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This research reports have been presented and disscussed at the CNR- UNESCO Workshop held in Ischia, Italy on 18-20 april 1999 and represent works in progress of ICAERI members.
Section 1
Immunopathology of HIV infection
1.1 Lymphocyte Activation During Acute SHIV89.6PD Infection in Macaques
Member: M. Wallace
Participants: Paul M. Waterman, Jacque L. Mitchen, Mahmoud Djavani, Parul Trivedi, Douglas Horejsh, Marta Dykhuizen, Moiz Kitabwalla, C. David Pauza
Host virus interactions control disease progression in HIV infected persons. These interactions evolve rapidly during acute infection and are key to the mechanisms for viral persistence and AIDS. We study rhesus macaques infected with SHIV89.6PD, a chimeric simian-human immunodeficiency virus, as a model of HIV infection. SHIV89.6PD can deplete CD4+ T cells from peripheral blood, spleen, and lymph nodes within two weeks after exposure and serves as a model for virulent, acute infection. Lymphocytes isolated from macaques during the interval of acute SHIV89.6PD infection have lost the capacity to proliferate in response to phytohemagglutinin (PHA).
T cell unresponsiveness to mitogen occurred within one week after mucosal inoculation yet prior to massive CD4+ T cell depletion and extensive virus
dissemination.
The lack of mitogen response is at least partially due to apoptosis of CD4+ T cells in vitro. An investigation of activation marker expression on peripheral blood T cells from infected macaques demonstrated increased surface expression of CD25, early activation marker CD69 and HLA-DR on CD4+ T cells during acute infection. All together, these observations indicate that activation induced cell death may explain both the hyporesponsiveness to mitogen and the rapid in vivo depletion of CD4+ T cells during acute infection. High levels of lymphocyte activation may in turn enhance SHIV89.6PD replication, thus increasing the loss of CD4+ T cells and diminishing the levels of virus-specific immunity that remain after acute infection. The level of surviving immunity dictates the capacity to control virus replication and disease progression. We describe this level of immune competence as the “host set-point” to show its pivotal role in AIDS pathogenesis.
1.2 “High fasL predisposes rhesus macaques to rapid AIDS progression”
Member: Maria Salvato
Participants: Cheng Yin, Dan Schmidt, Hideo Yagita, Dave Pauza
My lab has discovered that uninfected animals often have lytic activity to cells expressing SIV envelope protein and that this lytic activity is not MHC-restricted. We screened 56 uninfected monkeys and found half of them had high lytic activity and half had low MHC-unrestricted lytic activity to SIV env. We infected 11 animals and found that those with high lytic activity died more rapidly than those with low lytic activity (J. Virol. 1999, in press). This indicated that high lytic activity does not simply destroy virus-infected cells, but has a more devastating effect on the organism. We characterised the lytic activity to be mediated by FasL and its receptor
Fas, which was activated by the expression of env in target cells. Lysis could be blocked with antibody to FasL.
In a collaboration with Dr. Hideo Yagita (in Japan), we have tested the efficacy of an antibody treatment on AIDS disease progression. The antibody is Nok-2 which binds FasL, the mediator of cell death. Using four monkeys preselected for high FasL (which correlates with rapid AIDS progression), we have injected them intravenously prior to SIV challenge and for 5 weeks thereafter. The animals are in their 4th month of study and they have low antigenemia, and, as predicted, none show signs of rapid disease progression.
1.3 Activities of gamma/delta T-cells in vitro and in vivo: a basis for novel vaccines
Member: M. Malkovsky
Participants: V. Colizzi, F. Dieli, F. Martini, A. Pollak, A. Salerno, A. Sen, G. Sireci, M. Wallace, P. Wang, F. Poccia
Protection from exogenous invaders and elimination of endogenous aberrations are the main contributions of T cells to homeostasis. Vgamma9/Vdelta2 -encoded T-
cell receptors (TCR) have a unique ability to interact with a plethora of nonpeptidic phosphoantigens in an MHC-unrestricted manner. The biological function of this recognition is largely unclear. However, since Vgamma9/Vdelta2 T cells are the most potent cytotoxic effectors capable of killing target cells infected with
numerous viruses (including HIV, SIV, herpes viruses and many others), it is likely that they are involved in antiviral immunosurveillance. The findings of profound quantitative and functional changes in gamma/delta T cells associated with various infectious diseases (including those caused by viruses) are compatible with this hypothesis. For example, the overall level of gamma/delta T-cell activation at different stages of HIV infection may be clinically relevant. At an initial stage of HIV infection, the extremely potent antiviral cytotoxic activities of Vgamma9/Vdelta2 cells may limit the viral spread. At later stages of disease, Vgamma9/Vdelta2-cell dysfunction may contribute to the loss of resistance to opportunistic pathogens (such as atypical mycobacteria) and neoplasms (e.g., lymphomas) frequently associated with HIV infections.
Also, it is possible that chronic stimulation of gamma/deltaT cells may result in immunopathology. In particular, the massive immunologic activation that appears to be the major contributing element of AIDS pathogenesis could be, at least in part, driven by gamma/delta T cells overstimulated by repetitive exposures to HIV.
The main long-term objective of our work is to define the role of gamma/delta T lymphocytes in SIV/HIV infections and utilise this information in a) designing
nonpeptidic vaccines and b) AIDS immunotherapy trials. We have developed methods for in vivo sensitisation of rhesus macaque (Macaca mulatta)
Vgamma9/Vdelta2 T cells.
Specifically, intravenous injections of nonpeptidic antigens (DPG) induced an activated state of simian Vgamma9/Vdelta2 T cells which decreased after two months. The activation (proliferation and IFN-gamma and TNF-alpha production) was detectable with various phosphoantigens. Subsequent tests 12 months later have shown that four out of five DPG-treated monkeys developed anergy or hyporesponsiveness to nonpeptidic antigens.
