Targeting human langerin promotes HIV-1 specific humoral immune responses.

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4+ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC.

Modified Vaccinia Virus Ankara Vector Induces Specific Cellular and Humoral Responses in the Female Reproductive Tract, the Main HIV Portal of Entry.

The female reproductive tract (FRT) is one of the major mucosal invasion sites for HIV-1. This site has been neglected in previous HIV-1 vaccine studies. Immune responses in the FRT after systemic vaccination remain to be characterized. Using a modified vaccinia virus Ankara (MVA) as a vaccine model, we characterized specific immune responses in all compartments of the FRT of nonhuman primates after systemic vaccination. Memory T cells were preferentially found in the lower tract (vagina and cervix), whereas APCs and innate lymphoid cells were mainly located in the upper tract (uterus and fallopian tubes). This compartmentalization of immune cells in the FRT was supported by transcriptomic analyses and a correlation network. Polyfunctional MVA-specific CD8+ T cells were detected in the blood, lymph nodes, vagina, cervix, uterus, and fallopian tubes. Anti-MVA IgG and IgA were detected in cervicovaginal fluid after a second vaccine dose. Thus, systemic vaccination with an MVA vector elicits cellular and Ab responses in the FRT.

Distinct characteristics of endometrial and decidual macrophages and regulation of their permissivity to HIV-1 infection by SAMHD1.

UNLABELLED: In order to develop strategies to prevent HIV-1 (human immunodeficiency virus type 1) transmission, it is crucial to better characterize HIV-1 target cells in the female reproductive tract (FRT) mucosae and to identify effective innate responses. Control of HIV-1 infection in the decidua (the uterine mucosa during pregnancy) can serve as a model to study natural mucosal protection. Macrophages are the main HIV-1 target cells in the decidua. Here we report that in vitro, macrophages and T cells are the main HIV-1 targets in the endometrium in nonpregnant women. As reported for decidual macrophages (dM), endometrial macrophages (eM) were found to have an M2-like phenotype (CD68+ CD163+ CD206+ IL-10high). However, eM and dM may belong to different subpopulations, as they differently express certain markers and secrete different amounts of proinflammatory and anti-inflammatory cytokines. We observed strong expression of the SAMHD1 restriction factor and weak expression of its inactive form (pSAMHD1, phosphorylated at residue Thr592) in both eM and dM. Infection of macrophages from both tissues was enhanced in the presence of the viral protein Vpx, suggesting a role for SAMHD1 in the restriction of HIV-1 infection. This study and further comparisons of the decidua with FRT mucosae in nonpregnant women should help to identify mechanisms of mucosal protection against HIV-1 infection. IMPORTANCE: The female reproductive tract mucosae are major portals of HIV-1 entry into the body. The decidua (uterine mucosa during pregnancy) can serve as a model for studying natural mucosal protection against HIV-1 transmission. A comparison of target cells and innate responses in the decidua versus the endometrium in nonpregnant women could help to identify protective mechanisms. Here, we report for the first time that macrophages are one of the main HIV-1 target cells in the endometrium and that infection of macrophages from both the endometrium and the decidua is restricted by SAMHD1. These findings might have implications for the development of vaccines to prevent HIV-1 mucosal transmission.

Role of the human vaginal microbiota in the regulation of inflammation and sexually transmitted infection acquisition: Contribution of the non-human primate model to a better understanding?

The human vaginal microbiota has a central role in the regulation of the female reproductive tract (FRT) inflammation. Indeed, on one hand an optimal environment leading to a protection against sexually transmitted infections (STI) is associated with a high proportion of Lactobacillus spp. (eubiosis). On the other hand, a more diverse microbiota with a high amount of non-Lactobacillus spp. (dysbiosis) is linked to a higher local inflammation and an increased STI susceptibility. The composition of the vaginal microbiota is influenced by numerous factors that may lead to a dysbiotic environment. In this review, we first discuss how the vaginal microbiota composition affects the local inflammation with a focus on the cytokine profiles, the immune cell recruitment/phenotype and a large part devoted on the interactions between the vaginal microbiota and the neutrophils. Secondly, we analyze the interplay between STI and the vaginal microbiota and describe several mechanisms of action of the vaginal microbiota. Finally, the input of the NHP model in research focusing on the FRT health including vaginal microbiota or STI acquisition/control and treatment is discussed.

Local Innate Markers and Vaginal Microbiota Composition Are Influenced by Hormonal Cycle Phases.

