streptomycin stock solution is prepared by dissolving lyophilized antibiotic salt in distilled
H2O (dH2O) at a concentration of 100 mg/ml. Sterilize by filtration through a 0.22 µm filter
and store aliquots at -20 °C.
Ionomycin stock solution is prepared by dissolving the corresponding calcium salt in
dimethyl sulfoxide, DMSO, at a concentration of 1mg/ml. Store aliquots at -20 °C, shielded
from light.
Phorbol 12-myriststate 13-acetate, PMA, is dissolved in DMSO at a concentration of 0.1
mg/ml. Aliquots stored at -20 °C, protected from light.
Phytohemagglutinin, PHA, is dissolved in sterile PBS at a concentration of 0.5 mg/ml.
Aliquots stored at -20 °C, and at 4 °C after first thawing.
24
Matrix for mass spectrometry: 3,5-dymethoxy-4-hydroxy-cinnamic acid (sinapinic acid) is
dissolved in 0.1% trifluoroacetic acid, acetonitrile:water (1:1, v/v) solution, at a concentration
of 30 g/l. Store at -20 °C.
DEPC-treated H2O: RNAse free distilled water is prepared by adding 1µl/ml of diethylpyrocarbonate, DEPC. Shake bottle and let stand overnight under chemical hood before
autoclaving 120 min at 120 °C.
Phosphate-buffered saline (PBS)
137 mM NaCl
2,7 mM KCl
10 mM Na2HPO4
2 mM KH2PO4
Adjust pH to 7.4 with HCl. Sterilize by filtration. Store at room temperature
Ethidium bromide stock solution (10mg/mL) is stored at 4 °C, protected from light.
TE
20 mM
1mM
pH 7.5
TAE 20X
800 mM
Tris base
100 mM
CH3COONa
20mM
EDTA
Adjust pH to 7.4 with CH3COOH
Agarose gel loading buffer (6x)
0.25 %
0.25 %
30 %
Tris-HCl
EDTA
Bromophenol blue
Xylen cianol
Glycerol
Agarose gel for RNA and DNA electrophoresis
1% Agarose gel has been used routinely for RNA electrophoresis, while 1.5-2% agarose gels
have been used for analysis of PCR products. The gel is prepared as follows:
- Agarose
1-2 g
- TAE 20x
5ml
- dH2O
to 100 ml
- Melt in microwaves, let it cool and add 5µl ethidium bromide stock solution.
- Pour the solution in the gel-caster, let it harden.
Use TAE 1x as running buffer. Substitute dH2O with DEPC H2O for RNA electrophoresis.
dNTPs 10 mM
dATP, dCTP, dGTP,dTTP (100mM) 10 µl each and add dH2O to 100 µl.
Sybr Green I (Diatech) is stocked as 1000-fold dilution in TE pH 7,6; aliquots are stored at 20°C; after thawing, they are kept at 4 °C, shielded from light.
TaqMan probes and LUX primers are stored at -20 °C, protected from light, as mono or
double-use aliquots.
25
HCV core protein, HCV C, was a kind gift from Penelope Mavromara and Urania
Georgopoulou (Hellenic Pasteur Institute, Athens). The recombinant protein, genotype 1, has
an N-terminal His tag and was purified, under native conditions, as a mixture of truncated
products, with the highest molecular weight corresponding to the first 120 amino acids of the
native HCV C (191 aa total length). Stock solution in PBS, at a concentration of 300ng/ml, is
stored at -20 °C.
Hepatitis B virus core antigen, HBcAg (from IBT, Germany). Stock solution, with a
concentration of 0.75 mg/ml in 100mM NaCO3 pH 9.3, is stored at -20 °C.
3.1.6 Oligonucleotides for RT-PCR
An oligo-(dT)18 has been used to prime first strand cDNA synthesis. Stock solution, 500
µg/ml, is stored at -20 °C.
Sequences of the primers used for real time RT-PCR are given below.
HNP forward : 5’-GCAGAATACCAGCGTGCATTGCAGGAG-3’ and
HNP reverse: 5’-CAGCAGAATGCCCAGAGTCTTCCC-3;’
GAPDH for: 5’-TGGGCTACACTGAGCACCAG-3’ and
GAPDH rev: 5’-CAGCGTCAAAGGTGGAGGAG-3;
GAPDH-certified LUX primer set, JOE-labelled(Invitrogen).
TBP for: 5’-GCACAGGAGCCAAGAGTGAAG-3’
TBP rev: 5’-TCACAGCTCCCCACCATGTTC-3’
LL37 for: 5’-CATCATTGCCCAGGTCCTCA-3’
LL37 rev FAM-labelled: 5’-caaccTCCGAGGACCGCTGGTTG-3’
IL-15 for: 5’-TGCAAAGAATGTGAGGAACTGG-3’
IL-15 rev FAM-labelled:
5’-cactcgCACATTTGAAATGAAATGCCGAGTG-3’
IL-2 for FAM-labelled: 5’-gacttagCCTGTCTTGCATTGCACTAAGTC-3’
IL-2 rev: 5’-TGAGCATCCTGGTGAGTTTGG-3’
The above LUX primer pairs, for LL37, IL-2 and IL-15, were designed using LUX-primer
design programme, available at www.invitrogen.com; bold and underlined case indicates the
fluorophore-marked nucleotide in the labelled primer; lower case letters indicate the 5’ nonannealing regions involved in the intramolecular hairpin structure.
Primers and FAM-labelled TaqMan probes, produced by Diatech laboratories (Jesi), for
amplification and detection of HNP and GAPDH transcripts, have also been used (sequences
available on request). In part of the study, Diatech primers have been used also in
combination with Sybr Green I dye, without TaqMan probe.
26
3.2
METHODS
3.2.1 PBMC preparation
Peripheral blood mononuclear cells, PBMC, were isolated from EDTA-anticoagulated
peripheral blood (~20ml) by Ficoll density gradient centrifugation according to the procedure
given below.
- Dilute freshly drawn blood with 2-4 volumes of PBS.
- Carefully layer 35ml of the diluted blood over 15 ml Ficoll in a 50ml conical tube and
centrifuge 30 min at 400g at 20 °C.
- Transfer the interphase cells (lymphocytes and monocytes) to a new conical tube, fill it
with PBS and centrifuge at 300g for 10 min at 20°C. Remove supernatant carefully.
-Wash cells from platelets: resuspend pellet in 20 ml PBS. From this suspension a small
aliquot is used for counting in a hemocytometer; the remaining is divided into two
aliquots and centrifuged at 300g for 10 min at 20°C. Discard super.
- Freeze one aliquot for subsequent peptide extraction; resuspend cells of the second
aliquot in appropriate lysis buffer (TRIzol), for subsequent RNA extraction.
Alternatively, PBMC are directly used for in vitro experiments.
- Store samples at -80 °C.
3.2.2 Granulocytes preparation
Use freshly drawn EDTA-anticoagulated blood.
- Prepare the density gradient by layering 10 ml histopaque 1119 and 10 ml histopaque
1077 (Sigma) in a conical tube.
- Layer the blood (15-20 ml) over the solution and centrifuge at 400 g for 30 min at
20°C.
- Collect the white cell layer just above the red blood cells (that is the lowest cell ring),
transfer to a new tube and add PBS to 30 ml; centrifuge 10 min at 300g for 10 min at
20 °C. Repeat this wash.
- Resuspend in 5 ml PBS, use an aliquot for hemocytometer reading and centrifuge the
remaining to recover the granulocytes as before.
- Resuspend the cells in TRIzol, for subsequent RNA extraction, and store at -80 °C.
3.2.3 In vitro treatment of PBMC
Freshly isolated PBMC were resuspended in RPMI 1640, supplemented with 10 % fetal
bovine serum and 80µg/ml streptomycin. PBMC final concentration was in the range of 1-1.5
x106 cell/ ml. Cells were cultured in 18-well round-bottomed polystyrene plates, at 37°C in a
humidified atmosphere with 5% CO2 in air, in the absence (unstimulated controls) or in the
presence of:
- 2 µg/ml of recombinant HCV C, or
- 2,5 µg/ml of HBcAg.
To analyse whether α-defensins expression is mediated by the transcription factor NFAT,
part of the PBMC were pre-incubated with:
1 µg/ml cyclosporin A, CsA, at 37°C for 30 min, before HCV C addition.