In addition, we have investigated the role of adhesion/costimulatory molecules in Vgamma9/Vdelta2 responses to phosphoantigens. The disruption of the CD2/LFA-3 interaction by blocking antibodies resulted in inhibition of Vgamma9/Vdelta2 proliferation and TNF-alpha secretion. However, the ability of Vgamma9/Vdelta2 T cells to lyse susceptible targets was not blocked by CD2 or LFA-3 antibodies. In contrast, the LFA-1/ICAM-1 interaction was required for cytotoxic activities, but its disruption did not influence substantially the DNA synthetic response of and TNF-alpha secretion by Vgamma9/Vdelta2 T cells.
1.4 MHC-unrestricted lymphocytes in natural immunity against HIV-infection
Member: F. Poccia
Participants: C. Agrati, C. Gioia, R. Casetti,V .Colizzi
The reactivities of Vg9Vd2 T cells induced by different stimuli are controlled by receptors for MHC class I molecules. Specifically, these cells express the inhibitory CD94/NKG2-A receptor for HLA class I molecules. The Vg9Vd2 T-cell regulation through the CD94 receptor may be important for the potentially dual function in innate immunity, i.e., NK-like and TCR ligand-induced cytolitic activities. gd CTLs are activated by HIV infection and represent one of the most important killer of virus-infected cells. The cytopatic effect of HIV may induce the release of nonpeptidic compounds able to stimulate the gd TCR. However, the cytolitic pathway is TCR-independent but involves specific NK-receptors including the CD94/NKG2-A. Moreover, gd T cell activation with nonpeptidic phosphoantigens results in potent inhibition of HIV replication through soluble-released factors. The phosphoantigen-activated gd T cells produce a substantial amount of b-chemokines (MIP-1a, MIP-1b and RANTES), known to represent the natural ligand for CCR5 HIV coreceptor. Accordingly, anti-b-chemokine antibodies neutralised the inhibition of monocytotropic HIV strain by gd T cell released factors. Finally, a T-tropic HIV strain using the CXCR4 co-receptor for virus entry was also potently inhibited. Altogether, gd T lymphocytes may play a relevant antiviral role through both cytolitic and noncytolitic pathways: a) the release of
soluble antiviral factors including chemokines; and b) a direct cytotoxic effect induced by TCR-stimulation and controlled by NK receptors.
1.5 gd T anergy in HIV-infected persons with opportunistic infections and recovery after highly active antiretroviral therapy
Member: F.Martini
Participants: R. Urso,C. Gioia, A. De Felici, P. Narciso, A. Amendola, M. G. Paglia, V. Colizzi, F. Poccia
gd T lymphocytes recognise nonpeptidic microbial antigens without
antigen processing and MHC restriction, representing an early
defence-line against invader pathogens. Since a defective response to nonpeptidic antigens was observed in HIV+ persons, the aims of this study were: a) to analyse the involvement of gd T cell anergy on the increased susceptibility to other pathogens; b) to evaluate the role of highly active antiretroviral therapy (HAART) on gd T cell functions.The gd T cell distribution and functional reactivity was analysed in 47 HIV+ persons: 28 asymptomatic (HIV-ASY) and 19 with opportunistic infections/co-infections (HIV-OIC).
In a longitudinal study the recovery of gd T cell reactivity after HAART was finally analysed. The gd T cell distribution was significantly altered in HIV-OIC patients when compared to HIV-ASY ones.
Specifically, the Vd2/Vd1 ratio was inverted as a consequence of a decrease in Vd2 T cell number. Moreover, IPP-stimulated Vd2 T cells from HIV-OIC group displayed a cytokine profile different from controls and HIV-ASY groups, with a major defect in IFN-g production.
The inability of Vd2 T cells to secrete IFN-g may contribute to the Th1 hyporesponsiveness and to the increased susceptibility to other pathogens in HIV patients.
Although Vd2 T cell anergy was observed in HIV infected persons independently of the viral load, HAART restored gd T cell reactivities to nonpeptidic antigens, extending the recovery of cell-mediated immunity to nonpeptidic microbial antigens.
Recent observations indicates that at least two mechanisms may be involved in the induction of Vg9Vd2 T cell anergy: a) an absence of TCR-mediated recognition through a process of clonal selection and b) the triggering of negative signals through inhibitory NK receptors.
1.6 Changes in the gamma delta T-cell repertoire after HIV-1 infection
Member: C. D. Pauza
Participants: Yin, C. Rakasz, E. Evans, P. Ruckwardt, T. Waterman, PM, Enders, P. Poccia, F. Martini, F. Malkovsky.
A principal consequence of chronic HIV-1 infection is declining immune competence with increasing susceptibility to opportunistic pathogens. Earlier research in this field concentrated on the direct effects of HIV-1 within infected CD4+ T cells and macrophages. These studies provided important insights into the control of virus replication but did not account for phenotypic changes occurring throughout these cell populations (among both infected and uninfected cells), and in other lymphocytic or monocytic cells that were not preferred targets for HIV-1 infection. Recent studies from our laboratories and others, showed profound effects of virus infection on macrophages, CD8+ T cells, B cells, and gamma delta T cells in addition to CD4+ T cells. In studies described here, we concentrate on gamma delta T cells to illustrate the substantial changes that can occur after HIV-1 infection, even when the cell subset is not a preferred target for infection.
We evaluated the population of T cells bearing the Vgamma9/Vdelta2 T cell receptor. MHC-unrestricted responses to pyrophosphate antigens are an important functional property of this T cell subset and are known to be altered as a consequence of HIV-1 infection. Among uninfected individuals, Vgamma9 shows a limited repertoire due to predominant use of a single J chain sequence (JP). Cells expressing the JP segment are likely to respond to pyrophosphate
antigens.
During HIV-1 infection, the Vgamma9/Vdelta2 population is decreased numerically and shows changes in the response to pyrophosphate antigens.
Examination of the expressed Vgamma9 sequences showed a substantial shift from the pattern in uninfected individuals, resulting in lower frequencies of cells
expressing the JP segment of the T cell receptor gene complex. Changes to the Vgamma9 repertoire were not accompanied by substantial effects on Vdelta1,
Vdelta2, or Vdelta3 sequences. Overall, the Vgamma9 repertoire became more diverse as a result of HIV-1 infection and these changes were accompanied by
alterations in functional properties of these cells.
