Phylogéographie des principales vipères européennes (Vipera ammodytes, V. aspis et V. berus), structuration génétique et multipaternité chez Vipera berus

Résumé: Les vipères du genre Vipera sont des serpents venimeux distribués dans la totalité du Paléarctique. Malgré cette répartition considérable, elles sont extrêmement menacées, leur déclin étant principalement dû à la destruction et à la fragmentation de leur habitat ainsi qu'à la persécution humaine. Afin d'apporter de nouveaux éléments dans le contexte de la protection de ce groupe de reptiles, nous avons utilisé durant ce travail de thèse différents marqueurs moléculaires pour étudier la structuration génétique à petite et à large échelle chez trois espèces appartenant au genre Vipera. La première étude, une phylogéographie moléculaire de la vipère ammodytes (Vipera ammodytes), a montré dans l'ensemble de l'aire de répartition une forte structuration génétique provenant d'isolements antérieures au Pléistocène. La présence d'un nombre important de clades dans le centre des Balkans suggère que cette région a fourni de nombreux refuges isolés durant les glaciations. Ces dernières ont également eu un impact considérable sur la diversité génétique au sein de la majorité des clades, suite à d'importants goulots d'étranglement durant le Pléistocène. L'étude de la phylogéographie de la vipère aspic (Vipera aspis) a montré une différenciation génétique entre les populations présentes de chaque côté des Alpes, mais également une forte structuration interne avec la mise en évidence d'un refuge en France. Cette étude est la première à établir clairement l'utilisation d'un refuge français pour un vertébré terrestre. La troisième partie de cette thèse a étudié la phylogéographie de la vipère péliade (Vipera berus), espèce cible de ce travail. En plus de la mise en évidence d'un groupe génétique inattendu (localisé dans le nord de l'Italie, le sud de l'Autriche, le nord de la Slovénie et l'extrême sud-est de la Suisse), la variabilité génétique au sein du groupe nordique (comprenant les animaux de l'entier de l'aire de répartition de l'espèce à l'exception des individus du groupe italien et les animaux provenant des Balkans) est suffisamment importante pour conclure à l'utilisation de refuges glaciaires nordiques durant les dernières glaciations, en complément des refuges habituellement décrits pour la majorité des espèces animales (soit les péninsules ibérique, italienne et balakanique). Ces résultats nous ont conduit à effectuer une étude morphologique (quatrième partie) comparant les vipères péliades du "clade italien" et du "clade nordique" décrits ci-dessus. Seules de petites différences morphologiques ont pu être mises en évidence, malgré une séparation de ces groupes estimée à plus d'un million d'années. Une étude à plus petite échelle, centrée sur le Massif jurassien et certaines populations alpines et françaises, a été entreprise afin d'estimer leur diversité génétique et d'évaluer la structuration génétique entre les populations à l'aide de marqueurs microsatellites (cinquième partie). Une importante structuration a été observée entre les populations distantes de plus de 3 kilomètres, la structuration entre les populations plus proches étant plus limitée. De plus, une diversité génétique plus faible dans les populations jurassiennes et alpines comparativement aux populations du massif central et de la côte atlantique a été constatée, probablement due à une perte de diversité génétique lors de la recolonisation post-glaciaire. La sixième étude s'est intéressée au succès reproducteur des mâles de vipères péliades en conditions naturelles. Une corrélation entre la taille des mâles et leur succès reproducteur a été relevée, les individus de plus grande taille ayant un succès reproducteur plus élevé. Le taux de multipaternité a aussi été investigué, démontrant que la proportion de pontes issues de plusieurs pères est élevée (69%) malgré la faible densité de vipères observée sur le site étudié. Finalement, aucun lien entre le nombre de pères au sein d'une ponte et la mortalité des jeunes à la naissance n'a pu être mis en évidence, contrastant avec des travaux précédents. En conclusion, l'observation de la structuration très marquée chez les vipères péliades devrait permettre d'affiner les méthodes de protection de l'espèce dans le massif jurassien. A plus large échelle, l'importante structuration génétique observée chez les vipères ammodytes, aspic et péliade résultant de l'utilisation de nombreux refuges glaciaires, complémentaires aux refuges habituellement utilisés par les espèces animales, démontre