27
PBMC have been incubated also with:
- 0.05 µg/ml phorbol 12-myristate 13 acetate, PMA;
- 1.5 µg/ml ionomycin;
- 3 µg/ml phytohemagglutinin, PHA.
These drugs were used alone or variously combined, with or without 30 min preincubation with CsA; control non-stimulated cells were incubated in RPMI alone,
contemporarily.
After 3 hours and 30 min incubation, PBMC were recovered by mild centrifugation and
used for subsequent peptide and RNA extraction procedure.
The HNP mRNA and peptide levels, detected in the differently stimulated cells, were
normalized to those observed in the unstimulated control cells.
3.2.4 Peptide extraction
Low molecular weight proteins of the granule fraction were obtained from PBMC,
according to the following protocol.
- Thaw PBMC and resuspend in 0.34 M sucrose (pH 7.4).
- Disrupt cells by rapid sonication (cell breakage is confirmed by light microscopy) and
centrifuge at 200g for 10 min to remove the cell debris.
- Centrifuge supernatant at 27,000g for 30 min at 4 °C. Resuspend the pellet in 5% cold
acetic acid, sonicate on ice, and let overnight at 4 °C for peptide extraction.
- Clear the peptide extract by centrifugation at 27,000g for 30 min at 4 °C.
- Dry the extract in a vacuum centrifuge, and dissolve in 20% ethanol for antibacterial
activity determination.
3.2.5 Antibacterial activity
The antibacterial activity of the PBMC peptide extracts was tested against Bacillus
megaterium Bm11, using the inhibition zone assay (Hultmark et al., 1983).
- Thin plates of 1% agarose in LB broth, containing approximately 4x105 cells are made:
bacteria are grown in LB broth till an optical density of 1 at 590 nm, corresponding to
a mid-logarithmic phase. The bacterial cells are hence diluted in LB medium and
approximately 2x105 colony forming units are added to 6 ml LB/1% agarose and
poured onto a Petri dish.
- Small wells (3 mm diameter) are punched onto the LB-agar layer and 3 µl samples are
loaded into the wells.
- Incubate overnight at 30 °C; measure the diameters of the inhibition zones and express
the resulting antibacterial activity as cecropin A units (CAU)/106 PBMC, using a
standard curve. One cecropin A unit corresponds to the activity of 1 ng of cecropin A
on B. megaterium Bm 11 (Simmaco et al., 1998).
3.2.6 ELISA
HNP levels in PBMC peptide extract were measured using a human HNP ELISA test kit
(Hbt, Walter Occhiena), based on the sandwich principle. Instructions from the manufacturer
were followed and the main steps of the procedure are reported below:
- incubation of standards and sample peptide extracts for 1hr at RT, followed by 3
washes;
28
- incubation with secondary biotinilated-antibody for 1 hr at RT, followed by 4
washes;
- incubation with streptavidin-peroxidase conjugate for 1hr at RT, followed by 4
washes;
- incubation with substrate reagent for 20-30 min in the dark, at RT;
- stop of the reaction;
- measurement of absorbance at 450 nm in a microplate reader.
The sensitivity of this test ranges from 40 to 10,000 pg/ml.
3.2.7 Mass spectrometry
Peptide extracts were analyzed by matrix assisted laser desorption/ionization time-offlight mass spectroscopy (MALDI-ToF MS) using a Voyager DE mass spectrometer
(PerSeptive Biosystem), using external calibration. The calibrator mixture is composed by:
angiotensin I (1,297.51 Da), adrenocorticotropic hormone (ACTH), fragment 1-17 (2,094.46
Da), ACTH, clip 18-39 (2,094.46), ACTH, fragment 7-38 (3,660.19) and bovine insulin
(5,734.59 Da).
Sample preparation is as follows:
- mix 1 µl of peptide extract with 4 µl of the matrix solution (sinapinic acid
solution);
- deposit a droplet (0.5-1 µl) of the matrix/sample mixture on the stainless steel plate
and let it dry.
- analyse by MALDI ToF, using up to 500 shots per spectrum.
3.2.8 RNA preparation
-Total RNA was purified from PBMC using TRIzol reagent (Invitrogen). The main steps
of the procedure are reported below.
- Homogenize up to 107 cells in 1ml TRIzol, by repetitive pipetting and vortexing, and
incubate at RT for 5 min;
- add 200 µl chloroform, shake vigorously and incubate at RT for 3 min;
- centrifuge at 12,500 g for 15 min at 4 °C and transfer the upper aqueous phase to a
new tube; precipitate by adding 0.5 ml isopropanol and keeping at -20 °C for 15
min. Centrifuge at 12,500 g for 15 min at 4 °C;
- wash the pellet with 1 ml ice-cold 75% ethanol, centrifuge again at 8,000 g for 5 min
at 4°C; discard supernatant, blot the excess of liquid and let the pellet dry for 10-15
min at RT.
- Resuspend the RNA pellet in 20-50 µl of DEPC H2O and store at -80 °C.
Part of the RNAs was extracted using Qiagen RNeasy mini kit, following the instructions
on the accompanying handbook.
RNA recovery and purity were controlled by spectrophotometric absorption
measurements at 260 nm and 280 nm; RNA integrity is checked by electrophoresis and
visualization on 1% agarose gel. Part of the RNA preparations was analysed by capillary
electrophoresis on RNA 6000 Nano LabChip Kit using the Bioanalyser (Agilent
Technologies), and following the manual instructions. This highly sensitive method is
particularly useful when handling small amounts of RNA sample, in fact it allows qualitative
29
and quantitative analysis of as little as 50 ng of total RNA. Figure 3.1 shows representative
conventional RNA gel electrophoresis and Bioanalyzer chip electrophoresis.
A.
B.
Fig 3.1 Examples of total RNA analysis by gel electrophoresis (A) and by capillary
electrophoresis on the Bioanalyzer chip (B). In A, the two major bands correspond to
28S and 18S ribosomal RNAs, a lower fainter band corresponds to 5S rRNA. In lane 5,
an upper band due to genomic contamination is evident. B is the elecropherogram
profile of an RNA sample : peaks at about 40 sec and 47 sec represent 18S and 28S,
respectively, while the peak on the left is the marker; the sharper the peaks, the better
the quality of the RNA.
DNase I treatment
In order to remove any genomic contamination, RNA was treated with RNase free DNase
I, prior to retrotranscription. Major steps of the protocol are described below.
- Incubate total RNA (5 µg) with DNase I (5 units), in the presence of 20 mM Tris HCl,
pH 8.2, 50 mM KCl, 1 mM MnCl2, at 25 ˚C for 30 min, in a final volume of 50 µl;
- stop the reaction by adding 2,5 mM EDTA (pH 8.0) and heating 5 min at 75˚C.
RNA is subsequently re-extracted using TRIzol, and, after that, its integrity is checked
again by gel electrophoresis and/or chip electrophoresis on the Bioanalyzer (Fig 3.1).
30
cDNA synthesis
First strand cDNAs are synthesized from total RNA using SuperScript II RT (Invitrogen)
with oligo-(dT)18, according to the manufacturer’s protocol:
- preheat at 65 °C for 5 min the RT-reaction mixture, including up to 2 µg of total RNA,
25 µg/ul oligo-dT primer and 0.5 mM dNTPs;
- add 0.01 M DTT and 1x First-Strand Buffer; incubate the mix at 42 °C for 1 min;
- add 200 units of SuperScript II and incubate at 42 ˚C for 50 min;
- inactivate reverse transcriptase by heating at 70 ˚C for 15 min.
cDNAs are stored at -20 °C.
3.2.9 Purification of DNA fragments from agarose gel
Specificity of PCR was checked by gel electrophoresis of the amplification products.
PCR-bands are compared to those of a molecular weight standard. The band corresponding to
the expected size is excised from the gel and purified using an agarose gel DNA extraction kit
(Roche), based on a silica matrix with high affinity for DNA. DNA is eluted from the matrix
using appropriate volumes of dH2O, usually 20-50 µl. DNA concentration is determined by
spectrophotometer reading at 260 nm. The purified amplicon is then serially diluted, to be
used as template and to generate the standard, reference curve.