The pattern of increased diversity among Vgamma9-expressing cells was observed in HIV-1 patients that represented a large spectrum of clinical disease stages (n = 18). These changes provide direct evidence for HIV-1 infection causing substantial changes in a non-target cell population. Ongoing studies of the changes in Vgamma9/Vdelta2 T cells during antiretroviral therapy will show whether these changes are reversible, and whether we might manipulate the gamma delta
population as one component in a strategy for immune reconstitution after HIV-1 infection.
1.7 HLA-E influence on antiviral cytotoxic activity and HIV-1 pathogenesis
Member: F. Poccia
Participants: F. Di Pietro, A. Amendola, C. Agrati, F. Martini, G. Mancino, M. Lopez-Botet, V. Colizzi
HLA influence on human pathogen sensitivity have been described and a strong HLA correlation with accelerated AIDS progression was recently reported
(Carrington M. et al., Science 1999). Since a rapid HIV-1 disease progression is correlated with decreased NK cell activity, we analysed whether certain HLA
molecules may be involved in the regulation of NK cell activity. PMBC from healthy donors and HIV-infected persons at different stage of disease progression (CDC
-A, -B or -C) were analysed for HLA expression and NK specificity (CD161, CD154, CD94 and NKB1). The expression of HLA molecules was analysed by flow
cytometry using a panel of monoclonal antibodies: pan HLA class I (W6/32), HLA-C (L31), HLA-G (HBF4) and HLA-E (3D12). In vitro HIV infection was performed on CEMx174 and U937 lines with HIV-1Lai and monitored as p24 release in the supernatants. The expression of HLA class I molecules was found to increase during HIV-1 disease progression and to correlate with CD4+ T cell numbers. Specifically, HLA-E was increased on the surface of CD4+ T cells and CD14+ monocytes from HIV-infected patients. We than analysed the influence of in vitro HIV-1 infection on the expression of HLA molecules on CEMx174 and U937 lines.
Although the total HLA class I expression was significantly reduced by in vitro HIV-1 infection, the expression of HLA-E remains stable on the surface of infected cells. Altogether, these data shows that HIV may discriminate among HLA class I alleles, inducing the down-regulation of HLA-dependent CTL epitopes and preserving the expression of HLA motifs with NK inhibitory activity. Thus, HLA allele expression may influence the regulation of natural cytotoxicity and AIDS pathogenesis.
1.8 HIV-1 nef interferes with antigen-specific proliferation of antigen-specific T helper cell lines
Member: C. Montesano
Participants: M. Guckian, J. B. Marriot, V. Colizzi, F. Cook, T. Reinhart, A. G. Dalgleish
The HIV-1 nef gene is known to play a prominent role in HIV
replication and pathogenesis. Nef protein has been shown to mediate down-regulation of cell surface CD4 and enhance of viral replication in primary T cells. The aim of the study was to investigate the effects of exogenous Nef protein on function of antigen presenting cells and on activation of antigen specific T helper cell lines. Cytokine production and expression of surface markers by recombinant Nef (rNef) treated antigen presenting cells was investigated. The proliferative response of PPD- and Tetanus Toxoid- specific T-cell lines to antigen pulsed PBMCs was studied. Autologous PBMCs were pulsed with specific antigen in the presence of recombinant Nef protein. A dramatic Nef-dependent decrease of antigen-specific proliferation was observed when PBMCs were simultaneously pulsed with antigen and rNef. This could be increased by preincubation of PBMCs with rNef. The decrease of antigen-specific proliferation was shown to be Nef-specific, as pulsing PBMCs in the presence of unrelated antigens failed to reduce antigen-specific cell proliferation.
The mechanism by which rNef protein reduces antigen-specific proliferation of treated PBMCs has not been elucidated. However, it is likely that the observed
results reflect changes in cell activation. This could result in release of a soluble factor or changes in surface antigen expression (e.g. down-regulation of a
costimulatory molecule). It is interesting to note that a proliferative defect is observed in CD4+ T cells isolated from asymptomatic HIV-infected individuals.
1.9 Accumulation of TNFa-Producing T Cells as a Consequence of Defective Regulation by Apoptosis During HAART. Relation with the Lipodystrophy Syndrome
Member: M-L. Gougeon
Participants: E. Ledru, H. Lecoeur, N. Christeff
We previously showed by single cell analysis, that chronic HIV infection is associated with an imbalance in the representation of Th1 cytokine T cell producers, characterised by the progressive disappearance of IL-2 producers, correlated with the progressive shrinkage of the CD4+CD45RA+ T cell compartment, and the decrease in TNFa-producing T cells, both correlated with a gradual increased susceptibility to activation-induced apoptosis (E. Ledru et al. J. Immunol. 1998, 160:3194-3206). We asked in this study whether the cytokine pattern can be normalised under HAART (2 RTI + 1 PI). Single cell analysis of cytokine producers was performed by flowcytometry in T cells after 16hr-stimulation with PMA+Ionomycine. The susceptibility to apoptosis of Th subsets was analysed concomittantly with the 7-AAD dye. In addition, seric concentrations of cytokines and lipids were determined. A longitudinal follow-up was performed on 15 HIV+ patients treated during 18 months with HAART. In addition, 39 HIV+ patients with = 9 months of HAART, 11 without lipodystrophy (LD-) and 18 patients with lipodystrophy (LD+) were compared. After 18 months of HAART, the percentages of IL-2 producing cells was increased in CD8+ but not in CD4+ T cells. The proportion of IL-2+ CD4+CD45RA+ cells remained lower in HAART-treated patients compared to healthy donors. Strikingly, a dramatic increase in the representation of both CD4 and CD8 TNFa-positive T cells was detected after 9 months of HAART, reaching proportions above normal values. This TNFa+ T cell expansion was found correlated to a dramatic decrease in the apoptosis rate of TNFa-producing cells, suggesting that under HAART, the physiological control by apoptosis of the T cell pool producing proinflammatory cytokines is lost. In addition, this upregulation of TNFa production was found to be associated with the LD syndrome. Indeed, LD+ patients showed a marked CD8 lymphocytosis mainly related to an increase in the number of CD8+TNFa+ T cells, which was correlated to the dyslipidemia (increase in cholesterol, increase in the ApoB/ApoA atherogenic ratio) characteristic of this syndrome.