Background: The female reproductive tract (FRT) mucosa is the first line of defense against sexually transmitted infection (STI). FRT environmental factors, including immune-cell composition and the vaginal microbiota, interact with each other to modulate susceptibility to STIs. Moreover, the menstrual cycle induces important modifications within the FRT mucosa. Cynomolgus macaques are used as a model for the pathogenesis and prophylaxis of STIs. In addition, their menstrual cycle and FRT morphology are similar to women. The cynomolgus macaque vaginal microbiota is highly diverse and similar to dysbiotic vaginal microbiota observed in women. However, the impact of the menstrual cycle on immune markers and the vaginal microbiota in female cynomolgus macaques is unknown. We conducted a longitudinal study covering three menstrual cycles in cynomolgus macaques. The evolution of the composition of the vaginal microbiota and inflammation (cytokine/chemokine profile and neutrophil phenotype) in the FRT and blood was determined throughout the menstrual cycle. Results: Cervicovaginal cytokine/chemokine concentrations were affected by the menstrual cycle, with a peak of production during menstruation. We observed three main cervicovaginal neutrophil subpopulations: CD11bhigh CD101+ CD10+ CD32a+, CD11bhigh CD101+ CD10- CD32a+, and CD11blow CD101low CD10- CD32a-, of which the proportion varied during the menstrual cycle. During menstruation, there was an increase in the CD11bhigh CD101+ CD10+ CD32a+ subset of neutrophils, which expressed higher levels of CD62L. Various bacterial taxa in the vaginal microbiota showed differential abundance depending on the phase of the menstrual cycle. Compilation of the factors that vary according to hormonal phase showed the clustering of samples collected during menstruation, characterized by a high concentration of cytokines and an elevated abundance of the CD11bhigh CD101+ CD10+ CD32a+ CD62L+ neutrophil subpopulation. Conclusions: We show a significant impact of menstruation on the local environment (cytokine production, neutrophil phenotype, and vaginal microbiota composition) in female cynomolgus macaques. Menstruation triggers increased production of cytokines, shift of the vaginal microbiota composition and the recruitment of mature/activated neutrophils from the blood to the FRT. These results support the need to monitor the menstrual cycle and a longitudinal sampling schedule for further studies in female animals and/or women focusing on the mucosal FRT environment.

Women for science and science for women: Gaps, challenges and opportunities towards optimizing pre-exposure prophylaxis for HIV-1 prevention.

Preventing new HIV infections remains a global challenge. Young women continue to bear a disproportionate burden of infection. Oral pre-exposure prophylaxis (PrEP), offers a novel women-initiated prevention technology and PrEP trials completed to date underscore the importance of their inclusion early in trials evaluating new HIV PrEP technologies. Data from completed topical and systemic PrEP trials highlight the role of gender specific physiological and social factors that impact PrEP uptake, adherence and efficacy. Here we review the past and current developments of HIV-1 prevention options for women with special focus on PrEP considering the diverse factors that can impact PrEP efficacy. Furthermore, we highlight the importance of inclusion of female scientists, clinicians, and community advocates in scientific efforts to further improve HIV prevention strategies.

A Case Study to Dissect Immunity to SARS-CoV-2 in a Neonate Nonhuman Primate Model.

Most children are less severely affected by coronavirus-induced disease 2019 (COVID-19) than adults, and thus more difficult to study progressively. Here, we provide a neonatal nonhuman primate (NHP) deep analysis of early immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in blood and mucosal tissues. In addition, we provide a comparison with SARS-CoV-2-infected adult NHP. Infection of the neonate resulted in a mild disease compared with adult NHPs that develop, in most cases, moderate lung lesions. In concomitance with the viral RNA load increase, we observed the development of an early innate response in the blood, as demonstrated by RNA sequencing, flow cytometry, and cytokine longitudinal data analyses. This response included the presence of an antiviral type-I IFN gene signature, a persistent and lasting NKT cell population, a balanced peripheral and mucosal IFN-γ/IL-10 cytokine response, and an increase in B cells that was accompanied with anti-SARS-CoV-2 antibody response. Viral kinetics and immune responses coincided with changes in the microbiota profile composition in the pharyngeal and rectal mucosae. In the mother, viral RNA loads were close to the quantification limit, despite the very close contact with SARS-CoV-2-exposed neonate. This pilot study demonstrates that neonatal NHPs are a relevant model for pediatric SARS-CoV-2 infection, permitting insights into the early steps of anti-SARS-CoV-2 immune responses in infants.