l'intérêt de l'analyse phylogéographique des reptiles pour la compréhension des phénomènes de colonisation et d' extinction des populations durant la fin du Tertiaire et le Quaternaire. La mise en évidence chez les différentes espèces de vipères étudiées de nombreux groupes génétiques distincts (ESUs) devrait conduire à des modifications de la taxonomie ainsi qu'au statut de protection de ces espèces. Abstract: The vipers of the genus Vipera are venomous snakes widespread throughout the Palaearctic regions. Despite a large distribution area, several species are extremely threatened, especially due to the destruction and fragmentation of their habitats, as well as by human persecution. In order to increase the knowledge on these species and to improve their protection, several molecular markers have been used to investigate the genetic structure on small and large scales, within three species of the genus Vipera. The first study, a molecular phylogeography of the nose-horned viper (Vipera ammodytes), showed a considerable structuring throughout the distribution area, due to isolation into refugia before the Pleistocene. A high number of clades in the centre of the Balkans suggests that this region harboured numerous isolated glacial refugia during the last glaciation. Moreover, low genetic diversity within several clades implies that most populations of nose-horned vipers have suffered bottlenecks during the Pleistocene. The study of the phylogeography of the asp viper (Vipera aspis) showed genetic differentiation between populations on each side of the Alps, as well as considerable internal genetic structure, suggesting the use of a glacial refugium in France. This study is the first to establish firmly the occurrence of a French refugia for a terrestrial vertebrate. The third part of this work involved a phylogeographic study of the adder (Vipera berus), the target species of this thesis. Three clades were revealed: a Balkan clade (corresponding to the subspecies V. b. sachalinensis), an unexpected Italian clade (limited to northern Italy, southern Austria, northern Slovenia and southeasternmost corner of Switzerland) and a Northern clade clade (including adders of the whole distribution area excepted animals from the Balkan and the Italian clades). The genetic variability within the Northern clade is sufficiently high to conclude that a northern glacial refugia during the last glaciation, in addition to those refugia already described for the main species (Iberian, Italian and Balkan peninsula). These results motivated a morphological study (part four) comparing the adders from the Italian and the Northern clades describe above. Only small morphological differences have been found, despite the split between these two clades have taken place more than 1 million years ago. A study on a local scale, focused on the Jura Mountains, on a few populations in the Alps and France was, performed to estimate the genetic diversity and the genetic structure between populations using microsatellite markers (part five). Considerable structure was observed between populations separated by more than 3 kilometres, whereas the structure between closer populations is less marked. Moreover, lower genetic diversity in the populations from Jura Mountains and Alps was noticed compared to populations from Massif Central of Atlantic coast. Such loss of genetic variation probably followed post-glacial recolonisation. The sixth study focused on the reproductive success of male adders in the wild. A positive correlation between body length and reproductive success was observed. Multiple paternity was also observed in most of clutches (69%) despite the low density of adders in the study area. Finally, no relationship was found between the number of fathers in a clutch and the survival of offspring at birth, contradicting previous studies. To conclude, the observation of a significant genetic structure in Vipera berus will enable recommendations to be made to improve protection of this species in the Jura Mountain. On a larger scale, the considerable genetic structure found within Vipera ammdoytes, V. aspis and V. berus, resulting from isolation in additional glacial refugia to those already described for other species, demonstrates the relevance of phylogeographic studies of reptiles to better understand the colonisation and disappearance during the last Tertiary and the Quaternary. The observation of several groups of evolutionary significant units (ESUs) within the three studied species might lead to a revision of the taxonomy, as well as their conservation status.

