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Depot medroxyprogesterone acetate (DMPA) is an injectable hormonal contraceptive used by millions of women worldwide. However, experimental studies have associated DMPA use with genital epithelial barrier disruption and mucosal influx of human immunodeficiency virus (HIV) target cells. We explored the underlying molecular mechanisms of these findings. Ectocervical biopsies and cervicovaginal lavage (CVL) specimens were collected from HIV-seronegative Kenyan sex workers using DMPA (n = 32) or regularly cycling controls (n = 64). Tissue samples were assessed by RNA-sequencing and quantitative imaging analysis, whereas protein levels were measured in CVL samples. The results suggested a DMPA-associated upregulation of genes involved in immune regulation, including genes associated with cytokine-mediated signaling and neutrophil-mediated immunity. A transcription factor analysis further revealed DMPA-associated upregulation of RELA and NFKB1 which are involved in several immune activation pathways. Several genes significantly downregulated in the DMPA versus the control group were involved in epithelial structure and function, including genes encoding keratins, small proline-rich proteins, and cell-cell adhesion proteins. Pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development, including keratinization and cornification processes. The cervicovaginal microbiome composition (Lactobacillus dominant and non-Lactobacillus dominant) had no overall interactional impact on the DMPA associated tissue gene expression. Imaging analysis verified that DMPA use was associated with an impaired epithelial layer as illustrated by staining for the selected epithelial junction proteins E-cadherin, desmoglein-1 and claudin-1. Additional staining for CD4+ cells revealed a more superficial location of these cells in the ectocervical epithelium of DMPA users versus controls. Altered protein levels of SERPINB1 and ITIH2 were further observed in the DMPA group. Identification of specific impaired epithelial barrier structures at the gene expression level, which were verified at the functional level by tissue imaging analysis, illustrates mechanisms by which DMPA adversely may affect the integrity of the genital mucosa.

Depot medroxyprogesterone acetate (DMPA) is the most common hormonal contraceptive used by women in sub-Saharan Africa, however, it has been epidemiologically associated with HIV infections. To assess whether DMPA has an effect on the number and activation of HIV target cells, this study assessed the levels and phenotype of blood- and mucosal-derived HIV target cells among women using DMPA. Thirty-five HIV uninfected women from the Pumwani Sex Worker cohort from Nairobi, Kenya were enrolled in the study (15 using DMPA and 20 not using hormonal contraception). Blood (plasma and peripheral blood mononuclear cells) and cervicovaginal (lavage, cervical cells, and ectocervical biopsies) samples were collected. Cellular phenotype and activation status were determined by flow cytometry, cytokine levels were assessed by bead array and image analysis assessed cell number and phenotype in situ. In blood, the proportion of HIV target cells and activated T cells was lower in DMPA users versus those not using hormonal contraceptives. However, analysis of cervical mononuclear cells showed that DMPA users had elevated levels of activated T cells (CD4+CD69+) and expressed lower levels of the HIV co-receptor CCR5 on a per cell basis, while tissue samples showed that in the ectocervix, DMPA users had a higher proportion of CD4+CCR5+ T cells. This study demonstrates that DMPA users had higher levels of activated T cells and HIV target cells in the genital tract. The increased pool of mucosal HIV target cells provides new biological information about the potential impact of DMPA on HIV susceptibility.

Mucosa‐associated invariant T (MAIT) cells are unconventional T lymphocytes defined by their innate‐like characteristics and broad antimicrobial responsiveness. Whether MAIT cells are part of the tissue‐resident defense in the oral mucosal barrier is unknown. Here, we found MAIT cells present in the buccal mucosa, with a tendency to cluster near the basement membrane, and located in both epithelium and the underlying connective tissue. Overall MAIT cell levels were similar in the mucosa compared to peripheral blood, in contrast to conventional T cells that showed an altered representation of CD4+ and CD8+ subsets. The major mucosal MAIT cell subset displayed a tissue‐resident and activated profile with high expression of CD69, CD103, HLA‐DR, and PD‐1, as well as a skewed subset distribution with higher representation of CD4–/CD8– double‐negative cells and CD8αα+ cells. Interestingly, tissue‐resident MAIT cells had a specialized polyfunctional response profile with higher IL‐17 levels, as assessed by polyclonal stimulus and compared to tissue nonresident and circulating populations. Furthermore, resident buccal MAIT cells were low in perforin. Together, these data indicate that MAIT cells form a part of the oral mucosal T cell compartment, where they exhibit a tissue‐resident‐activated profile biased toward IL‐17 production.