Decidual soluble factors participate in the control of HIV-1 infection at the maternofetal interface.

BACKGROUND: Maternofetal transmission (MFT) of HIV-1 is relatively rare during the first trimester of pregnancy despite the permissivity of placental cells for cell-to-cell HIV-1 infection. Invasive placental cells interact directly with decidual cells of the uterine mucosa during the first months of pregnancy, but the role of the decidua in the control of HIV-1 transmission is unknown. RESULTS: We found that decidual mononuclear cells naturally produce low levels of IL-10, IL-12, IL-15, TNF-α, IFN-α, IFN-γ and CXCL-12 (SDF-1), and large amounts of CCL-2 (MCP1), CCL-3 (MIP-1α), CCL-4 (MIP-1ß), CCL-5 (Rantes), CXCL-10 (IP-10), IL-6 and IL-8. CCL-3 and CCL-4 levels were significantly upregulated by in vitro infection with R5 HIV-1 but not X4. Decidual CD14+ antigen presenting cells were the main CCL-3 and CCL-4 producers among decidual leukocytes. R5 and X4 HIV-1 infection was inhibited by decidual cell culture supernatants in vitro. Using HIV-1 pseudotypes, we found that inhibition of the HIV-1 entry step was inhibited by decidual soluble factors. CONCLUSION: Our findings show that decidual innate immunity (soluble factors) is involved in the control of HIV-1 infection at the maternofetal interface. The decidua could thus serve as a mucosal model for identifying correlates of protection against HIV-1 infection.

Neonatal Immune System Ontogeny: The Role of Maternal Microbiota and Associated Factors. How Might the Non-Human Primate Model Enlighten the Path?

Interactions between the immune system and the microbiome play a crucial role on the human health. These interactions start in the prenatal period and are critical for the maturation of the immune system in newborns and infants. Several factors influence the composition of the infant's microbiota and subsequently the development of the immune system. They include maternal infection, antibiotic treatment, environmental exposure, mode of delivery, breastfeeding, and food introduction. In this review, we focus on the ontogeny of the immune system and its association to microbial colonization from conception to food diversification. In this context, we give an overview of the mother-fetus interactions during pregnancy, the impact of the time of birth and the mode of delivery, the neonate gastrointestinal colonization and the role of breastfeeding, weaning, and food diversification. We further review the impact of the vaccination on the infant's microbiota and the reciprocal case. Finally, we discuss several potential therapeutic interventions that might help to improve the newborn and infant's health and their responses to vaccination. Throughout the review, we underline the main scientific questions that are left to be answered and how the non-human primate model could help enlighten the path.

SARS-CoV-2 infection in nonhuman primates alters the composition and functional activity of the gut microbiota.

The current pandemic of coronavirus disease (COVID) 2019 constitutes a global public health issue. Regarding the emerging importance of the gut-lung axis in viral respiratory infections, analysis of the gut microbiota's composition and functional activity during a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection might be instrumental in understanding and controling COVID 19. We used a nonhuman primate model (the macaque), that recapitulates mild COVID-19 symptoms, to analyze the effects of a SARS-CoV-2 infection on dynamic changes of the gut microbiota. 16S rRNA gene profiling and analysis of ß diversity indicated significant changes in the composition of the gut microbiota with a peak at 10-13 days post-infection (dpi). Analysis of bacterial abundance correlation networks confirmed disruption of the bacterial community at 10-13 dpi. Some alterations in microbiota persisted after the resolution of the infection until day 26. Some changes in the relative bacterial taxon abundance associated with infectious parameters. Interestingly, the relative abundance of Acinetobacter (Proteobacteria) and some genera of the Ruminococcaceae family (Firmicutes) was positively correlated with the presence of SARS-CoV-2 in the upper respiratory tract. Targeted quantitative metabolomics indicated a drop in short-chain fatty acids (SCFAs) and changes in several bile acids and tryptophan metabolites in infected animals. The relative abundance of several taxa known to be SCFA producers (mostly from the Ruminococcaceae family) was negatively correlated with systemic inflammatory markers while the opposite correlation was seen with several members of the genus Streptococcus. Collectively, SARS-CoV-2 infection in a nonhuman primate is associated with changes in the gut microbiota's composition and functional activity.

Activation of Toll-Like Receptors Differentially Modulates Inflammation in the Human Reproductive Tract: Preliminary Findings.