3.2.10 Quantitative real time RT-PCR
Instrumentations and principles
Quantitative real-time PCR was performed in the Rotor-Gene 3000 (Corbett Research). In
this real time PCR instrument, a thermal cycler and a fluorescence-detection system are
controlled by a software which monitors real time product accumulation, by measuring the
increase of fluorescence at each cycle; the product of amplification, or amplicon, is detected
by specific fluorogenic probes and/or DNA binding dyes (see figure 3.2). This type of
technique provides high sensitivity and allows quantitative analysis.
FLUORESCENCE
CYCLE
Fig 3.2. Typical fluorescence profile during a real time PCR run in the Rotor Gene 3000.
Generation and accumulation of the amplicon determines an exponential increase in
fluorescence as the cycles progress.
31
Three different types of detection chemistries were used: Sybr Green I dye, TaqMan
probe, and LUX- primers. Fig 3.3 illustrates their features.
2
1
A
3
1) Intercalating dye: Sybr
Green I
2) Dual-labelled probe :
TaqMan
B
3) LUX primer
Fig 3.3. Types of detection chemistries, and corresponding principles, used in real time PCR,
in the present thesis. 1) Sybr Green I is a DNA-intercalating dye, whose fluorescence
increases upon DNA binding. 2) TaqMan probe is an oligonucleotide with a
fluorophore at its 5’ end and a quencher at its 3’ end: due to the exonuclease activity
of Taq polymerase, the probe is degraded, the fluorophore released into the solution
and separated from the quencher, allowing a higher fluorescence emission. 3) LUX
primers are oligonucleotides labelled with a single fluorophore at their 3’ end:
fluorescence is quenched by the intrinsic hairpin structure and increased upon
primer extension (LUX: Light Upon eXtension).
Fluorescence acquisition and calibration were performed on the following channels,
according to the fluorophores used:
- Sybr (source 470 nm, detector 585 nm);
- FAM/Sybr (source 470 nm, detector 510nm);
- JOE (source 530nm, detector 585 nm)
Quantitation methods
The two standard curves method was used to obtain either relative or absolute
quantification of the genes of interest, comparing their level of expression to that of the
housekeeping gene: fig 3.4 (Corbett Research user manual).
32
Fig 3.4. Representative fluorescence profile and corresponding standard curve obtained by
amplifying a six log-range dilution series of a standard DNA, with known absolute
concentration. Cycle threshold, Ct, values obtained for the unknown samples are
related to the standard curve and so concentration can be calculated.
For relative quantification, the standard curve is generated, by using, as template, serial
dilutions of a cDNA sample; the units, used to describe the dilution series, are relative and
based on the dilution factor of the standard curve. For absolute quantification, the standard
curve is generated from serial dilutions of the purified specific amplicon, whose concentration
has been spectrophotometrically determined; the units used to describe the dilution series are
expressed as “copy number/µl”.
The relative quantification method was adopted for a first part of the study on the
expression of HNPs, and for IL-2 gene. The absolute quantitation method was used for HNP
genes profile in most of the HCV-patients, including those in therapy, and the in vitro
experiments, as well as for LL37 and IL-15.
Assay conditions
PCR mix and cycling conditions (such as oligonucleotides and fluorophore concentration,
MgCl2 concentration, annealing and fluorescence acquisition temperatures, cycle number,
etc.) were pre-optimized for each particular primer pair and target gene, so to improve
efficiency and specificity of the reaction. A typical PCR reaction mix contained two-to-twenty
ng of template cDNA, corresponding to 2-5 µl of diluted cDNA, in a final reaction volume of
20-25 µl.
For Sybr Green I-detection, the PCR mix includes:
-2.5 U of Taq polymerase (Diatech)
-0.2 mM dNTPs
33
-1.5-2.5 mM MgCl2
-1x PCR buffer
-Sybr Green I (1:40,000-1:20,000 dilution)
-50-125 nM forward and reverse primers
When using LUX primers, the mix is essentially the same as above, but it contains a
Platinum hot start polymerase (Invitrogen) and no Sybr Green I.
Concentrations of each component, in the mix used with TaqMan probes, have been
optimized in Diatech laboratories.
Typical cycling conditions in a Sybr Green or LUX primer-based reaction are:
- initial denaturation at 95 °C for 5-3 min
- 40-30 cycles comprising:
20 sec denaturation at 94 °C;
20-30 sec annealing at 55-61 °C;
20-30 sec extension at 72 °C ( with or without acquisition of
fluorescence);
15 sec at 80-85 °C, for acquisition of fluorescence;
- 1 min final extension at 72 °C;
- melt analysis (see below).
Typical cycling conditions in a TaqMan probe-based reaction are:
- initial denaturation at 95 °C for 5-3 min;
- 40 cycles comprising:
20 sec denaturation at 95 °C;
60 sec annealing and extension at 60 °C, with fluorescence acquisition.
Fluorescence data are acquired either during the extension phases or in a subsequent phase
at higher temperature, according to the melting point of the specific amplicons.
When using Sybr Green I and/or LUX primers, a melt curve is generated after each PCR
run, and subsequently analyzed to check for specificity of the amplification products: samples
are heated at 70 °C for 2 min and then slowly heated at 0,2 °C /sec up to 99 °C with a
continuous acquisition of the fluorescence.
IL-2
IL-15
Fig 3.5. A representative melt curve analysis. In this example, two different melting
temperatures were detected for IL-2 and IL-15 amplification products. PCR were
run using specific LUX primers.
Different amplification products will melt (=denature) at different temperatures, based on
their lengths and G/C contents; the melting peaks result from the differential of the
fluorescence (y axis) relatively to the temperature (x axis), as illustrated in figure 3.5. The
melting peaks reflect the products amplified during the reaction, and are analogous to the
34
bands on an electrophoresis gel. Melting curve analysis allows qualitative monitoring of the
reaction, and permits to adjust the fluorescence acquisition temperature so to exclude
aspecific, primer-dimer signals.
Each PCR assay included duplicates of each cDNA sample, no-template controls, and two
standard curves, for the housekeeping gene (GAPDH), and for the gene of interest,
respectively, constructed so to cover a 3 to 6 LOG-range (1-1,000,000). Data were analyzed
using the Rotor-Gene Analysis Software V 4.6.
3.2.11 Statistical analysis
Statistical analysis was performed using Student's t test for parametric data and Wilcoxon
rank sum test for non parametric data. The correlation between α-defensin levels and the
degree of hepatic fibrosis was assessed by the Pearson’s correlation coefficient. Any p
value<0.05 was considered significant.
35
4 RESULTS AND DISCUSSION
4.1 Expression profiling of HNPs in HCV patients
Quantitative real time reverse transcription PCR (qRT-PCR): validation of the
housekeeping genes
Quantitative gene expression assays require a parameter for normalization of the data: this
can be either the number of cells, the RNA quantity, or an internal control gene. The approach
based on an endogenous control gene, to which normalize the mRNA fraction, is the most
frequently used, and it has been adopted even in this study.
In a quantitative RT-PCR experiment, the reference gene is simultaneously amplified with
the target gene or gene of interest (GOI): the quantitative values obtained for the GOI can
then be normalised to this internal standard; this operation allows minimisation of errors due
to sample variations (variations in the amount of starting material, samples obtained from
different individuals, different efficiencies in enzymatic activities, etc.) and makes possible
comparison of the data obtained from different samples and sets of experiments.
The expression of an ideal internal standard, also referred to as house-keeping gene
(HKG), should not vary among different individuals or tissues under investigation, nor in
response to experimental treatment or pathological states (Bustin, 2002). In addition, the
HKG should also be expressed at about the same level as the gene/s under study.
Validation of the candidate reference genes, in order to confirm their presumed stability of
expression, is an essential prerequisite to any serious quantitative real time RT-PCR
experiment. In the following study, we considered two putative housekeeping genes: the
moderately abundant level-expressed glyceraldehydes-3-phosphate-dehydrogenase (GAPDH)
(accession number BC029618, NM_002046), coding for the fundamental glycolytic pathway
enzyme, and the low level-expressed TATA box binding protein (TBP) (accession M55654,
NM_003194), encoding the general RNA polymerase II transcription factor.