Functional alterations in the Th1 subsets are still observed after HAART. The physiological regulation by apoptosis of pro-inflammatory cells is abolished, leading to
an excess in TNFa-production, which may have detrimental metabolic effects and be implicated in the lipodystrophy syndrome. However, TNFa overproduction may
beneficial by favouring the clearance of HIV from residual latently infected cells.
1.10 Cytokine - treatments reduce spontaneous apoptosis in HIV infected patients undergoing HAART
Member: G. D'Offizi
Participants: A. Amendola, F. Poccia, V. Galati, M. Pierdominici, M. Marziali, F. Martini, P. Narciso
Accelerated programmed cell death or apoptosis is an alternative mechanism for cell death besides necrosis and it has received increased attention in HIV
pathogenesis where it plays an important role.
T-cell apoptosis in HIV infection can be related to a variety of mechanisms: induction by specific viral proteins (Env, Tat, Vpr), interaction of envelope protein with CD4 molecule and interaction of cytokines with the Fas and the Fas-ligand. Apoptosis has been linked to active HIV replication and disease progression, and the process seems to be most evident in CD4+ cells. It has therefore been proposed that limiting apoptosis may represent a therapeutic modality to control HIV infection.
Highly active antiretroviral therapy (HAART) has dramatically reduced rates of morbidity and mortality from HIV infection. HAART has resulted in significant reduction of viral load and rise of CD4+ cells. The aim of our study was to evaluate the effects of HAART and low dosage of recombinant interleukin 2 (IL2) compared to HAART alone on spontaneous apoptosis in patients with asymptomatic HIV infection. We enrolled 22 outpatients with HIV infection, naive for antiretroviral therapy,
with >400 and <600/cmm CD4+ and HIV RNA >5000 copies/ml. Six patients were treated with HAART (arm 1); eight patients with HAART and IL2 (1,000,000 UI/
daily of rIL2 subcutaneously 5 days/week at alternative weeks (arm 2); eight patients with HAART and IL2 after the recruitment of totipotent stem cells by
granulocyte colony-stimulating factor (G-CSF, Filgrastim) (arm 3). Spontaneous apoptosis was evaluated before (t0) and after the treatment (t28 w) in all patients by
flow-cytometry analysis studying morphology of apoptotic T cells and the permeability of cellular membrane (ethidium bromide). Yet, spontaneous apoptosis was
studied in addition of IL2 in vitro. In parallel, phenotypic characterisation of PBMCs was performed by flow cytometry at t0 and t28 w. The data at t0 showed in all
patients an increased level of apoptosis, compared to HIV negative controls (41%±11 versus 24%±3.5% respectively, P=0.01). After six months of therapy, we
observed a substantial reduction of apoptosis in patients treated with HAART and IL2 (arm 2 and 3, P=0.01), compared to patients with HAART alone (arm 1,
P=0.05). The addition of IL2 in vitro significantly reduced spontaneous apoptosis at t0 and t24 in all arms (P<0.03). At t24, reduced levels of apoptosis were associated to: i) increased CD4+ T cell counts, especially CD4+ naive T cells (CD45RA+CD62L+) in all patients; ii) decreased expression of Fas receptor (CD95), particularly in CD45RA+CD62L+ naive T cells (arm 1) and also CD8+ T cells in treated patients; iii) a substantial reduction in viral load in all patients; iv) increased percentage of PBMC expressing IL2 receptor (CD25), in particular CD4+ T cells, CD8+ T cells, NK cells and CD19+ B cells (arm 2, 3). Altogether, these observations suggest that, in HIV therapy, addition of type 1 cytokines (IL2) to HAART may contribute not only to a significant reduction of spontaneous apoptosis of T cells, but also to a marked reconstitution of the CD4 cell compartment, beside the viral load reduction.
1.11 Nerve Growth factor is an autocrine factor essential for the survival of macrophages infected by human immunodeficiency virus
Member: C. F. Perno
Participants: M.C. Caroleo, L. Aloe, S. Aquaro, M. Piacentini, N. Costa, A. Amendola, A. Micera, R. Caliò, E. Garaci
Monocyte-derived macrophages (M/M) are able to survive infection by human immunodeficiency virus (HIV), and produce viral particles for a long period of time.
The factors that let M/M survive HIV infection are still unknown. Nerve growth factor (NGF) is a neurotrophin able to exert specific effects upon the survival of
bone-marrow derived cells such as memory B-lymphocytes. For this reason, we assessed whether NGF may be able to affect cell survival upon infection by HIV.
Human M/M infected in vitro with HIV-1 were treated with antibody neutralising the trophic effect of NGF. Apoptosis and virus production have thus been tested.
M/M infected by HIV (but not control- uninfected-M/M) produce substantial levels of endogenous NGF, that are associated with enhanced expression of the high affinity NGF receptor (p140 trkA) on M/M surface. Treatment of HIV-infected human M/M with anti-NGF antibody blocking the biological activity of NGF leads to a marked decrease of the expression of p140 trkA high affinity receptor, to a concomitant increased expression of p75NTR low affinity receptor for NGF, and to the occurrence of apoptotic death of M/M. These phenomena are typical only of HIV-infected M/M treated with neutralising anti-NGF antibody, since neither the infection with HIV alone, nor the treatment of uninfected M/M with anti-NGF antibody, are able to affect the expression of p75NTR receptor or the apoptosis in infected M/M. In addition, and concomitant with the modulation of receptor expression and apoptosis, the treatment with anti-NGF antibody markedly downmodulates the production of HIV by M/M. Taken together, these findings suggest a novel role of NGF as an autocrine survival factor which rescues human M/M from the cytopathic effect caused by HIV infection, and causes the long-lasting virus production typically found in these cells.