Immunological correlates of natural resistance to HIV have been identified in HIV-exposed seronegative (HESN) individuals and include a low-inflammatory genital mucosal status. The cervicovaginal epithelium has not been studied for such correlates despite constituting an important barrier against sexual HIV transmission. To fill this gap in knowledge, we collected samples of blood, cervical mononuclear cells, cervicovaginal lavage, and ectocervical tissue from Kenyan HESN sex workers (n = 29) and controls (n = 33). The samples were analyzed by flow cytometry, protein profiling, 16S rRNA gene sequencing, in situ image analysis, and tissue-based RNA sequencing. A significantly higher relative proportion of regulatory T cells in blood (B7+CD25hiFoxP3+CD127loCD4+ and B7+Helios+FoxP3+CD4+), and a significantly lower proportion of activated cervical T cells (CCR5+CD69+CD4+ and CCR5+CD69+CD8+), were found in the HESN group compared with the controls. In contrast, there were no statistically significant differences between the study groups in cervicovaginal protein and microbiome compositions, ectocervical epithelial thickness, E-cadherin expression, HIV receptor expression, and tissue RNA transcriptional profiles. The identification of an intact ectocervical microenvironment in HESN individuals add new data to current knowledge about natural resistance to sexual transmission of HIV.

The female genital tract (FGT) mucosa is a critically important site for immune defense against microbes. Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population that recognizes microbial riboflavin metabolite antigens in an MR1-dependent manner. The role of MAIT cells in the FGT mucosa is unknown. Here, we found that MAIT cells and MR1+ antigen-presenting cells were present in the upper and lower FGT, with distinct tissue localization of MAIT cells in endometrium vs. cervix. The MAIT cells from the FGT and blood displayed a distinct phenotype with expression of interleukin (IL)-18Rα, CD127, α4β7, PD-1, as well as the transcription factors promyelocytic leukemia zinc finger (PLZF), RORγt, Helios, Eomes, and T-bet. Their expression levels of PLZF and Eomes were lower in the FGT compared with blood. When stimulated with Escherichia coli, MAIT cells from the FGT displayed a bias towards IL-17 and IL-22 expression, whereas blood MAIT cells produced primarily IFN-γ, TNF, and Granzyme B. Furthermore, both FGT- and blood-derived MAIT cells were polyfunctional and contributed to the T-cell-mediated response to E. coli. Thus, MAIT cells in the genital mucosa have a distinct IL-17/IL-22 profile and may have an important role in the immunological homeostasis and control of microbes at this site.

Depot medroxyprogesterone acetate (DMPA) is the most common hormonal contraceptive used by women in sub-Saharan Africa, however, it has been epidemiologically associated with HIV infections. To assess whether DMPA has an effect on the number and activation of HIV target cells, this study assessed the levels and phenotype of blood- and mucosal-derived HIV target cells among women using DMPA. Thirty-five HIV uninfected women from the Pumwani Sex Worker cohort from Nairobi, Kenya were enrolled in the study (15 using DMPA and 20 not using hormonal contraception). Blood (plasma and peripheral blood mononuclear cells) and cervicovaginal (lavage, cervical cells, and ectocervical biopsies) samples were collected. Cellular phenotype and activation status were determined by flow cytometry, cytokine levels were assessed by bead array and image analysis assessed cell number and phenotype in situ. In blood, the proportion of HIV target cells and activated T cells was lower in DMPA users versus those not using hormonal contraceptives. However, analysis of cervical mononuclear cells showed that DMPA users had elevated levels of activated T cells (CD4+CD69+) and expressed lower levels of the HIV co-receptor CCR5 on a per cell basis, while tissue samples showed that in the ectocervix, DMPA users had a higher proportion of CD4+CCR5+ T cells. This study demonstrates that DMPA users had higher levels of activated T cells and HIV target cells in the genital tract. The increased pool of mucosal HIV target cells provides new biological information about the potential impact of DMPA on HIV susceptibility.