Fixed amounts of template cDNA, corresponding to approximately 10-20 ng of starting
RNA, were used to drive real time RT-PCR of both potential housekeeping genes; RNA
samples were representative of a population including PBMC from both healthy and diseased
individuals. The genes exhibited a marked difference in abundance: GAPDH transcript
resulted far more abundant (Ct values ≈ 20) than TBP (Ct values ≈32).
The Ct values obtained are conveniently represented on a graph as relative concentrations.
Figures 4.1 and 4.2 show the results obtained for GAPDH and TBP genes, respectively.
36
Gapdh relative concentration
GAPDH mRNA calculated concentration using 1020 ng of cDNA template in RT-PCR
120
100
80
60
40
20
A
H
cu IV
C te
hr
on A
ic
ot C
B
he ir
r rh
in os
fe is
m ctio B
ea n
n s
va
lu
e
C
C
irr
ho
t
H
ic
C
C
C
hr
on
ic
H
ea
lth
y
C
co
n
tro
ls
0
Fig. 4.1. Relative concentrations obtained for GAPDH transcripts using cDNA samples from
healthy controls and different types of diseased patients. The mean value ± standard
deviation is also shown. HCC, hepatocellular carcinoma.
TBP mRNA calculated concentration using 10-20
ng of cDNA template in RT-PCR
TBP mRNA relative
concentration
100
80
60
40
20
0
H
ea
s
C
ol
ic
tr
n
n
o
co
hr
y
C
h
lt
C
tic
ho
r
ir
C
H
CC
H
IV
C
ic
on
r
h
B
A e
te valu
u
c
A an
e
m
Fig. 4.2. Relative concentrations obtained for TBP transcripts using cDNA samples from healthy
controls and different types of diseased patients. The mean value ± SD is also shown.
HCC, hepatocellular carcinoma.
The observed variability was evaluated using Student’s T test: no statistically significant
variation was found (P> 0.05, comparing values from diseased patients, versus those from
healthy controls). The variance observed among different samples may be compatible with
intrinsic variability deriving from errors in spectrophotometric determinations of RNA
concentration, different efficiencies in RT reactions, pipetting inaccuracies, differences in
handling and storage of biological samples (cells, cDNA), etc. To further confirm this
evaluation, the ratios between the observed RNA levels of GAPDH and TBP were calculated
and plotted: if the two putative HKGs are truly invariant, this ratio should be constant for the
37
different samples examined. Fig. 4.3 shows the results: the medium value of the ratios is 1.2 ±
0.7 (mean ± SD) and the plot shows an acceptable level of inter-samples variability.
Gapdh mRNA/TBP mRNA)
Ratio between GAPDH and TBP mRNA levels
in different patients and healthy controls
5
4
3
2
1
0
patients
healthy
controls
m ean
ratio
Fig. 4.3. Values obtained dividing GAPDH mRNA level by TBP mRNA level for
cDNAs from diseased patients and healthy controls.
In order to prove that either of the above genes could be used as internal references, the
transcriptional level of the gene of interest, that is HNP, was analysed in a limited set of
samples using simultaneously both genes for the normalization of the data. The results, shown
in Fig. 4.4, suggest that the same expression trend is maintained using either of the proposed
HKGs. The graph indicates also that the variation in the level of expression of HNP, among
different individuals, is much higher than the inter-sample variability observed when
comparing the levels of the two candidate HKGs: this allows us to neglect GAPDH and TBP
variability and to consider them as good HKGs.
38
HNP relative transcription using GAPDH and TBP
as internal reference genes
HNP/TBP
HNP/GAPDH
relative mRNA level
10000
1000
100
10
1
sample
Fig. 4.4.
HNP expression profile using two alternative housekeeping genes. The dotted bars
refer to values obtained from healthy controls, all the other data are from diseased
patients.
Given that both genes are equally valid as internal standards, we decided to use GAPDH
for the rest of the study: this choice is motivated by its higher expression level (thus
producing a stronger and clear fluorescence signal, even at low template concentrations) and
by the better efficiency in its RT-PCR reaction.
Quantitative real time RT-PCR analysis of HNP genes
The expression profile of HNP genes was studied at the transcriptional level, using two
different real time qRT-PCR approaches.
In a first set of HCV-patients the relative abundance of the HNP transcripts was
determined using a Sybr Green detection based method. In this case the ratios HNP mRNA
/GAPDH mRNA levels obtained from each analysed sample (both patients and healthy
controls) were normalized to that of a calibrator sample arbitrarily designated among the
healthy controls. The results of this approach are outlined in Fig. 4.5.
39
Healthy
controls
Acute
Chronic
Cirrhotic
HCC
1
10
100
1000
Relative HNP mRNA level
Fig. 4.5. HNP relative transcriptional level in PBMC from healthy controls and patients at
different HCV infection stages. HCC, hepatocellular carcinoma. Relative quantitation
and Sybr Green detection-based method. Bar charts show mean values ± SEM
(standard error of the mean) for each subgroup of patients.
The expression of HNP genes was further analysed in a larger group of patients and
healthy individuals by a real time RT-PCR approach, based on a more specific and sensitive
TaqMan detection system and using an absolute quantitation method. The results obtained by
this approach are outlined in Fig. 4.6.
Comparing the data obtained using these two alternative approaches a correlation factor of
0.82 was obtained, which is indicative of a good agreement.
40
Healthy
controls
Acute
Chronic
Cirrhotic
HCC
1
10
100
1000
HNP mRNA level (HNP/GAPDH)
Fig. 4.6. HNP transcriptional level in PBMC from healthy controls and patients at different
HCV infection stages. HCC, hepatocellular carcinoma. Absolute quantitation
method and TaqMan probe detection-based system. Mean values and standard
errors for each subgroup of patients are shown.
The transcriptional level of HNP genes was found to be significantly higher in all HCV
infection stages with respect to the level observed in the healthy controls: p(healthy/acute) <
0.001; p(healthy/chronic) = 0.004, p(healthy/ cirrhotic) = 0.02; p(healthy/HCC) = 0.02.
No significant correlation was found between HNP mRNA levels and percentage of
contaminating granulocytes and/or monocytes in the starting PBMC preparation.
ELISA
The concentration of the HNPs in peptide extracts obtained from PBMC of healthy and
diseased individuals was specifically determined by ELISA. Fig. 4.7 outlines the result of this
analysis: the concentration values were normalized to the number of starting cells. The
concentration of HNPs resulted significantly higher in PBMC from patients at every HCV
infection stages when compared to the range of concentrations observed in PBMC from
healthy individuals. Values are as follows: p(healthy/acute)<10-6; p(healthy/chronic)<0.001;
p(healthy/cirrhotic)<10-5; p(healthy/HCC)<0.0005.
41
Healthy
controls
Acute
Chronic
Cirrhotic
HCC
0,0
0,4
0,8
1,2
1,6
6
HNP level (ng/10 cell)
Fig. 4.7. HNP level, as detected by ELISA, in peptide extracts from PBMC of healthy controls
and patients at different HCV infection stages. Mean values and corresponding
standard errors are reported for each class. HCC, hepatocellular carcinoma.
HNP levels did not correlate with aspartate aminotransferase (AST) or alanine
aminotransferase (ALT) serum concentrations; besides, higher amounts of α-defensins were
detected in subjects with HCV-RNA>100,000 IU/mL, but there was no correlation with HCV
viral load or different HCV genotypes.
Antibacterial activity
The presence of the α-defensins in the peptide extracts can be assessed by evaluating their
distinctive biological activity, that is their antimicrobial property. To this aim, the
antimicrobial capacity of the whole peptide extract was tested against a laboratory strain of
Gram-positive bacteria, Bacillus megaterium Bm11, using an inhibition zone assay. The
diameter of the zone where the growth of the microorganism has been inhibited is converted
into cecropin A units. This type of test lacks the specificity of the ELISA, since also low
molecular weight antimicrobial agents, other than defensins, might contribute to the
antimicrobial capacity of the extract; nevertheless, results obtained with both these
approaches were found to be in good agreement with each other (R=0.82); moreover, the
antimicrobial assay provides a useful measure of the α-defensins which are present in a
biologically active form.
The mean values of antimicrobial activity found in PBMC extracts from healthy controls
and HCV patients at different stages are reported in Fig. 4.8.