Between Immunology And Tolerance: Controlling Immune Responses Employing Tolerogenic Dendritic Cells

Dendritic cells (DCs) are the most efficient antigen presenting cells, they provide co-stimulation, are able to secrete various proinflammatory cytokines and therefore play a pivotal role in shaping adaptive immune responses. Moreover, they are important for the promotion and maintenance of central and peripheral tolerance through several mechanisms like the induction of anergy or apoptosis in effector T cells or by promoting regulatory T cells. The murine CD8α+ (MuTu) dendritic cell line was previously derived and described in our laboratory. The MuTu cell line has been shown to maintain phenotypical and functional characteristics of endogenous CD8α+ DCs. They are able to cross-present exogenous antigens to CD8+ T cells and produce interleukin (IL-) 12 upon engagement of Toll like receptors. The cell line constitutes an infinite source of homogenous, phenotypically well-defined dendritic cells. This allows us to investigate the role and potential of specific molecules in the induction as well as regulation of immune responses by DCs in a rational and standardized way. In a first project the MuTu dendritic cell line was transduced in order to stably express the immunosuppressive molecules IL-10, IL-35 or the active form of TGF-β (termed IL-10+DC, IL-35+DC or actTGFβ+DC). We investigated the capability of these potentially suppressive or tolerogenic dendritic cell lines to induce immune tolerance and explore the mechanisms behind tolerance induction. The expression of TGF-β by the DC line did not affect the phenotype of the DCs itself. In contrast, IL-10+ and IL-35+DCs were found to exhibit lower expression of co-stimulatory molecules and MHC class I and II, as well as reduced secretion of pro-inflammatory cytokines upon activation. In vitro co-culture with IL-35+, IL10+ or active TGFβ+ DCs interfered with function and proliferation of CD4+ and CD8+ T cells. Furthermore, IL-35 and active TGF-β expressing DC lines induced regulatory phenotype on CD4+ T cells in vitro without or with expression of Foxp3, respectively. In different murine cancer models, vaccination with IL-35 or active TGF-β expressing DCs resulted in faster tumor growth. Interestingly, accelerated tumor growth could be observed when IL-35-expressing DCs were injected into T cell-deficient RAG-/- mice. IL-10expressing DCs however, were found to rather delay tumor growth. Besides the mentioned autocrine effects of IL-35 expression on the DC line itself, we surprisingly observed that the expression of IL-35 or the addition of IL-35 containing medium enhances neutrophil survival and induces proliferation of endothelial cells. Our findings indicate that the cytokine IL-35 might not only be a potent regulator of adaptive immune responses, but it also implies IL-35 to mediate diverse effects on an array of cellular targets. This abilities make IL-35 a promising target molecule not only for the treatment of auto-inflammatory disease but also to improve anti-cancer immunotherapies. Indeed, by applying active TGFβ+ in murine autoimmune encephalitis we were able to completely inhibit the development of the disease, whereas IL-35+DCs reduced disease incidence and severity. Furthermore, the preventive transfer of IL-35+DCs delayed rejection of transplanted skin to the same extend as the combination of IL-10/actTGF-β expressing DCs. Thus, the expression of a single tolerogenic molecule can be sufficient to interfere with the adequate activation and function of dendritic cells and of co-cultured T lymphocytes. The respective mechanisms of tolerance induction seem to be different for each of the investigated molecule. The application of a combination of multiple tolerogenic molecules might therefore evoke synergistic effects in order to overcome (auto-) immunity. In a second project we tried to improve the immunogenicity of dendritic cell-based cancer vaccines using two different approaches. First, the C57BL/6 derived MuTu dendritic cell line was genetically modified in order to express the MHC class I molecule H-2Kd. We hypothesized that the expression of BALB/c specific MHC class I haplotype (H-2Kd) should allow the priming of tumor-specific CD8+ T cells by the otherwise allogeneic dendritic cells. At the same time, the transfer of these H-2Kd+ DCs into BALB/c mice was thought to evoke a strong inflammatory environment that might act as an "adjuvant", helping to overcome tumor induced immune suppression. Using this so called "semi-allogeneic" vaccination approach, we could demonstrate that the delivery of tumor lysate pulsed H-2Kd+ DCs significantly delayed tumor growth when compared to autologous or allogeneic vaccination. However, we were not able to coherently elucidate the cellular mechanisms underlying the observed effect. Second, we generated MuTu DC lines which stably express the pro-inflammatory cytokines IL-2, IL-12 or IL-15. We investigated whether the combination of DC vaccination and local delivery of pro-inflammatory cytokines might enhance tumor specific T cell responses. Indeed, we observed an enhanced T cell proliferation and activation when they were cocultured in vitro with IL-12 or IL-2-expressing DCs. But unfortunately we could not observe a beneficial or even synergistic impact on tumor development when cytokine delivery was combined with semi-allogeneic DC vaccination.