Invariant NKT (iNKT) cells are innate-like T cells that respond rapidly with a broad range of effector functions upon recognition of glycolipid Ags presented by CD1d. HIV-1 carries Nef- and Vpu-dependent mechanisms to interfere with CD1d surface expression, indirectly suggesting a role for iNKT cells in control of HIV-1 infection. In this study, we investigated whether iNKT cells can participate in the innate cell–mediated immune response to HIV-1. Infection of dendritic cells (DCs) with Nef- and Vpu-deficient HIV-1 induced upregulation of CD1d in a TLR7-dependent manner. Infection of DCs caused modulation of enzymes in the sphingolipid pathway and enhanced expression of the endogenous glucosylceramide Ag. Importantly, iNKT cells responded specifically to rare DCs productively infected with Nef- and Vpu-defective HIV-1. Transmitted founder viral isolates differed in their CD1d downregulation capacity, suggesting that diverse strains may be differentially successful in inhibiting this pathway. Furthermore, both iNKT cells and DCs expressing CD1d and HIV receptors resided in the female genital mucosa, a site where HIV-1 transmission occurs. Taken together, these findings suggest that innate iNKT cell sensing of HIV-1 infection in DCs is an early immune detection mechanism, which is independent of priming and adaptive recognition of viral Ag, and is actively targeted by Nef- and Vpu-dependent viral immune evasion mechanisms.

PROBLEM Susceptibility to HIV is associated with the menstrual cycle and vaginal microbiome, but their collective impact on vaginal inflammation remains unclear. Here, we characterized the cervicovaginal proteome, inflammation, and microbiome community structure and function during the menstrual cycle. METHOD OF STUDY Cervicovaginal secretions were collected from regularly cycling women (n = 16) at median day 10, 16, and 24 of each menstrual cycle and analyzed by mass spectrometry, 16S rRNA gene sequencing, and a multiplex bead array immunoassay. Follicular, ovulatory, and luteal phases were defined by serum sex hormone levels. RESULTS Ovulation showed the largest mucosal proteome changes, where 30% and 19% of the 406 human proteins identified differed compared to the luteal and follicular phases, respectively. Neutrophil/leukocyte migration pathways were lowest during ovulation and peaked in the luteal phase, while antimicrobial and epithelial barrier promoting proteins were highest during ovulation. Vaginal microbial community structure and function did not vary significantly during the menstrual cycle, with the majority consistently Lactobacillus-dominant (63%) or non-Lactobacillus-dominant (25%). Fluctuations in the epithelial barrier protein RPTN between the ovulatory and luteal phase were amplified in women with Gardnerella vaginalis and anaerobic bacteria and reduced when Lactobacillus was dominant. CONCLUSION This small study demonstrates that sex hormones modulate neutrophil/leukocyte inflammation, barrier function, and antimicrobial pathways in the female genital tract with the strongest changes occurring during ovulation. The data further suggest a microbiome context for hormone-driven changes in vaginal immunity which may have implications for HIV susceptibility.

Background Genital mucosa is the main portal of entry for various incoming pathogens, including human immunodeficiency virus (HIV), hence it is an important site for host immune defenses. Tissue-resident memory T (TRM) cells defend tissue barriers against infections and are characterized by expression of CD103 and CD69. In this study, we describe the composition of CD8+ TRM cells in the ectocervix of healthy and HIV-infected women. Methods Study samples were collected from healthy Swedish and Kenyan HIV-infected and uninfected women. Customized computerized image-based in situ analysis was developed to assess the ectocervical biopsies. Genital mucosa and blood samples were assessed by flow cytometry. Results Although the ectocervical epithelium of healthy women was populated with bona fide CD8+ TRM cells (CD103+CD69+), women infected with HIV displayed a high frequency of CD103-CD8+ cells residing close to their epithelial basal membrane. Accumulation of CD103-CD8+ cells was associated with chemokine expression in the ectocervix and HIV viral load. CD103+CD8+ and CD103-CD8+ T cells expressed cytotoxic effector molecules in the ectocervical epithelium of healthy and HIV-infected women. In addition, women infected with HIV had decreased frequencies of circulating CD103+CD8+ T cells. Conclusions Our data provide insight into the distribution of CD8+ TRM cells in human genital mucosa, a critically important location for immune defense against pathogens, including HIV.