1.12 Migration and Infectious Diseases in the Mediterranean Area.
Member: Nicola Petrosillo
Participants: Giuseppe Ippolito and Nicola Petrosillo
In recent years, migration has become a determining factor in health and social development throughout the world. More and more people are travelling greater distances, more rapidly, than ever before. People tend to relocate as a result of war events and “ethnic cleansing”, poverty and the search for a better life, ecological changes or just plain curiosity. At the same time, whether people migrate permanently or temporarily - or return to visit their places of origin, as they often do - modern transportation has facilitated such movement to the extent that attempts to regulate and study migration represent a major challenge. Clearly, migration is extremely complicated. When considering the epidemiological standpoint, one obvious complication is that diseases migrate as people migrate. However, the living conditions of migrants (overcrowded living, high-risk behaviour such as prostitution, etc.) are such that disease acquisition becomes a concern of at least equal importance. Furthermore, the task of treating diseases among migrants, whether the disease was acquired before, during, or after migration, is compounded by language and cultural differences. Matters are further complicated by the fact that some migrants are clandestine. Although several European countries (including Italy) provide health care at no cost, a sick clandestine may be afraid to make his presence known by approaching the national health service, and therefore goes both untreated and unknown.
Each year, in Europe alone, it is estimated that over 100 million border crossings take place. Because of the recent war events in former Yugoslavia, the Middle East, and migration resulting from other events and conditions around the Mediterranean basin, Southern European countries have witnessed a constantly increasing flow of migrants. Of great concern are the diffusion of tuberculosis (TB), HIV/AIDS, STD and hepatitis as migration continues to increase in the EU. Indeed, the World Health Organisation announced in 1993 that tuberculosis constitutes a global emergency, predicting up to 10 million new cases by the beginning of the 21st century. TB is often related to HIV and typically surrounds poverty. Low education levels, poor nutrition and housing, overcrowding, and limited access
to preventive and curative medical services are the ideal conditions for TB to thrive. As most migration, both into and within the EU involves people moving from less to more economically developed regions, countries around the Mediterranean basin are seeing changes in their TB profiles. The role that population mobility has played in the spread of HIV infection in EU countries remains unclear. In Italy, 4.6% of AIDS cases notified in 1996-1997 concerned people from countries outside the European Union. Sexual transmission accounted for 78.6% of all these cases, 48.1% of which were heterosexual, and 30.5% of which were homosexual; 17.1% of the notified cases involved intravenous drug users. Migrants tend to be pushed to the margins of society, due both to their language and cultural status of “other”, and often due to the subcultures that they create to protect themselves against the normal, mainstream society - a similar dynamic to that experienced by the poor, and homosexuals and drug users, whose habits are commonly perceived as different and unusual. In this light, one begins to understand the societal conditions in which migrants live, and just how dangerous these conditions can be in terms of infectious diseases such as HIV/AIDS.
In conclusion, implications of migration and mobility for health care systems make the challenge of making population mobility healthy a dramatic and ethical need.
Section 2
Immunopathology of M.tuberculosis infection
2.1 Apoptosis regulation in infectious diseases: role of “tissue” transglutaminase
Member: A. Amendola
Participants: M. Piacentini, C. Rodolfo, S. Oliverio, R. Nardacci
Apoptosis is the active process of cell death that results in the deletion of individual cells from viable tissues. Alteration of the cell death program has been shown to play an important role in the pathogenesis of the most important human diseases, including cancer and AIDS. We have identified one of the effector elements of the apoptotic cell death program: the “tissue” transglutaminase (tTG). “Tissue” transglutaminase (tTG) is a Ca2+-dependent enzyme that cross-links proteins through e(g-glutamyl) lysine bonds and selectively accumulates to high levels in both in vivo and in vitro cells undergoing apoptotic cell death. The activation of tissue transglutaminase inside apoptotic cells results in extensive intracellular protein cross-linkage leading to the formation of stable intracellular polymers which stabilise the dying cell before its elimination by phagocytosis. Recently it has been evidenced that, in addition to the cross-linking activity, tTG may also act as GTP-binding protein (tTG-GTP) which, by a receptor-stimulated mechanism - GTP binding, might prevent the activation of the genetic death program. In fact, not only the Ca2+-dependent cross-linking activity of tTG is blocked in the GTP-complexed form, but by modulating the level of intracellular second messengers such as PIP2's product, the enzyme activates the protein kinase C pathway which is known to inhibit apoptosis. Resting T lymphocytes from healthy donors do not express tTG.
However, peripheral blood mononuclear cells (PBMCs) and lymphoid tissues from HIV-infected individuals display very high levels of tTG expression with respect to seronegative individuals. In asymptomatic individuals, 80% of the circulating CD4+ T cells synthesise tTG protein and the number of these cells matches the level of apoptosis in the PBMCs of the same patients. In lymph nodes from HIV-infected individuals, the accumulation of tTG protein was not limited to the T cells, but the enzyme was also localised in a large number of polymorphic follicular dendritic-like cells and in the syncytia, showing distinctive morphological and biochemical features of apoptosis, as well as lymphocytes.
The significant and progressive enhancement of e(g-glutamyl)lysine isodipeptide levels in the plasma of HIV-infected patients indicates that, in these individuals, tTG enzyme is activated. In M-CSF-treated HIV-infected monocytes-macrophage (M/M) cultures that undergo massive apoptosis and express high tTG levels, the ratio between virions released in the medium and intracellular viral particles is much lower than in untreated cultures with no apoptotic figures. An in vitro infection of monocytes with MTB, but not with HIV, induced an increased number of apoptotic cells. The induction of apoptosis by MTB required viable bacteria and was restricted to the virulent strain (H37Rv) Immunocytochemical analysis showed that while those cells which undergo apoptosis express tTG, the cells surviving to MTB infection expressed Bcl-2. Together, these findings suggest a protective role for apoptosis and tTG enzyme activation in infectious disease, as the reduction of the spreading of infectious virions from dying infected cells or syncytia could contribute in slowing down the spread of the infection.