Synthetic long peptide booster immunization in rhesus macaques primed with replication-competent NYVAC-C-KC induces a balanced CD4/CD8 T-cell and antibody response against the conserved regions of HIV-1.

The Thai trial (RV144) indicates that a prime-boost vaccine combination that induces both T-cell and antibody responses may be desirable for an effective HIV vaccine. We have previously shown that immunization with synthetic long peptides (SLP), covering the conserved parts of SIV, induced strong CD4 T-cell and antibody responses, but only modest CD8 T-cell responses. To generate a more balanced CD4/CD8 T-cell and antibody response, this study evaluated a pox-vector prime/SLP boost strategy in rhesus macaques. Priming with a replication-competent NYVAC, encoding HIV-1 clade C gag, pol and nef, induced modest IFNγ T-cell immune responses, predominantly directed against HIV-1 Gag. Booster immunization with SLP, covering the conserved parts of HIV-1 Gag, Pol and Env, resulted in a more than 10-fold increase in IFNγ ELISpot responses in four of six animals, which were predominantly HIV-1 Pol-specific. The animals showed a balanced polyfunctional CD4 and CD8 T-cell response and high Ab titres.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

Valeur diagnostique du rapport CD103+CD4+/CD4+ pour différencier la sarcoïdose d'autres causes de lymphocytose alvéolaire.

Introduction: CD103 is a specific integrin present on some CD4+ lymphocytes of the mucosal immune system. It has been hypothesized that most CD4+ lymphocytes in pulmonary sarcoidosis do not originate from mucosal sites but from redistribution from the peripheral blood, and therefore do not bear the CD103 integrin. Several studies have suggested that a low CD103+ percentage among bronchoalveolar lavage (BAL) CD4+ lymphocytes discriminates between sarcoidosis and other causes of lymphocytic alveolitis, but contradictory data exist. Methods: We reviewed 1151 consecutive patients with BAL lymphocytosis >10% and flow cytometry performed between 2006 and 2014. 944 cases were excluded due to poor BAL quality (n= 97), unavailable clinical data (n= 760), or unclear diagnosis (n= 87). The remaining 207 patients were grouped into 9 diagnostic categories. To assess the discriminative value of the CD103+CD4+/CD4+ ratio to distinguish sarcoidosis from the other entities, area under ROC curves (AUC) were determined. Results: Sarcoidosis patients (n=53) had a lower CD103+CD4+/CD4+ ratio than the other diagnostic categories. AUC was 62% for sarcoidosis compared to all other patients and 69% for sarcoidosis compared to other interstitial lung diseases. When combining CD103+CD4+/CD4+ and CD4+/CD8+ ratios, AUC increased to 76% and 78% respectively. When applying published cut-offs from 4 previous studies to our population, AUC varied between 54 and 73%. Conclusions: The CD103+CD4+/CD4+ ratio does not accurately discriminate between sarcoidosis and other causes of lymphocytic alveolitis, neither alone nor in combination with CD4+/CD8+ ratio, and is not a relevant marker for the diagnosis of sarcoidosis.

Chemokines, lymphocytes, and HIV

Chemokines are members of a family of more than 30 human cytokines whose best-described activities are as chemotactic factors for leukocytes and that are presumed to be important in leukocyte recruitment and trafficking. While many chemokines can act on lymphocytes, the roles of chemokines and their receptors in lymphocyte biology are poorly understood. The recent discoveries that chemokines can suppress infection by HIV-1 and that chemokine receptors serve, along with CD4, as obligate co-receptors for HIV-1 entry have lent urgency to studies on the relationships between chemokines and lymphocytes. My laboratory has characterized Mig and Crg-2/IP-10, chemokines that are induced by IFN-<FONT FACE="Symbol">g</font> and that specifically target lymphocytes, particularly activated T cells. We have demonstrated that the genes for these chemokines are widely expressed during experimental infections in mice with protozoan and viral pathogens, but that the patterns of mig and crg-2 expression differed, suggesting non-redundant roles in vivo. Our related studies to identify new chemokine receptors from activated lymphocytes resulted in the cloning of STRL22 and STRL33. We and others have shown that STRL22 is a receptor for the CC chemokine MIP-3<FONT FACE="Symbol">a</font>, and STRL22 has been re-named CCR6. Although STRL33 remains an orphan receptor, we have shown that it can function as a co-receptor for HIV-1 envelope glycoproteins, and that it is active with a broader range of HIV-1 envelope glycoproteins than the major co-receptors described to date. The ability of STRL33 to function with a wide variety of envelope glycoproteins may become particularly important if therapies are instituted to block other specific co-receptors. We presume that investigations into the roles of chemokines and their receptors in lymphocyte biology will provide information important for understanding the pathogenesis of AIDS and for manipulating immune and inflammatory responses for clinical benefit