42
Healthy
controls
Acute
Chronic
Cirrhotic
HCC
0
300
600
900
1200
6
Antimicrobial activity (CAU/10 cell)
Fig. 4.8. Antimicrobial activity of peptide extracts from PBMC of healthy individuals and
patients at different stages of HCV infection. The antimicrobial activity is expressed
as cecropin A units normalized to cell number. Mean values and corresponding
standard errors are reported for each class. HCC, hepatocellular carcinoma.
The results indicate that in all stages of HCV infection, the level of antimicrobial activity
is
significantly
greater
than
in
healthy
controls:
P(healthy/acute)<10-5;
-5
P(healthy/chronic)<0.01; P(healthy/cirrhotic)<10 ; P(healthy/HCC)<0.001.
MALDI ToF analysis
The mass spectral analysis of the peptide extracts from PBMC of healthy controls and
HCV-patients revealed the presence of the three most abundant HNP isoforms. A
representative mass spectrum is shown in Fig 4.9: a cluster of three peaks with the expected
molecular masses for HNP-1, 2, and 3 was observed in each sample, including those from
healthy controls.
43
3372 HNP-2
% Intensity
3443 HNP-1
3487 HNP-3
m/z
Fig. 4.9. Mass spectral analysis. Representative MALDI-ToF mass spectrum of peptide extract
from PBMC. The three major peaks correspond to molecular masses compatible with
those expected for HNP-1, 2 and 3.
The MALDI ToF analysis confirmed that the peptide extracts contained mostly defensins,
since little or no signal of comparable intensity was observed in the spectral region spanning
from 3 kDa to 10 kDa, where other antimicrobial peptides, such as cathelicidins, might be
detected (see below).
4.2 HNPs expression in other hepatic pathologies
The expression of HNP genes was studied also in PBMC from patients affected by liver
pathologies other than hepatitis C. A quantitative real time RT-PCR analysis allowed us to
compare the HNP transcriptional levels between patients in acute stages of infection by
hepatitis C, A and B viruses (HCV, HAV and HBV) and in HCV and HBV chronic and
cirrhotic stages. Fig. 4.10 outlines the results of this study: HNP expression level in acute A
and chronic B stages is similar to that of the healthy controls, whereas it increases in acute
and cirrhotic B patients, though the extent of the increase seems less relevant than that of the
corresponding HCV stage.
The HNP transcription was compared between patients with hepatocellular carcinoma
associated to HCV infection and two patients with hepatic carcinoma of non-viral origin (Fig.
4.11). Due to the limited sample size, the result of this analysis may only suggest that the
highest expression level is a distinguishing feature of HCV-originated HCC.
All the above data, obtained by quantitative real time PCR, were confirmed by ELISA and
antimicrobial assays.
44
45
30
15
cirrhosis B
cirrhosis C
chronic B
chronic C
acute B
acute C
acute A
0
healthy controls
HNP transcriptional level (HNP/GAPDH)
60
Fig. 4.10. HNP transcriptional level in PBMC from healthy controls and from patients at
different stages of infection by hepatitis C, A and B viruses. Mean values and
corresponding standard errors for each class are indicated.
Healthy
controls
HCC (HCV-)
HCC (HCV+)
1
10
100
1000
relative HNP m RNA level
Fig. 4.11.
HNP trancriptional level in PBMC from healthy controls and from patients with
hepatocellular carcinoma (HCC) of viral (HCV+) and non-viral (HCV-) origin. Data
were obtained using relative quantitation method and Sybr Green detection.
Overall, these result indicate that, though an increased HNP expression level may be
associated to a generic pathological condition, a much more significant increase seems to be
characteristic of HCV-linked pathologies.
45
4.3 Expression profile of other immune-related genes in HCV-patients
To better understand the characteristics of the immune response to HCV, we decided to
study the expression of the genes encoding the cathelicidin antimicrobial peptide LL37 and
interleukin-15 (IL-15).
LL37 gene was chosen as a kind of negative control: this gene codes for a peptide that
shares with defensins the same fundamental role in human innate immunity, being an
important component of both the phagocyte and the epithelial defences systems; yet, the
structure of its promoter, led us to expect a rather different type of transcriptional regulation.
IL-15, a novel cytokine sharing many immunological activities with IL-2, is critical in the
defence against viral infections by activating NK cells (Waldmann and Tagaya Y, 1999). IL15 was a sort of positive control, as we actually expected an enhanced transcription for this
gene: in fact, it is known that the plasma concentration of this cytokine rises in HCV
infections and correlates strongly with disease progression; moreover, IL-15 promotes
transcription of α-defensin genes in NK cells, in vitro (Kakumu et al., 1997; Obata-Onai, et
al., 2002).
A quantitative real time RT-PCR, based on sensitive LUX primer detection and absolute
quantitation method, allowed us to study the expression of these genes at the transcriptional
level. The results obtained for each class of HCV patients, together with the trend of
expression already found for HNP genes, are shown in Figs. 4.12 and 4.13.
HNP/Gapdh
HNP or LL37 mRNA level .
120
(LL37/Gapdh)x100
100
80
60
40
20
0
healthy
controls
acute
chronic
cirrhotic
HCC
Fig 4.12. HNP and LL37 mean transcriptional levels in PBMC from healthy controls and
HCV patients at different stages.
46
HNP/Gapdh
(IL-15/Gapdh)x100
HNP or IL-15 mRNA level
120
100
80
60
40
20
0
healthy
controls
Fig. 4.13.
chronic
cirrhotic
HCC
HNP and IL-15 mean transcriptional levels in PBMC from healthy controls and
HCV patients at different stages.
No significant variation was found in LL37 expression (p>0.05 comparing healthy with
each class of diseased subjects); conversely, a relevant increase in transcription of IL-15 gene
was observed: p(healthy/chronic)<0.001, p(healthy/cirrhotic)<0.05, p(healthy/HCC)<0.05.
Moreover, the trend of IL-15 transcription seems to reflect that of HNP genes. IL-15
transcription was found to be significantly higher also in chronic B hepatitis patients (data not
shown), with a mean level that is very close to that found in HCV chronic subjects. It is
noteworthy, however, that in the case of chronic B hepatitis, the higher IL-15 expression is
not accompanied by a parallel increase of α-defensins.
The absolute level of transcription of both LL37 and IL-15 was found to be about 100 fold
lower than HNP genes: this feature can be partly explained considering that cytokines act at
very low concentrations, resembling, for this aspect, the function of hormones. For LL37, the
low level of expression can be due either to the lack of appropriate stimuli, or to very poor
expression by the types of cells (mostly lymphocytes) contained in the PBMC preparation.
The low expression of LL37 was confirmed also by the lack of any peak corresponding to its
molecular mass (≈4.5 kDa), when analyzing the peptide extracts by MALDI ToF.
The expression of HNP, LL37 and IL-15 was studied in an acute HCV patient undergoing
spontaneous viral clearance: the greatest modulation was observed for HNP and IL-15 genes,
with little or no variation for LL37 (Fig 4.14). This finding suggests that both IL-15 and HNP
are somehow associated to HCV pathology, whereas LL37 is not.
47
mRNA level
50
HNP/Gapdh
40
(LL37/Gapdh)x100
IL-15/Gapdh
30
20
10
0
acute C
aviremic acute C
Fig. 4.14. Transcriptional levels of HNP, LL37 and IL-15 in an acute C patient before
and after viral clearance.
HNP or LL37 mRNA
level
A strong modulation for LL37 gene was found only when studying control patients with
bacterial infections, such as meningitis (Fig. 4.15), suggesting the involvement of this AMP in
the immune response to this kind of disease. In that case, transcription of both HNP and LL37
was found to rise significantly.
This finding implies that HNP transcriptional enhancement is not specific of immune
response to HCV infection. Moreover, it may explain the raise in the plasmatic level of
defensins, observed during bacterial infections (Ihi et al., 1997), as the result of a de novo
synthesis of these peptides, rather than as a consequence of a more intense neutrophil activity.
10000
HNP/Gapdh
(LL37/Gapdh)x100
1000
100
10
1
healthy controls
Fig. 4.15.
bacterial infection
HNP and LL37 transcriptional levels in PBMC from healthy controls and
from patients with bacterial infections.
4.4 HNPs expression in HIV patients
In this study, we included also a control group of ten patients with HIV infection. The
expression of both HNP and LL37 genes was studied by qRT-PCR in PBMC isolated from
these individuals: results are shown in Fig. 4.16.