2.2 Apoptosis in immunity to infections
Member: G. Mancino
Participants: R.Placido, M. Santucci, V. Colizzi
Apoptosis was studied at the level of T lymphocytes in the model of HIV infection ex vivo and in vitro. Main findings were the presence of apoptosis in lymphnodes and the association between apoptosis, heat shock proteins and tissue trasglutaminase (tTG) expression). Apoptosis was also studied at the level of monocytes/macrophages (M/M) infected with Mycobacterium tuberculosis (MTB). Apoptosis was found at the level of alveolar macrophages isolated from ex vivo samples of patients with different forms of tuberculosis and also in M/M infected in vitro with high doses of MTB. The level of apoptosis on ex vivo samples correlated with enhanced expression of tTG. MTB induces apoptosis on in vitro infected human monocytes/macrophages (M/M) through the activation of purinergic receptor P2X7, a new family of plasma membrane ligand-gated ion channels that has attracted increasing interest for its remarkable cytotoxic activity and for the selective expression on immune cells. Infection of M/M with MTB significantly increases the expression of P2X7 purinergic receptors, enhances the production/release of ATP from infected cells; these events are parallel with the subsequent induction of M/M apoptosis, and intracellular killing of mycobacteria.
Stimulation of M/M with bacterial endotoxin (LPS) activates purinergic receptor P2X7 and influences the ability to undergo apoptosis, but also to produce and secrete cytokines like Il-1b or Il-6, known to be involved in the chronic-granulomatous inflammatory responses.
2.3 Inhibition of “tissue” Transglutaminase Increases Cell Survival by Preventing Apoptosis
Member: A. Amendola
Participants: S. Oliverio, C. Rodolfo, A. Spinedi
Treatment of human promonocytic cell line, U937, with all trans retinoic acid (RA) commits these cells to apoptosis which can be triggered by simply increasing intracellular calcium levels by the ionosphere A23187. RA treatment of U937 cells is characterised by a decrease of Bcl-2 and marked induction of the “tissue” Transglutaminase (tTG) gene. tTG is a Ca2+-dependent enzyme that catalyses e(g-glutamyl)lysine crosslinkages, binds guanine nucleotides, hydrolyses GTP and ATP and has been identified as the 74 kDa a subunit associated with the 50 kDa b subunit of the GTP-binding protein Gh.
To investigate at what extent the overexpression of the tTG gene is a key biochemical event in the death program, we studied the effect on apoptosis of the RA-dependent induction of tTG in cell lines derived from promonocytic U937-derived cell clones transfected with the human tTG gene in antisense orientation. We show that inhibition of tTG expression in U937 clones determines a pronounced decrease in apoptosis induced by several stimuli, including Mycobacterium tuberculosis infection.
These findings demonstrate that the crosslinking of intracellular proteins catalysed by tTG represents an important biochemical event of the gene-regulated cell death in monoblasts. In addition, our data indicate that the apoptotic program in promonocytic cells is strictly regulated by RA and that a key role is played by the free intracellular calcium concentration.
2.4 Immunological evaluation of tubercular patients and their contact persons.
Member: A. Sanduzzi
Participants: F. Perna, I. Marchetiello, A. Panico, G. Fiorentino, S. Maione, A. Ponticiello
Although Tuberculosis (TB) is, obviously, an infective disease, it shows a low infectious pattern. The contact persons of TB patients are easily infected by Mycobacterium tuberculosis but rarely develop an active disease. In order to verify an immunological background of this peculiar feature, we evaluated 90 pulmonary TB patients (74 males and 16 females, mean age 37,7, HIV-negative, all Caucasians out of 3) and their 277 contact persons, living in Southern Italy, to assess the immunological aspects of both populations. So, we analysed the tuberculin skin test by Mantoux method and the serological response to A60 antigen; besides, we studied by flow cytometry the following blood parameters:
- T-lymphocyte subsets (CD3, CD4, CD8, CD3 gd)
- B-lymphocytes;
- NK subsets ( CD16-56, CD16-CD57)
- Activation markers (HLA-DR, CD25, CD28, CD57).
The results of the study are summarised as follows:
• a positive Mantoux test was found in 97,3% of the TB group and
44 % of the contacts group;
• among the contacts, only 11 % showed a high IgG titre Vs A60;
• high CD3 in TB group;
• normal CD4/CD8 ratio in both groups;
• very high values of “early activated” CD4 (CD4-CD25) in TB group;
• very high “late activated” CD4 ( CD4 HLA-DR) in PPD+ contacts
and in TB cases;
• high activated CD8 both in TB cases and in PPD+ contacts;
• among CD8, low values of “MHC-restricted cytotoxic” (CD28) in
the TB group ;
• high “activated NK” (CD 57) in contacts;
• low B-lymphocytes in TB cases and in PPD+ contacts.
In conclusions, contact persons’ group shows a good lymphocyte activation which could be responsible for the low rate of active disease among contacts, that we observed.
2.5 Human monocytes lose surface CD14 expression in the course of apoptosis induced with high doses of Mycobacterium tuberculosis
Member: M. Fraziano
Participants: M. Fraziano, M. Santucci, M. Amicosante, C. Montesano, M. Casarini, S. Giosuè, A. Bisetti, V. Colizzi
Apoptosis has been recently described in monocytes/macrophages during active pulmonary tuberculosis and in the course of Mycobacterium tuberculosis (MTB) in vitro infection. However, apoptotic signals delivered by MTB in monocytes/macrophages are not at the moment completely clarified. In this context, a direct correlation between the amount of MTB in the sputum and apoptosis in BAL cells coming from patients with active pulmonary tuberculosis has been found and confirmed during in vitro MTB infection.