Cellular and matrix interactions during the development of T lymphocytes

The thymus contains an extensive extracellular matrix. Although thymocytes express integrins capable of binding to matrix molecules, the functional significance of the matrix for T cell development is uncertain. We have shown that the matrix is associated with thymic fibroblasts which are required for the CD44+ CD25+ stage of double negative (CD4-8-) thymocyte development. The survival of cells at this stage is dependent on IL-7 and we propose that the role of fibroblasts is to present, via the matrix, IL-7 to developing T cells.

The role of CD8+ T cells during allograft rejection

Organ transplantation can be considered as replacement therapy for patients with end-stage organ failure. The percent of one-year allograft survival has increased due, among other factors, to a better understanding of the rejection process and new immunosuppressive drugs. Immunosuppressive therapy used in transplantation prevents activation and proliferation of alloreactive T lymphocytes, although not fully preventing chronic rejection. Recognition by recipient T cells of alloantigens expressed by donor tissues initiates immune destruction of allogeneic transplants. However, there is controversy concerning the relative contribution of CD4+ and CD8+ T cells to allograft rejection. Some animal models indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas in others CD4-depleted mice reject certain types of allografts. Moreover, there is evidence that CD8+ T cells are more resistant to immunotherapy and tolerance induction protocols. An intense focal infiltration of mainly CD8+CTLA4+ T lymphocytes during kidney rejection has been described in patients. This suggests that CD8+ T cells could escape from immunosuppression and participate in the rejection process. Our group is primarily interested in the immune mechanisms involved in allograft rejection. Thus, we believe that a better understanding of the role of CD8+ T cells in allograft rejection could indicate new targets for immunotherapy in transplantation. Therefore, the objective of the present review was to focus on the role of the CD8+ T cell population in the rejection of allogeneic tissue.

Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T lymphocytes

The multidrug resistance P-glycoprotein is a transmembrane efflux pump expressed by lymphocytes and is involved in their cytolytic activity. In the present study, we investigated the age-related changes of P-glycoprotein function in normal peripheral blood lymphocytes. Blood samples from 90 normal volunteers (age range, 0 to 86 years) were analyzed. P-glycoprotein function was assessed by the flow cytometric rhodamine 123 assay. P-glycoprotein function was highest in cord blood and progressively declined with age in peripheral blood T CD4+ and CD8+ cells. In contrast, P-glycoprotein function did not vary with age in CD19+ B or CD16+CD56+ natural killer cells. These data suggest that the decline in P-glycoprotein function in T CD4+ and CD8+ lymphocytes as a function of age may contribute to the decrease in T cell cytolytic activity with aging.

Contribution of CD4+ T cells to the early mechanisms of ischemia- reperfusion injury in a mouse model of acute renal failure