The female reproductive tract (FRT) is the main site of entry of sexually transmitted infections (STIs). Toll-like receptors (TLRs) that recognize pathogenic motifs are widely expressed in the FRT. TLR stimulation induces immune activation and local production of inflammatory mediators. In the FRT, this response should also be compatible with reproductive functions and symbiosis with host microbiota. With a view to develop efficient mucosal vaccines to prevent STI acquisition, the role of TLR ligands in the FRT needs to be explored. We have therefore investigated the cytokine profiles of the different compartments of the FRT (vagina, endocervix, ectocervix, and uterus) before and after stimulation of mononuclear cells from human tissue specimens. The comparison with PBMCs allowed us to highlight the FRT specificities. We first characterized the main immune cell populations in each compartment and observed that their distribution was different through the compartments. The CD45+ cells represented a maximum of 11% in the FRT in contrast to 96% in PBMCs. We identified two main populations among the CD45+ cells in the four compartments of the FRT: CD3+ T cells (CD4+ and CD8+) and CD14+ APCs. B cell populations (CD19+) were much less frequent than T cells in all the FRT regions and were equally distributed. NK CD56+ cells were detected in all compartments and were more abundant in the uterus. Stimulation of the mononuclear cells was then performed with TLR agonists: R848 for TLR7/8, Poly I:C for TLR3, LPS for TLR4 and ODN CpG for TLR9. Cytokine levels in unstimulated cultures of cells isolated from all FRT compartments were higher than in cultures of unstimulated PBMCs. In contrast, after stimulation with TLR agonists, cytokine responses induced by TLR agonists were moderate in the FRT and significantly lower than in PBMCs. These responses were varied with different TLR ligands and FRT compartments. The cytokine profile induced by TLR activation in the FRT supports the role of these tissues in genital anti-microbial immunity and in the control of inflammation while allowing maintenance of its reproductive function.

Proteasome-independent degradation of HIV-1 in naturally non-permissive human placental trophoblast cells.

BACKGROUND: The human placenta-derived cell line BeWo has been demonstrated to be restrictive to cell-free HIV-1 infection. BeWo cells are however permissive to infection by VSV-G pseudotyped HIV-1, which enters cells by a receptor-independent mechanism, and to infection by HIV-1 via a cell-to-cell route. RESULTS: Here we analysed viral entry in wild type BeWo (CCR5+, CXCR4+) and BeWo-CD4+ (CD4+, CCR5+, CXCR4+) cells. We report that HIV-1 internalisation is not restricted in either cell line. Levels of internalised p24 antigen between VSV-G HIV-1 pseudotypes and R5 or X4 virions were comparable. We next analysed the fate of internalised virions; X4 and R5 HIV-1 virions were less stable over time in BeWo cells than VSV-G HIV-1 pseudotypes. We then investigated the role of the proteasome in restricting cell-free HIV-1 infection in BeWo cells using proteasome inhibitors. We observed an increase in the levels of VSV-G pseudotyped HIV-1 infection in proteasome-inhibitor treated cells, but the infection by R5-Env or X4-Env pseudotyped virions remains restricted. CONCLUSION: Collectively these results suggest that cell-free HIV-1 infection encounters a surface block leading to a non-productive entry route, which either actively targets incoming virions for non-proteasomal degradation, and impedes their release into the cytoplasm, or causes the inactivation of mechanisms essential for viral replication.

Dynamics of Vaginal and Rectal Microbiota Over Several Menstrual Cycles in Female Cynomolgus Macaques.

The composition of the microbiota in cynomolgus macaques is only partially characterized, although this animal model is often used to study pathogenesis and preventive strategies against infections. We thus performed, for the first time, a longitudinal characterization of the vaginal and rectal microbiota of five cycling female cynomolgus macaques. Samples were collected weekly for 15 weeks and the V3/V4 regions of the16S rRNA gene sequenced. Sequences were analyzed with QIIME for OTU detection and taxonomic assignment. Progesterone levels were also determined to evaluate hormonal influence on bacteria relative abundance. The rectal and vaginal bacterial composition in cynomolgus macaques is polymicrobial and clearly distinct, with larger individual variability in the vagina. Rectal microbiota profiles were consistent between animals, whereas they were highly variable and animal-specific in the vagina. In the rectum, the most abundant taxa were Ruminococcaceae, Prevotella, and Clostridiales. In the vagina, the most abundant genera were Sneathia, Porphyromonas, Prevotella, and Fusobacterium. Lactobacillus were found at relative abundances higher than 1% in only one animal and were not predominant. Comparison of the vaginal cynomolgus macaque microbiota with that of humans showed similarity to community state type IV-A usually associated with dysbiosis. In the vagina, the relative abundance of 12 bacterial genera was found to be associated with progesterone levels. Our study provides a detailed characterization of the rectal and vaginal microbiota in female cynomolgus macaques and opens new perspectives of this animal model.