As CD14 expression has been recently reported to mediate early effector mechanisms of life or death in monocytes, we analysed CD14 expression modifications in cells undergoing to MTB induced apoptosis. Flow cytometry analysis by FITC-Annexin V and either PE-anti-CD14 or PE-anti-HLA class I monoclonal antibody double stainining showed that CD14 expression was strongly down-regulated at day 3 from in vitro infection with the virulent strain MTB H37Rv, according to a dose-dependent effect, while HLA-class I (A, B, C) was unaffected. Moreover, a direct correlation between cell death induction, CD14 down-regulation and the virulence of mycobacteria has been observed. In fact, the infection with M. avium does not induce neither apoptosis nor modifications in membrane CD14 expression. Finally, kinetic studies showed Annexin V positivity already at 1 hour from MTB exposure, while CD14 membrane expression was lost later on.
These results show that apoptosis occurs at very early stages of infection and is depending upon the amount of mycobacteria phagocyted by monocytes. Finally, CD14 down-regulation is an intermediate event occurring after phosphatidylserine traslocation to outer surface of cell membrane, and it is differently regulated according to the virulence of mycobacteria.
2.6 ab and gd T cell responses in childhood tuberculosis
Member: F. Dieli
Participants: G. Sireci, C. Di Sano, G. Friscia, A. Salerno
We have analysed the ab and gd T cell responses in the peripheral blood of children affected by active tuberculosis (TB) and healthy PPD+ and PPD- children. No difference was found between healthy and diseased children in the in vitro proliferative response to PPD. In contrast, only a small fraction (18%) of PPD+ TB patients responded to peptide p38G derived from the 38 kDa protein of Mycobacterium tuberculosis.
This p38G-specific reduced response in vitro in TB children, reversed during chemotherapy. Analysis of the whole gd T cell population and of its Vg9/Vd2 subset showed comparable frequencies in PPD+ TB children and in healthy PPD+ and PPD- children. Vg9/Vd2 cells from TB children responded to 5 different phosphoantigens at an extent similar to that detected in healthy PPD+ children, but very poorly in healthy PPD- children. In vitro expansion of Vg9/Vd2 T cells strongly decreased during therapy.
The ab and gd T cell responses were also analysed in the cerebrospinal fluid (CSF) of children affected by tuberculous meningitis (TBM) and in control children. The repertoire of gd T cells from the CSF of TBM patients was characterised by the predominance of Vg9/Vd2 T lymphocytes, which accounted for more than 80% of gd T cells. Vg9/Vd2 cells from CSF of TBM children responded to different synthetic and natural (mycobacterial) phosphoantigens and produced discrete amounts of IFN-g and TNF-a.
The in vitro expansion of Vg9/Vd2 T cells from CSF and peripheral blood of TBM patients prominently decreased following chemotherapy and similarly the proportion of ex-vivo unstimulated Vg9/Vd2 T cells in CSF of TBM patients decreased to levels detected in the CSF of control subjects. Vd2 CD4+ T cell lines from the site of disease responded to PPD, and to the 16- and 38-kDa proteins and derived epitopes in vitro, but four months after chemotherapy the proliferative response to PPD and to the 16- or 38-kDa proteins and derived peptides decreased in CD4+ T cell lines from the site of disease, while increased in lines from the peripheral blood.
2.7 Nonpeptidic phosphoantigens elicit delayed-type hypersensitivity and antimycobacterial T cell reactivity in non-human primates 3
Member: M.Mattei
Participants: R. Casetti, G. Perretta, A. Taglioni, A. Amendola, F. Martini, M. Piacentini, M. Malkovsky, V. Colizzi, F. Poccia
Delayed-type hypersensitive (DTH) response mounted by the majority of people exposed to M. tuberculosis (MTB) controls the infection and later protects against re-infection. We analysed whether nonpeptidic phosphoantigens could elicit anti-mycobacterial DTH.
Since murine gd T cells exhibit a different antigen specificity than human counterparts, the study was performed in monkeys (Macaca fascicularis and mulatta). Monoclonal antibodies know to cross-react between monkeys and humans were used in flow cytometry. Cytokine production (TNF-a) was measured by ELISA.
Immunohistochemistry was performed to confirm DTH reaction. We observed that primate Vg9/Vd2 T cells recognise nonpeptidic mycobacterial antigens without MHC restriction, cross-reacting with isopentenyl pyrophosphate, monoethyl pyrophosphate and diphosphoglyceric acid. Intravenous injection of nonpeptidic antigens induced an immediate release of TNFa in the sera followed by an activated state of simian Vg9/Vd2 T cells. Skin-test reactions revealed a DTH response to nonpeptidic mycobacterial antigens in sensitised animals. Experiments of in vitro killing of mycobacteria by gd T cells from the immunised animals are in progress.
These data show that nonpeptidic antigens elicit antimycobacterial responses mediating a DTH response. Therefore, these properties may be useful to effectively monitor the exposure to M.tuberculosis, suggesting new strategies for the design and development of nonpeptidic vaccines and/or adjuvants based on nonpeptidic T cell antigens.
2.8 Expression of CCR5 is Increased in Human Monocyte Derived- and in Alveolar- Macrophages in the Course of in vivo and in vitro Mycobacterium tuberculosis Infection
Member: M. Fraziano
Participants: G. Cappelli, M. Santucci, F. Mariani, M. Amicosante, M. Casarini, S. Giosuè, A. Bisetti, V. Colizzi
Human Immunodeficiency Virus (HIV) replicates more efficiently in Mycobacterium tuberculosis infected macrophages than in uninfected controls. We investigated whether this may be partly explained by changes in expression of CCR5 in the course of mycobacterial infection, as this molecule has been shown to be a coreceptor for HIV entry.
Since the lung is the preferential organ of HIV replication in the course of tuberculosis, we preliminarily analysed b-chemokine receptor expression in alveolar macrophages coming from patients with active tuberculosis by flow cytometry, by using Mip-1a ligand-biotin/avidin FITC detection system. An increased MIP-1a receptor (MIP-1aR) expression in alveolar macrophages from infected patients was observed while no detectable expression could be revealed in uninfected controls.
Since MIP-1a can bind also to CCR1 and CCR4, the presence of CCR5 mRNA was investigated in Broncho Alveolar Lavage (BAL) cells and detected in alveolar macrophages from tuberculosis patients only. The study was then extended on in vitro MTB infected macrophages. Monocyte derived macrophages (MDM) were let to differentiate for 7 days before MTB H37Rv infection, and CCR5 expression was monitored using a specific monoclonal antibody at day 1, 6, and 11 after infection.