Renal ischemia-reperfusion (IR) injury is the major cause of acute renal failure in native and transplanted kidneys. Mononuclear leukocytes have been reported in renal tissue as part of the innate and adaptive responses triggered by IR. We investigated the participation of CD4+ T lymphocytes in the pathogenesis of renal IR injury. Male mice (C57BL/6, 8 to 12 weeks old) were submitted to 45 min of ischemia by renal pedicle clamping followed by reperfusion. We evaluated the role of CD4+ T cells using a monoclonal depleting antibody against CD4 (GK1.5, 50 µ, ip), and class II-major histocompatibility complex molecule knockout mice. Both CD4-depleted groups showed a marked improvement in renal function compared to the ischemic group, despite the fact that GK1.5 mAb treatment promoted a profound CD4 depletion (to less than 5% compared to normal controls) only within the first 24 h after IR. CD4-depleted groups presented a significant improvement in 5-day survival (84 vs 80 vs 39%; antibody treated, knockout mice and non-depleted groups, respectively) and also a significant reduction in the tubular necrosis area with an early tubular regeneration pattern. The peak of CD4-positive cell infiltration occurred on day 2, coinciding with the high expression of ßC mRNA and increased urea levels. CD4 depletion did not alter the CD11b infiltrate or the IFN-g and granzyme-B mRNA expression in renal tissue. These data indicate that a CD4+ subset of T lymphocytes may be implicated as key mediators of very early inflammatory responses after renal IR injury and that targeting CD4+ T lymphocytes may yield novel therapies.

Imbalance of naive and memory T lymphocytes with sustained high cellular activation during the first year of life from uninfected children born to HIV-1-infected mothers on HAART

The immune consequences of in utero HIV exposure to uninfected children whose mothers were submitted to highly active antiretroviral therapy (HAART) during gestation are not well defined. We evaluated 45 HIV-exposed uninfected (ENI) neonates and 45 healthy unexposed control (CT) neonates. All HIV-infected mothers received HAART during pregnancy, and the viral load at delivery was <50 copies/mL for 56.8%. Twenty-three ENI neonates were further evaluated after 12 months and compared to 23 unexposed healthy age-matched infants. Immunophenotyping was performed by flow cytometry in cord and peripheral blood. Cord blood lymphocyte numbers did not differ between groups. However, ENI neonates had a lower percentage of naive T cells than CT neonates (CD4+, 76.6 vs 83.1%, P < 0.001; CD8+, 70.9 vs 79.6%, P = 0.003) and higher percentages of central memory T cells than CT neonates (CD4+, 13.9 vs 8.7%, P < 0.001; CD8+, 8.6 vs 4.8%, P = 0.001). CD38 mean fluorescence intensity of T cells was higher in ENI neonates (CD4+, 62.2 vs 52.1, P = 0.007; CD8+, 47.7 vs 35.3, P < 0.001). At 12 months, ENI infants still had higher mean fluorescence intensity of CD38 on T cells (CD4+, 34.2 vs 23.3, P < 0.001; CD8+, 26.8 vs 19.4, P = 0.035). Despite effective maternal virologic control at delivery, HIV-exposed uninfected children were born with lower levels of naive T cells. Immune activation was present at birth and remained until at least 12 months of age, suggesting that in utero exposure to HIV causes subtle immune abnormalities.

Expression of the chemokine receptor CXCR4 on lymphocytes of leprosy patients

Leprosy is caused by Mycobacterium leprae, which induces chronic granulomatous infection of the skin and peripheral nerves. The disease ranges from the tuberculoid to the lepromatous forms, depending on the cellular immune response of the host. Chemokines are thought to be involved in the immunopathogenesis of leprosy, but few studies have investigated the expression of chemokine receptors on leukocytes of leprosy patients. In the present study, we evaluated 21 leprosy patients (M/F: 16/5) with a new diagnosis from the Dermatology Outpatient Clinic of the University Hospital, Federal University of Minas Gerais. The control group was composed of 20 healthy members (M/F: 15/5) of the community recruited by means of announcements. The expression of CCR2, CCR3, CCR5, and CXCR4 was investigated by flow cytometry on the surface of peripheral blood lymphocytes. There was a decrease in percentage of CD3+CXCR4+ and CD4+CXCR4+ lymphocytes in the peripheral blood of leprosy patients (median [range], 17.6 [2.7-41.9] and 65.3 [3.9-91.9], respectively) compared to the control group (median [range], 43.0 [3.7-61.3] and 77.2 [43.6-93.5], respectively). The percentage of CD4+CXCR4+ was significantly lower in patients with the tuberculoid form (median [range], 45.7 [0.0-83.1]) of the disease, but not in lepromatous patients (median [range], 81.5 [44.9-91.9]). The CXCR4 chemokine receptor may play a role in leprosy immunopathogenesis, probably directing cell migration to tissue lesions in tuberculoid leprosy patients.