Seminal Plasma Exposures Strengthen Vaccine Responses in the Female Reproductive Tract Mucosae.

HIV-1 sexual transmission occurs mainly via mucosal semen exposures. In the female reproductive tract (FRT), seminal plasma (SP) induces physiological modifications, including inflammation. An effective HIV-1 vaccine should elicit mucosal immunity, however, modifications of vaccine responses by the local environment remain to be characterized. Using a modified vaccinia virus Ankara (MVA) as a vaccine model, we characterized the impact of HIV-1+ SP intravaginal exposure on the local immune responses of non-human primates. Multiple HIV-1+ SP exposures did not impact the anti-MVA antibody responses. However, SP exposures revealed an anti-MVA responses mediated by CD4+ T cells, which was not observed in the control group. Furthermore, the frequency and the quality of specific anti-MVA CD8+ T cell responses increased in the FRT exposed to SP. Multi-parameter approaches clearly identified the cervix as the most impacted compartment in the FRT. SP exposures induced a local cell recruitment of antigen presenting cells, especially CD11c+ cells, and CD8+ T cell recruitment in the FRT draining lymph nodes. CD11c+ cell recruitment was associated with upregulation of inflammation-related gene expression after SP exposures in the cervix. We thus highlight the fact that physiological conditions, such as SP exposures, should be taken into consideration to test and to improve vaccine efficacy against HIV-1 and other sexually transmitted infections.

Distinct efficacy of HIV-1 entry inhibitors to prevent cell-to-cell transfer of R5 and X4 viruses across a human placental trophoblast barrier in a reconstitution model in vitro.

BACKGROUND AND METHODS: HIV-1 cell-to-cell transmission is more efficient than infection of permissive cells with cell-free particles. The potency of HIV-1 entry inhibitors to inhibit such transmission is not well known. Herein, we evaluated the efficacy of this new class of antiretrovirals to block cell-to-cell transmission of HIV-1 in a model of reconstitution of the human placental trophoblast barrier in vitro. RESULTS: Our data show that CCR5 antagonists and T20 inhibit the passage of the virus across the BeWo cell monolayer in contact with PBMCs infected with an R5 (Ba-L) and a dualtropic (A204) HIV-1 with IC50s in the range of 100 - 5,000 nM for TAK779; 90 to 15,000 nM for SCH-350581 and 3,000 to 20,000 nM for T20. The CXCR4 antagonist AMD3100 is also effective against X4 HIV-1 infected PBMCs in our model with IC50 comprised between 4 nM and 640 nM. HIV-1 entry inhibitors are less efficient to block cell-to-cell virus transmission than cell-free HIV-1 infection of PBMCs and CCR5 antagonists do not prevent PBMC infection by dual tropic HIV-1 in contrast to cell-to-cell infection in our model.Surprisingly, T20 (and C34) do not block cell-to-cell transmission of X4 HIV-1 but, rather, increase 80 to 140 fold, compared to control without drug, the passage of the virus across the trophoblast barrier. Additional experiments suggest that the effect of T20 on BeWo/PBMC-X4 HIV-1 is due to an increase of effector-target cells fusion. CONCLUSION: Our results support further evaluation of HIV-1 coreceptor antagonists, alone or combined to other antiretrovirals, in a perspective of prevention but warn on the use of T20 in patients bearing X4 HIV-1 at risk of transmission.

Tumour necrosis factor-alpha stimulates HIV-1 replication in single-cycle infection of human term placental villi fragments in a time, viral dose and envelope dependent manner.