48
HNP or LL37 transcriptional
level
HNP/GAPDH
1000
(LL37/GAPDH)X100
100
10
1
HIV patients
healthy controls
Fig. 4.16. Mean HNP and LL37 transcriptional levels in PBMC from HIV+patients and
healthy controls.
Mean HNP/GAPDH values and standard errors are 149±39 for HIV positive patients, and
4.4±0.9 for healthy controls; means for LL37/GAPDH are 0.125±0.03 and 0.036±0.02 for
HIV patients and controls, respectively.
Both HNP and LL37 transcriptional levels were found significantly higher in cells from
HIV-infected patients when compared to the values of the healthy controls: p<10-6 for HNP,
and p<0.05 for LL37.
The overexpression of HNPs is of particular interest, since these peptides have been
shown to have clear anti-HIV 1 properties; what is more, HNPs have been identified as
components of the CD8+ T cell antiviral factor, which is thought to play a fundamental role in
delaying AIDS symptoms in the so called HIV-non-progressor patients. More recently,
Trabattoni et al. (2004) have examined α-defensin expression in PBMC and CD8+ T cells and
found that the expression level was up to 10-fold higher in HIV-exposed seronegative patients
than in low risk healthy controls.
On the whole, our preliminary data appear to confirm that HNPs might have an important
function in immune response to this kind of viral infection; besides, they suggest that αdefensin overexpression is an index of immune activation in response to exposure to HIV,
rather than a genetically predetermined feature. More studies, are needed to understand HNP
specific role in this pathology, as well as the mechanism/s that might elicit their expression.
4.5 HNPs expression in selected cell subsets
Our PBMC preparations typically consisted of T and B lymphocytes, with a low
percentage of monocytes (≈10%) and less than 5% of neutrophils. Sensitive techniques, such
as double immuno-staining and RT-PCR, have already been used to demonstrate the
expression of HNP-1 and 3 by monocytes, B cells and NK cells (Agerberth et al., 2000). We
therefore sought to verify whether HNP are expressed also by the other cell types composing
PBMC. By qRT-PCR, we assessed the expression of HNP and LL37 genes in CD4+ and CD8+
T lymphocytes (purity >95%), and in neutrophils (Fig. 4.17). The α-defensins are expressed
by all these cell types with the lowest level found in neutrophils; conversely, LL37, whose
transcriptional level is much lower than HNP, is mainly expressed in neutrophils, with very
little mRNA in CD4+ and CD8+ T lymphocytes.
49
HNP/Gapdh
HNP or LL37 mRNA level
8
(LL37/Gapdh)x100
7
6
5
4
3
2
1
0
PBMC
Fig. 4.17.
CD4+
CD8+
Neutrophils
Transcriptional levels of HNP and LL37 genes in the whole PBMC and selected
cellular subsets from healthy individuals.
These results agree with our expectations: HNPs are massively expressed by neutrophils
only over a limited period of time corresponding to the stage of granulocyte precursor; once
differentiated, neutrophils contain high levels of defensins peptides but transcription of their
genes, at least in healthy individuals, is low and comparable to that of T lymphocytes.
Overall, these data support that peripheral leukocytes retain substantial ability to transcribe
HNP genes and to synthesize HNP peptides, in accordance with other recent works ( Fang et
al., 2003).
On the other hand, LL37 is poorly expressed by T lymphocytes and the low level we have
found in PBMC is likely to be largely due to contaminating neutrophils; with respect to the
low expression level in T cells, our data agree with previous findings (Agerberth et al., 2000).
Transcription of HNP in CD8+ cells has been questioned by many authors (Lehrer, 2004),
though Levy and colleagues have recently excluded that this type of T lymphocytes might
produce defensins (Mackewicz, et al. 2003). Yet, we are quite confident of our results:
considering the purity of the CD8+ cells (>95%), the intense signals obtained in real time
PCR, both using sensitive Taq Man probe and confirming specificity further by Sybr Green
melt curve analysis, cannot be solely due to an original contamination of the cells.
4.6 Correlation between HNP level and liver damage
In 90 patients with chronic HCV infection the HNP expression was studied in relation to
the histological damage, as determined by liver biopsy.
The antibacterial activity of peptide extracts from PBMC of patients with chronic C
hepatitis was found to rise progressively in correlation with liver fibrosis (Fig. 4.18). In
patients with moderate stage of fibrosis (stage 0-2), the median value of antibacterial activity
was 612 CAU/106 cells (mean=627, SD=140, range=400-888), whereas in those with severe
stage of fibrosis or cirrhosis (stage 3-4), the median value was 835 (mean=891, SD=221,
range=426-1342). Moreover, in patients with hepatocellular carcinoma, the median value of
antibacterial activity was 1104 CAU/106 (mean=1260, SD=560, range=630-2360). By using
the Wilcoxon rank sum test, we found higher CAU levels in patients with stage 0-2 compared
with controls (p=0.046, z=–1.992); in patients with stage 3-4 higher than in those with stage
0-2 (p<0.001, z=-3.563); in patients with hepatocellular carcinoma higher than in stage 3-4
(p=0.012, z=-2.51).
50
3000
6
CAU/10 cells
2000
1000
0
controls stage 0-2 stage 0-4 HCC
Fig 4.18. Antibacterial activity in peptide extracts from PBMC of healthy controls and patients
with a different stage of liver fibrosis. The antibacterial activity is expressed as
cecropin A units per cell number (CAU/106 cells). The top and the bottom of boxes
represent the 25th and 75th percentiles. The line in the middle of the box is the median.
The whiskers represent the values within 1.5 times the interquartile range from the
upper or lower quartile.
Our data also showed a very strong linear correlation between the stage of fibrosis and the
CAU values (r=0.706, p<10-6).
A similarly strong correlation was found even when considering the HNP levels as
detected by ELISA (Fig 4.19). HNP levels from healthy controls showed values ranging from
0.1 to 0.4 ng/106 cells (median value=0.2, mean value= 0.217, SD=0.09). In patients with
stage of fibrosis 0-2, the median value was 0.6 ng/106 cells (mean=0.72, SD=0.45,
range=0.25-2.5), whereas in those with stage of fibrosis 3-4, the median value was 1.1
(mean=1.29, SD=0.59, range=0.5-2.6). Moreover, in patients with hepatocellular carcinoma,
the median value of HNP levels was 1.78 ng/106 cells (mean=1.94, SD=1.14, range=1-4). By
using the Wilcoxon rank sum test, we found higher HNP levels in patients with stage 0-2
compared with controls (p=0.08, z=–2.669); in patients with stage 3-4 higher than in those
with stage 0-2 (p<0.001, z=-3.580); in patients with hepatocellular carcinoma higher than in
stage 3-4 (p=0.049, z=-1.961). We found a statistically significant linear correlation between
HNP levels and stage of fibrosis (r=0.59, p<106).
51
5
HNP level ng/106 cells
4
3
2
1
0
-1
controls stage 0-2 stage 0-4 HCC
Fig 4.19.
α-Defensin levels, as detected by ELISA, in PBMC of healthy controls and patients
with a different stage of liver fibrosis. The top and the bottom of boxes represent
the 25th and 75th percentiles. The line in the middle of the box is the median. The
whiskers represent the values within 1.5 times the interquartile range from the
upper or lower quartile
We also measured the α-defensin mRNAs in the same PBMC samples and found a direct
correlation between the antimicrobial activity and the number of HNP transcripts (r=0.51,
p<10-6) and between the HNP levels and the number of HNP transcripts (r=0.493, p<0.001).
On the basis of this correlation between HNP levels and liver damage, α-defensins might
be prospectively used as helpful biological markers to stage disease progression.
4.7 In vitro experiments on PBMC
Effect of HCV C, Cyclosporin A and the core protein of hepatitis B virus
In order to assess the potential effect of HCV core protein (HCV C) on enhancing the
expression of HNPs, we performed a series of in vitro experiments in which freshly isolated
PBMC were incubated in RPMI medium with or without the addition of a recombinant HCV
C (2 µg/ml). To verify the possibility of an involvement of NFAT in the transcriptional
activation of the α-defensin, HCV C stimulated PBMC were pre-treated with cyclosporin A
(CsA, 1 µg/ml), an immunosuppressive drug that specifically inhibits the NFAT pathway, by
preventing calcineurin-dependent dephosphorylation. Moreover, the specificity of the HCV C
effect was established by incubating PBMC with the recombinant core protein of the hepatitis
B virus, HBcAg (2.5 µg/ml). The response of the cells was investigated at both transcriptional
and peptide level, by real time quantitative RT-PCR and ELISA, respectively. The results of
these experiments are shown on Figs. 4.20. and 4.21.