The most immediate and evident effect of mucosal exposure to semen in vivo is a local release of proinflammatory mediators accompanied by an influx of leukocytes into the female genital mucosa (FGM). The implication of such response in HIV-1 transmission has never been addressed due to limitations of currently available experimental models. Using human tissue explants from the uterine cervix, we developed a system of mucosal exposure to seminal plasma (SP) that supports HIV-1 replication. Treatment of ectocervical explants with SP resulted in the upregulation of inflammatory and growth factors, including IL-6, TNF, CCL5, CCL20, CXCL1, and CXCL8, and IL1A, CSF2, IL7, PTGS2, as evaluated by measuring protein levels in explant conditioned medium (ECM) and gene expression in tissue. SP treatment was also associated with increased recruitment of monocytes and neutrophils, as observed upon incubation of peripheral blood leukocytes with ECM in a transwell system. To evaluate the impact of the SP-mediated response on local susceptibility to HIV-1, we infected ectocervical explants with the CCR5-tropic variant HIV-1BaL either in the presence of SP, or after explant pre-incubation with SP. In both experimental settings SP enhanced virus replication as evaluated by HIV-1 p24gag released in explant culture medium over time, as well as by HIV-1 DNA quantification in explants infected in the presence of SP. These results suggest that a sustained inflammatory response elicited by SP soon after coitus may promote HIV-1 transmission to the FGM. Nevertheless, ectocervical tissue explants did not support the replication of transmitted/founder HIV-1 molecular clones, regardless of SP treatment. Our system offers experimental and analytical advantages over traditional models of HIV-1 transmission for the study of SP immunoregulatory effect on the FGM, and may provide a useful platform to ultimately identify new determinants of HIV-1 infection at this site., Author summary The majority of HIV-1 transmissions worldwide occur via unprotected vaginal intercourse, with semen and the female genital mucosa (FGM) being the main vector and recipient of virus acquisition in women. It is well known that mucosal exposure to semen via vaginal intercourse or artificial fertilization results in transient inflammation of the FGM. This response is thought to be important to defend the host against eventual pathogens present in semen and clear the mucosa from residual sperm cells, as well as to promote fertilization. However its implication in HIV-1 transmission has never been addressed. Using human tissue explants from the uterine cervix, we developed a model of mucosal exposure to seminal plasma (SP) to study HIV-1 transmission to the FGM. Our model recapitulated the main features of the in vivo response to semen, namely upregulation of proinflammatory factors and leukocyte recruitment. SP treatment of explants enhanced HIV-1 replication, suggesting that a sustained inflammatory response elicited by SP soon after coitus may promote virus infection. Our experimental system may contribute to improve the efficacy of currently available prevention measures by understanding the mechanisms regulating immunity in the FGM, and leading to the identification of new determinants of HIV-1 transmission therein.

Antiproteases play diverse roles in nature, from regulating protease activity to innate defense against microorganisms. Recently, antiproteases have been shown to play important roles in HIV pathogenesis including, inhibiting HIV binding and replication and reducing activation and inflammation of susceptible cells. They have also been implicated as one of the initial host responders, in plasma, to control replication of HIV. More recently, antiproteases expressed at the mucosal surface have been linked to reduced susceptibility to HIV infection in HIV-exposed sero-negative individuals. These factors are expressed in the epithelial layer of the female genital tract, thus at the frontline of defense against mucosal infection. This review focuses on the specific antimicrobial roles of antiproteases, focusing on serpins and cystatins, with an emphasis on their known and potential roles in HIV infection. Their potential as therapeutic interventions to combat HIV is also discussed.

Studies using genital tissue samples from HIV-infected women might provide important information about HIV susceptibility and transmission. In this study, ectocervical biopsies were obtained from 20 HIV-seropositive (HIV+) Kenyan female sex workers (FSW) and 20 HIV-seronegative lower risk (HIV− LR) women. To control for the impact of sex work, 20 HIV− FSW were also recruited. Immune molecules were assessed in situ by immunohistochemistry and for mRNA expression by quantitative PCR. The HIV+ women were reportedly infected for a median of 3 y (1–21 y), with a median viral load of 11,735 copies/ml (20–648,000 copies/ml). These women had significantly lower CD4 blood cell counts than the HIV− LR women but comparable levels of CD4 expression in ectocervix. Whereas cellular markers were similar between the HIV+ group and the HIV− LR women, the HIV-binding molecules CCR5, dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin, and mannose receptor as well as the inflammatory markers CD69, IFN-γ, IL-6, and IL-22 were significantly upregulated in the HIV+ group. As compared with the HIV− FSW women, the HIV+ women had significantly upregulated levels of CD4, CD3, CCR5, Langerin, dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin, and mannose receptor as well as inflammatory cytokines. The CD4 cell depletion previously seen in the gut mucosa of HIV-infected individuals was thus not observed in the ectocervical mucosa. Stable CD4 cell expression and local immune activation in the lower female genital tract may promote viral replication and genital shedding and increase the risk of sexual HIV transmission.