BACKGROUND: The placenta plays an important role in the control of in utero HIV-1 mother-to-child transmission (MTCT). Proinflammatory cytokines in the placental environment are particularly implicated in this control. We thus investigated the effect of TNF-alpha on HIV-1 expression in human placental tissues in vitro. RESULTS: Human placental chorionic villi fragments were infected with varying doses of luciferase reporter HIV-1 pseudotypes with the R5, X4-Env or the vesicular stomatitis virus protein G (VSV-G). Histocultures were then performed in the presence or absence of recombinant human TNF-alpha. Luciferase activity was measured at different time points in cell lysates or on whole fragments using ex vivo imaging systems.A significant increase in viral expression was detected in placental fragments infected with 0.2 ng of p24 antigen/fragment (P = 0.002) of VSV-G pseudotyped HIV-1 in the presence of TNF-alpha seen after 120 hours of culture. A time independent significant increase of viral expression by TNF-alpha was observed with higher doses of VSV-G pseudotyped HIV-1. When placental fragments were infected with R5-Env pseudotyped HIV-1, a low level of HIV expression at 168 hours of culture was detected for 3 of the 5 placentas tested, with no statistically significant enhancement by TNF-alpha. Infection with X4-Env pseudotyped HIV-1 did not lead to any detectable luciferase activity at any time point in the absence or in the presence of TNF-alpha. CONCLUSION: TNF-alpha in the placental environment increases HIV-1 expression and could facilitate MTCT of HIV-1, particularly in an inflammatory context.

First Dominique Dormont International Conference on "Host-pathogen interactions in chronic infections - viral and host determinants of HCV, HCMV, and HIV infections".

The first Dominique Dormont International Conference on "Viral and host determinantsof HCV, HCMV, and HIV infections "was held in Paris, Val-de-Grâce, on December 3-4, 2004. The following is a summary of the scientific sessions of this meeting (http://www.congres-evenement.fr/ddormont).

Evaluation of Cytokeratin 7 as an accurate intracellular marker with which to assess the purity of human placental villous trophoblast cells by flow cytometry.

Villous trophoblast cells (TC) obtained from first trimester and term human placentae after trypsin/Percoll gradient isolation were immunodepleted of contaminant cells. The level of purity was assessed by the intracellular expression of the pan trophoblast marker cytokeratin-7 (CK7) and comparisons were made with the GB25 trophoblast-specific (cytotrophoblast+syncytiotrophoblast) cell surface marker. The presence of contaminating cells was traced with intracellular vimentin, or cell surface CD2, CD36, and CD163 markers and evaluated by flow cytometric analysis. The pattern of CK7 expression by trophoblast cells was also analyzed by immunofluorescence microscopy. Most batches of TC from first trimester or term placentae (92+/-3% and 96+/-2%, respectively) showed a high percentage of CK7 expressing cells, with less than 2% contaminating vimentin positive cells. In some batches of TC with a lower percentage (65+/-4%) of CK7-expressing cells, no vimentin was found, but a low percentage of CD36-expressing cells was evidenced, with no presence of CD2, and/or CD163-expressing cells. The intracellular CK7 signal correlated significantly with that of GB25 (p<0.05) cell surface expression in TC of term placentae. The choriocarcinoma BeWo and Jar cell lines also showed high levels (>92%) of CK7-expressing cells. Conversely, the control U87 astrocytoma cell line showed a high percentage (>90%) of vimentin but no CK7-expressing cells. These results provide evidence that the mutually exclusive pattern of intracellular CK7/vimentin expression of human TC can be used for evaluation by flow cytometry of the purity of primary human trophoblast cells.

Mother-to-child transmission of human immunodeficiency virus type 1 in relation to the season in Yaounde, Cameroon.

A public health program to prevent mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) by treatment with nevirapine has been ongoing in Yaounde, Cameroon since January 2000. After 24 months, plasma samples from 119 children born to HIV-1-positive mothers were tested for HIV-1 RNA between six and eight weeks after birth. Thirteen (10.9%) tested positive (95% confidence interval = 5.2-16.7%). Risk factors associated with MTCT in this study were maternal viral load (P < 0.05), low birth weight (chi2 for trend = 8.78, P = 0.01), and birth during the second half of the year. A high correlation was repeatedly observed between rainfall in a given month and the risk of MTCT of HIV-1 in children born three months later (r = 0.634, P < 0.001). Although we cannot rule out other tropical infections related to the rainy season, the role of malaria is highly suspected since the interval of three months we observed between the peaks of rainfall and the rate of transmission is consistent with the Plasmodium life cycle.

Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis), where maternal and fetal cells are in close contact. Toll-like receptors (TLRs) may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs) and NK cells (dNKs), the major decidual immune cell populations. We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3, and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8, and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1ß, IL-10, and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.