Upon incubation with HCV C, HNP transcription rose markedly (up to five-fold increase)
in PBMC from both diseased and healthy individuals. The experiment was repeated several
times on PBMC from healthy controls and twice in PBMC from chronic C patients, with
similar results. An enhancement of transcriptional level was detected also in PBMC from
52
Relative HNP transcriptional
level
acute C patients (data not shown). The increase in the transcription level was a fast response,
since it become evident already after 30 min incubation with HCV C, as detected in
preliminary time-course experiments. The same studies indicated that the maximal
transcriptional induction was reached after 3-4 h of incubation (Fig. 4.20).
6
PBMC from chronic C
patient
5
PBMC from healthy
control
4
3
2
1
0
HCV C
CsA
HBcAg
Relative HNP transcriptional level in HCV C, CsA and HBcAg–treated PBMC
from chronic C patient and healthy control. The relative transcriptional level of
HNP genes is defined as the ratio of the transcriptional level observed in the
stimulated cells normalized to that of the untreated cells (negative control).
Relative HNP level
Fig. 4.20.
CsA+ HCV C
2,5
PBMC from chronic C
patient
2,0
PBMC from healthy
control
1,5
1,0
0,5
0,0
HCV C
Fig. 4.21.
CsA + HCV C
CsA
HBcAg
Relative HNP concentration in peptide extracts from HCV C, CsA and HBcAgtreated PBMC from chronic C patient and healthy control. The HNPs level was
detected in PBMC peptide extracts by ELISA test. The relative HNP level was
obtained normalizing values observed in treated cells to those of the untreated cells
(negative control).
We could notice that the induction of HNP expression was generally greater in cells from
HCV patients, especially in acute stage, rather than in those from healthy individuals.
Nonetheless, since cells from both diseased and healthy individuals could respond to HCV C
induction, it is conceivable that this type of reaction is initiated by innate immune
53
mechanisms and is not exclusive of individuals who have experienced HCV infection and
mounted an adaptive immune response to it.
The induction of α-defensins expression was clearly inhibited upon CsA treatment, prior
to HCV C addition; while CsA alone kept the transcriptional levels close to those observed in
the untreated sample. These data suggest that the α−defensin synthesis induced by HCV C
and, presumably, by hepatitis C virus infection, may be mediated by the NFAT pathway.
Moreover, the up-regulation of the HNP genes seems a selective response to HCV C, as
no variation in α-defensins mRNA was observed in PBMC upon incubation with HBcAg, a
protein that shares with HCV C both the structural function and immunomodulatory
properties (Manigold et al., 2003; Hyodo et al., 2004).
Overall, the results obtained by qRT-PCR were confirmed by ELISA at the peptide level
(Fig. 4.21), even if the inducing effect of HCV C was less evident and a slight increase in
peptide level was discerned also upon treatment with the recombinant HBcAg.
The presence of the three major isoforms of HNPs, in the analysed samples, was
confirmed by MALDI-ToF spectrometry.
Effect of phorbol 12-myristate 13-acetate, ionomycin and phytohemagglutinin
The potential involvement of NFAT in transcriptional control of HNP genes was further
investigated by evaluating the expression of both HNP and IL-2 genes upon treating PBMC
with combinations of HCV C, CsA and canonical activators of NFAT cascade, such as
phorbol 12-myristate 13-acetate (PMA) and ionomycin (phorbol esters activate protein kinase
C, while ionophores, such as ionomycin, trigger intracellular calcium release, hence
concurring to activation of the NFAT pathway). The effect of the T cell mitogen,
phytohemagglutinin (PHA), was also studied: this lectin triggers T-cell activation and IL-2
production by binding non-specifically to the cell surface receptor complex; the combination
of PHA and PMA results in a greatly increased IL-2 production (Manger et al., 1986; Liu et
al., 1992). IL-2 gene was chosen as positive control, for it is classically known to be
transcriptionally regulated via NFAT.
Relative transcriptional levels of HNP and IL-2 genes were assessed by real time qRTPCR and were calculated dividing the level detected in treated cells by that observed in
untreated (negative control) cells: hence, values greater than 1 indicate an increased
transcription, while values equal to or minor than 1 indicate no variation or decreased
transcription, in relation to untreated cells. Transcriptional level of IL-2 gene was studied
using LUX primer-detection and a relative quantitation method. Data are reported on Fig.
4.22.
54
1000
IL-2
100
10
PH
A
A
A+
PH
sA
PM
M
+P
M
A+
io
n)
+C
A+
io
n
sA
+C
C+
P
V
(H
CV
C
HC
(P
M
A+
io
n)
V
HC
A+
io
n
V
HC
0,1
PM
C+
Cs
A
1
C
Relative mRNA level
HNP
Fig. 4.22. Relative transcriptional level of HNP and IL-2 genes in PBMC from acute C patient,
following incubation with HCV C, CsA, PMA, ionomycin (ion) and PHA, as
indicated on the x axis. Dotted boxes highlight data obtained combining NFAT
inducers with the inhibitor (CsA).
Both genes showed a similar trend of transcription. HCV C alone induced not only HNP,
but also IL-2; by adding CsA, transcriptional level decreased. NFAT inducing agents, PMA
and ionomycin, up-regulated the expression of both genes and the addition of CsA made the
level lower again: the effect of this treatment on IL-2 may appear obvious, since several other
works have already demonstrated it; yet, in our case it was a sort of cue reassuring us that the
conditions used in our in vitro system were correct.
The combination of HCV C and canonical activators resulted in enhanced transcription,
even if at level not so high as with PMA and ionomycin alone; pre-treatment with CsA again
inhibited the inducing effect. PHA, alone or combined with PMA, determined the
transcriptional activation of IL-2 and HNP.
On the whole, HNP and IL-2 transcription seem to vary accordingly, suggesting that a
common mechanism, possibly NFAT-mediated, might control both genes. However, addition
of HCV C to PMA and ionomycin did not result in a further enhance of transcription, which
rather decreased when compared to PBMC treated only with PMA and ionomycin: this might
be due to a perturbing effect of the combination of more drugs, possibly even a feed-back
mechanism of control; on the other hand, HCV C might trigger a transcriptional cascade, only
partly overlapping with that induced by PMA and ionomycin. In agreement with this latter
hypothesis, we found that a general inducer of T cells activation and proliferation, such as
PHA, can trigger HNP transcription. These observations induce us to speculate that rather a
complex mechanism, probably not only NFAT-mediated, might control HNP genes.
Another interesting feature is that IL-2 gene is much more strongly modulated in
comparison to HNP. IL-2 transcriptional level was found extremely low in unstimulated cells,
as it could be detected only in a very few naive PBMC samples (from both healthy and
diseased individuals) and, mainly, in those obtained from acute C and B hepatitis patients.
The transcriptional level was greatly enhanced, and thus easily detected, after appropriate cell
treatment. This finding corresponds to data already available in the scientific literature: IL-2,
in fact, seems to be produced at appreciable levels only in particular physiological conditions
and especially in acute inflammation states (Hartel et al., 1999).
55
Selectivity of HCV C induction
In order to understand the cellular response to HCV C treatment, the effect on the
expression of LL37 and IL-15 genes was investigated, at the transcriptional level, and
compared to the modulation already observed for HNP and IL-2. The expression of these
genes was monitored by real-time RT-PCR in HCV-treated PBMC from both healthy
individuals and acute C patients. The transcriptional levels observed in treated PBMC were
normalized to those found in untreated PBMC. Data are shown in Fig. 4.23.
Relative mRNA level
8
7
6
5
4
3
2
1
0
HNP
Fig. 4.23.
IL-2
IL-15
LL37
Relative transcriptional levels of HNP, IL-2, IL-15 and LL37 genes in HCV Ctreated PBMC.