Background There are strong theoretical arguments for initiating antiretroviral therapy ( ART) during primary HIV-1 infection ( PHI) to preserve HIV-1-specific T-cell responses and to decrease immune activation.Methods We assessed the degree of immune activation during PHI and after analytical treatment interruption (ATI) in plasma samples from 22 subjects by measuring 13 cytokines/chemokines with the Luminex system. Subjects initiated quadruple ART at PHI ( the QUEST cohort) and were classified as responders or nonresponders according to their HIV-1 viral load (VL) 6 months post-ATI.Results During PHI, nonresponders had higher levels of IIIV-1 RNA, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-10 and eotaxin than responders ( P

The correlates of protective immunity in HIV-1 infection include the endogenous production of compounds with anti-HIV-1 activity. These compounds can be produced independently of specific humoral or cellular immune responses. A model of compartmental inhibition of HIV-1 infection is the placenta, an organ that prevents transmission of HIV-1 to the fetus in the majority of HIV-1 pregnancies. Studies of this organ elucidated new compounds and mechanisms for prevention and treatment of HIV including the potent inhibitor of HIV-1, leukemia inhibitory factor (LIF). Besides coordinating the humoral and cellular immune responses, cytokines such as IFN-gamma exhibit intrinsic antiviral activity that represents the first line of defense against pathogens prior to the development of a specific immune response. The study of antiviral factors is particularly important in HIV/AIDS because of the direct destruction of the immune system by HIV-1. In this report, we focus on the identification and mechanism of endogenously produced anti-HIV factors and the overall function of these factors in the prevention and treatment of HIV/AIDS.

The most immediate and evident effect of mucosal exposure to semen in vivo is a local release of proinflammatory mediators accompanied by an influx of leukocytes into the female genital mucosa (FGM). The implication of such response in HIV-1 transmission has never been addressed due to limitations of currently available experimental models. Using human tissue explants from the uterine cervix, we developed a system of mucosal exposure to seminal plasma (SP) that supports HIV-1 replication. Treatment of ectocervical explants with SP resulted in the upregulation of inflammatory and growth factors, including IL-6, TNF, CCL5, CCL20, CXCL1, and CXCL8, and IL1A, CSF2, IL7, PTGS2, as evaluated by measuring protein levels in explant conditioned medium (ECM) and gene expression in tissue. SP treatment was also associated with increased recruitment of monocytes and neutrophils, as observed upon incubation of peripheral blood leukocytes with ECM in a transwell system. To evaluate the impact of the SP-mediated response on local susceptibility to HIV-1, we infected ectocervical explants with the CCR5-tropic variant HIV-1BaL either in the presence of SP, or after explant pre-incubation with SP. In both experimental settings SP enhanced virus replication as evaluated by HIV-1 p24gag released in explant culture medium over time, as well as by HIV-1 DNA quantification in explants infected in the presence of SP. These results suggest that a sustained inflammatory response elicited by SP soon after coitus may promote HIV-1 transmission to the FGM. Nevertheless, ectocervical tissue explants did not support the replication of transmitted/founder HIV-1 molecular clones, regardless of SP treatment. Our system offers experimental and analytical advantages over traditional models of HIV-1 transmission for the study of SP immunoregulatory effect on the FGM, and may provide a useful platform to ultimately identify new determinants of HIV-1 infection at this site.