An increased CCR5 expression in MTB infected macrophages was observed, with a peak at day 6 (64% in MTB infected versus 33% in control cultures) and a decrease at day 11 (25% in MTB infected versus 13% in control cultures). These results show that CCR5 expression is enhanced in the course of in vitro MTB infection and during active pulmonary tuberculosis.
2.9 Analysis at the single cell level of cytokine and chemokine expression by CD64+ monocytes in response to M. tuberculosis and modulation by IFN-g, IL-10 and IL-15. Consequences on AIDS pathogenesis.
Member: M. Bocchino,
Participants: E. Ledru, M. L. Gougeon
Cytokines and chemokines participate to the pathogenesis of human immunodeficiency virus (HIV) infection. To estimate the contribution of monocytes to their synthesis, we investigated by flow cytometry the proportion of CD64+ monocytes of healthy (n=18) and HIV-infected donors (n=70) producing interleukin (IL)-10, IL-12, tumor necrosis factor (TNF)-a and monocyte chemotactic protein (MCP)-1. Some HIV+ patients received anti-retroviral drugs, including HIV protease inhibitors (HIV-PIs).
Further, to get insight the mechanisms of immune protection in HIV-M. tuberculosis (Mtb) co-infection, the monokine pattern was also evaluated upon short-term Mtb and LPS stimulation. HIV infection was found associated with a dysregulation in the production of cytokines. An increased proportion of monocytes spontaneously producing IL-12 and TNF-a was detected in HIV+ patients compared to controls. No difference in the spontaneous IL-10 expression was observed in HIV+ vs healthy donors, whereas, surprisingly, monocytes from both controls and patients spontaneously produced MCP-1. Upon MTB challenge, the percentage of IL-12 expressing CD64+ monocytes was decreased in both groups upon Mtb-challenge, whereas that of TNF-a was not modified. In contrast, both IL-12 and TNF-a expression were increased by LPS.
The Mtb-induced IL-10 production was similar in controls and in patients, whereas LPS-induced IL-10 expression was significantly increased only in HIV-PIs receiving patients. MCP-1 expression was inhibited by Mtb and LPS stimulation in both groups of donors and not restored in patients undergoing therapy. Finally, exposure to exogenous IFN-g significantly restored IL-12 production in response to Mtb; IL-10 had an inhibitory effect, whereas IL-15 had no effect. Further, IFN-g also inhibited MCP-1 expression, which was increased by IL-10 and not modified by IL-15. Our results suggest that there is no alteration in the capacity of monocytes from HIV+ patients to produce, both spontaneously and in response to Mtb stimulation, a given cytokine or chemokine. In addition, modulation of monocytic activity could be induced by IFN-g, which stimulates IL-12 synthesis, essential for anti-mycobacterial specific immunity.
2.10 Mycobacterium tuberculosis gene expression: different patterns of gene transcription are displayed in synthetic medium, in in vitro infected human macrophages and in TBC patients’ alveolar macrophages
Member: F. Mariani
Participants: G. Cappelli, A. Sacchi, G. Riccardi, V. Colizzi
Regarding the concept of virulence of Mycobacterium tuberculosis (MTB) there is general agreement that it could reside in its capacity to survive and replicate in the human macrophage. The vaste majority of the studies on immunogenicity of MTB antigens employed recombinant proteins to challenge T lymphocytes and monocyte/macrophages, but to date none of those proteins showed to be protective for the human host. A few studies analysed the pattern of proteins effectively expressed by MTB during infection, and the present study is actually focused on gene expression of MTB in the following three different habitats of replication: Sauton synthetic medium, human in vitro infected macrophages, and alveolar macrophages from BAL of TBC patients.
Using RT-PCR on a group of 12 MTB-Complex-specific genes ( MT10Sa, ESAT6, 85A Ag, 85B Ag, 85C Ag, RPOb, inv1, inv2, ahpC, katG, 35kDa, Pab), we detected the mRNAs produced by MTB and found three groups of genes co-expressed either in synthetic medium, or in human macrophages and in both. Surprisingly, when we analysed the same pattern in TBC patients’ specimen, we found some interesting differences compared to the in vitro infection model, particularly regarding a major antigen of MTB. To study the mechanism of host-pathogen interaction, the pattern of expression of the following cytokines was investigated by RT-PCR in the in vitro infected macrophages and in the alveolar macrophages of TBC patients :IL-1a, IL-1b, IL-6, TNFa ,IL-10, IL-12, IL-16 and TGFb.
2.11 Repeated sequences of the microbial genome: role and function in the host parasite interaction.
Member: V. Parisi
Participants: E. Bersani, F. Pentini, V. De Fonzo, F. Mariani, V. Colizzi
Mycobacterium tuberculosis is the leading cause of adult death due to a single infectious agent world-wide, also in industrialised countries. One of the factors hampering the control of tuberculosis is the difficulty of differentiating among strains (essentially, virulent and non virulent), due to the highly conserved genome of M.tuberculosis, which has been completely sequenced in the case of H37Rv strain, such a difficulty has hindered understanding of the steps of the disease and of the molecular mechanism of defence against host. Recently, various polymorphic loci have been discovered in the genome, among which are the polymorphic G-C rich repetitive sequences (PGRS), linked to conserved open reading frames.
Various variable number of tandem repeats (VNTRs) are used by infectious agent to continuously change their surface antigens and escape the host immune surveillance, other VNTRs are involved in human disease. The polymorphic PGRSs are composed of motif sequences similar to the highly recombinogenic “Chi” sites of E. coli and there can be a smart recombinational mechanism allowing variability (as in the case of Trypanosomes).
The goal of our research is to test the presence of such VNTRs only in virulent strain(s) (H37Rv) and not in non virulent strains (H37Ra), as a cause of the failure of immune defences, their variability under the host immune stress, and their “use” (under “adaptive mutation” conditions in escape host defence.
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