The greatest modulation was observed for the two cytokine genes; to a minor extent, also
HNP transcription was enhanced, whereas no appreciable variation was detected in LL37
expression. These data, deriving from in vitro experiments, seem to agree with what was
already assessed when studying the expression profile in PBMC freshly isolated from HCV
patients: transcription of HNP and IL-15 was found significantly higher, while non-relevant
variation was noticed for LL37.
4.8. HNP expression profile in HCV patients under antiviral therapy
HNP expression was studied in PBMC from chronic C patients undergoing a
pharmacological treatment based on interferon-α (INF-α), an antiviral drug that inhibits viral
replication and stimulates the immune response of the host. The profile of expression was
monitored over a period of time ranging from 2 to 9 months after treatment beginning. Data
obtained by real time qRT-PCR are reported in Fig. 4.24. With the exception of patient 2, we
can notice a general trend toward a clear drop of α-defensin transcription, especially in the
first two-three months of therapy. Moreover, the expression levels tend to stabilize in the
range of values observed for the healthy controls.
56
H N P m R N A le v e l
75
p1
p2
60
p3
p4
45
p5
p6
30
15
Healthy
range
0
-1
0
1
2
3
4
5
6
7
8
9
10
months
Fig. 4.24.
HNP transcriptional profile in six chronic HCV patients (p1→
→p6) undergoing
therapy.
HNP mRNA level
(HNP/GAPDH)
150
120
90
60
30
0
before therapy
Fig. 4.25.
2 m onths after
therapy
HNP transcriptional modulation in an HCC patient under INFα/ribavirin-combined therapy.
A similar result was found in a patient with HCC, undergoing a more severe therapy,
based on INF-α combined with ribavirin (Fig. 4.25). The HNP transcriptional level was
measured before and after treatment.
These profiles of expression strengthen the hypothesis that α-defensins are somehow
associated to HCV pathogenesis, and that their expression may be stimulated just by the
presence of the virus. In an attempt to explain such a trend, we can therefore assume that INFα, by preventing HCV replication, might indirectly contribute to limit α-defensins expression.
In perspective, it would be worth evaluating the expression profile of HNP genes in
relation to the type of response that the patients develop as a consequence of the therapy.
57
5 CONCLUSIONS
The studies undertaken in the present thesis allow us to draw some relevant conclusions.
i) A significant increase of HNP expression in PBMC from HCV-infected individuals was
demonstrated. This was verified by multiple approaches based on real time qRT-PCR,
ELISA and biological activity analyses. The HNP over-expression might be either a direct
or indirect effect of the presence of the virus: in the former case, over-expression could be
mediated by HCV core protein, which was found to stimulate HNP genes transcription in
vitro. In the latter, the phenomenon could be cytokine-mediated, for instance IL-15mediated, as we found this cytokine being over-expressed in HCV-patients, as well as
being able to enhance HNP genes transcription in vitro. Possibly, both types of
mechanisms are involved in this up-regulation: a direct effect of the virus is particularly
likely during the stages of high viraemia, such as during acute infection; conversely, in the
phase of chronicization, cytokines and other immune factors may contribute to maintain
high HNP levels.
ii) The elevated levels of α-defensins, their correlation with liver fibrosis progression, and the
pattern of expression in patients under therapy, all suggest the involvement of these
antimicrobial peptides in HCV pathogenesis. It would be interesting to know the kind of
role played by HNPs in this context. HNPs might be protective and thus have a positive
function for the host organism, giving that they are antimicrobial and antiviral, though a
direct activity against HCV has not been documented yet. At the same time, HNPs might
be even disadvantageous for the host: at physiologic concentrations, in fact, α-defensins
promote tissue healing by triggering fibroblast proliferation and deposition of extracellular
matrix (Murphy et al., 1993; Oono et al., 2002). Therefore, it is possible that, at
pathological concentrations, that is when over-expressed, as in this case, α-defensins
might increase fibrogenesis and enhance substitution of functional hepatic tissue with
fibrotic non-functional tissue.
Whatever the function of α-defensins in HCV pathogenesis, their involvement raises
particular interest from a clinical point of view: firstly, it represents a further little step in
understanding the pathogenesis of this infection, which is still rather obscure, under
several aspects; secondly, it opens up perspectives for new, complementary diagnostic
tools and possible therapeutic targets, in a disease for which no plainly successful therapy
has been found, yet.
iii) Our data support the participation of NFAT to the transcriptional regulation of HNP
genes. NFAT is a major factor controlling several immune-related genes, including genes
encoding AMPs; however, its involvement in the control of α-defensin genes is a novel
aspect, since, so far, the regulation of their expression has been mainly studied in relation
to cell differentiation and maturation processes.
The position of the putative NFAT core site in the promoter of HNP genes is peculiar, in
that it is overlapping with an Ets-like regulatory element, which was demonstrated to be
essential for α-defensin expression during myeloid blood cell differentiation (Ma et al.,
1998). However, adjacent and/or overlapping Ets and NFκB/NFAT binding sites have
been identified in the promoters of several inducible lymphoid genes, suggesting that
interaction between members of these families of transcription factors might be an
evolutionary conserved mechanism for regulating immune-related genes (Bassuk et al.,
1997). We can therefore speculate that such a cooperative mechanism might operate also
for HNP genes.
It is noteworthy that in the promoter of the gene encoding the chemokine
CCL5/RANTES there is a potential NFAT site: interestingly, also this gene is over58
expressed in chronic hepatitis C, in such a way that positively correlates with the severity
of hepatic inflammation (Apolinario et al., 2002; Melchiorsen et al., 2003). The overexpression of CCL5 was detected in hepatic cells, which represent the main target of the
virus; it is plausible that, if HCV promotes HNP transcription via NFAT, we should
expect a more considerable effect on the dysregulation of these genes, just at the hepatic
level, that is in infiltrating T-lymphocytes, as well as hepatocytes.
iv) Our study confirms the immunomodulatory properties of the HCV core protein: in this
specific case, HCV C was shown to function - and possibly trigger NFAT pathway - even
exogenously with respect to the affected cells.
This is of particular concern, as this viral protein may be released in the blood stream of
infected patients (Kanto et al., 1995; Sabile et al., 1999); consequently, by taking
advantage of this strategic location, HCV C could modulate gene expression even in nondirectly infected-cells, and also in tissues and organs which are distant from the primary
site of infection.
59
6 ACKNOWLEDGMENTS
I wish to thank my tutor, Prof. Donatella Barra, whose extraordinary human and
professional qualities I could appreciate during the years I have spent in the Department of
Biochemical Sciences, University of Rome La Sapienza.
I am grateful to Prof. Maurizio Simmaco, for his constant and contagious enthusiasm and
for giving me the opportunity to work in a brand new laboratory, in a stimulating
environment.
I thank Dott. Maria Luisa Mangoni, for the technical and moral support, and for her
friendly and precious collaboration.
I thank the Coordinator Prof. Paolo Sarti and the teaching body of the Ph.D course, for
their contribution to my scientific formation.
I thank Prof. Antonio Aceti and his collaborators, especially Dott. Barbara Zechini, for
contributing to the realization of this study.
A big thank goes to all my lab mates, for their help and for having created a nice and
enjoyable atmosphere, which has been so important to me, particularly during my “rainy
days”.
Finally, I thank my family, my parents and Peppe, for their encouragement and sweet
patience: to them I dedicate this thesis.
60
7
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8
PUBLICATIONS
Mangoni ML, Fiocco D, Mignogna G, Barra D, and Simmaco M. (2003) Functional
characterization of the 1-18 fragment of esculentin -1b, an antimicrobial peptide from
Rana esculenta. Peptides, 24, 1771-1777
Simmaco M, Mangoni ML, Miele R, Borro M, Fiocco D, and Barra D. (2003) Defence
peptides in the amphibian immune system. Bacterial, Plant & Animal Toxins, 155-167.
Carzaniga R, Fiocco D, Bowyer P, and O’Connell RJ. (2002) Localization of melanin in
conidia of Alternaria alternata using phage display antibodies. Mol. Plant-Microbe
Interactions, 15, 216-224
Miele R, Borro M, Fiocco D, Barra D, and Simmaco M. (2000) Sequence of a gene from
Bombina orientalis coding for the antimicrobial peptide BLP-7. Peptides, 21, 16811686
69