Mucosa-associated invariant T (MAIT) cells are a class of non-classical T lymphocytes defined by their invariant Vα7.2-containing MR1-restricted T cell receptor, as well as high expression of CD161. At present, the characteristics of MAIT cells in the oral mucosa are poorly defined. In this study, we have analyzed buccal biopsies and matched blood samples from a cohort of healthy volunteers. MAIT cells were present in the buccal mucosa, with a tendency to cluster around the basal layer, a region also found to contain a higher frequency of MR1-expressing antigen-presenting cells. The frequency of MAIT cells was similar in the buccal mucosa compared to matched peripheral blood, in contrast to other major T cell subsets. However, the buccal mucosal MAIT cells displayed a tissue resident activated profile, with high CD69, HLA-DR and PD-1 expression, as well as a skewed subset distribution with higher representation of CD4/CD8 double-negative cells and of CD8αα+ cells within the CD8+ subset. The Jα chain usage in the MAIT cell TCR was more variable in oral mucosal MAIT cells than in peripheral blood MAIT cells. Functionally, the oral mucosal MAIT cells had lower perforin levels, indicative of a lower cytolytic potential. Interestingly, aspects of the oral MAIT cell population were associated with life-style factors such as consumption of bacterially fermented milk products and alcohol intake. Together, these data show that MAIT cells form a part of the oral mucosal T cell compartment, where they exhibit a tissue-resident activated profile. Thus, MAIT cells are positioned in the oral cavity and equipped to handle resident microbiota as well as invading pathogens.

The female genital tract is a portal of entry for sexual HIV transmission and a possible viral reservoir. In this study, the ectocervical CD8+ T cell distribution was explored in situ and was related to expression of CD3 and HLA-DR and presence of HIV RNA. For this purpose, ectocervical tissue samples and genital secretions were collected from HIV-seropositive (HIV+) Kenyan female sex workers (FSWs) (n = 20), HIV-seronegative (HIV−) FSWs (n = 17), and HIV− lower-risk women (n = 21). Cell markers were assessed by in situ staining and by quantitative PCR. HIV RNA expression in tissue was analyzed by in situ hybridization, and viral shedding was assessed by quantitative PCR. The HIV+FSW group had a higher amount of total cells and CD8+, CD3+, and HLA-DR+ cells compared with the HIV−FSW group and HIV− lower-risk women. The majority of CD8+ cells were CD3+ T cells, and the numbers of CD8+ cells correlated significantly with plasma and cervical viral load. HIV RNA expression in situ was found in 4 of the 20 HIV+FSW women but did not correlate with cervical or plasma viral load. Thus, the HIV+ women displayed high numbers of CD8+, CD3+, and HLA-DR+ cells, as well as a limited number of HIV RNA+ cells, in their ectocervical mucosa; hence, this localization cannot be neglected as a potential viral reservoir. The elevated levels of CD8+ T cells may play a role in the immunopathogenesis of HIV in the female genital tract.

PROBLEM: Sex hormones can influence the immune defenses of the female genital tract (FGT) and its susceptibility to infections. Here we investigated the effect of different hormonal contraceptives on the production of antimicrobial peptides (AMPs) in different compartments of the female genital mucosa (FGM) secretions and tissue. METHOD OF STUDY: Cervicovaginal secretions (CVS) and ectocervical tissue samples obtained from women using progesterone intrauterine devices (pIUD) (n = 23) and combined oral contraceptives (COC) (n = 23) were analyzed for the expression and in situ localization of HNP1-3 BD-2 LL-37 SLPI and trappin-2 by ELISA real-time PCR and immunohistochemistry. RESULTS: Women using COC had significantly lower mRNA levels of BD-2 and trappin-2 in ectocervical tissue than pIUD users. The two groups showed no differences in CVS concentration as well as similar in situ expression patterns in ectocervical tissue of all five AMPs. CONCLUSIONS: The use of hormonal contraceptives influences AMP expression differently in genital secretions compared to ectocervical tissue. This suggests that the impact of sex hormones on local immune defenses varies in different compartments of the FGM and likely in different locations across the FGT. (c) 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Most HIV+ individuals require lifelong highly active antiretroviral therapy (HAART) to suppress HIV replication, but fail to eliminate the virus in part because of residual replication in gut-associated lymphoid tissues (GALT). Naturally elicited HIV-specific CD8+ T cells generated in the acute and chronic infectious phases exhibit antiviral activity, but decrease in number after HAART. Therapeutic vaccines represent a potential strategy to expand cellular responses, although previous efforts have been largely unsuccessful, conceivably because of a lack of responding HIV-specific central-memory CD8+ T cells (Tcm). To determine whether patients receiving HAART possess CD8+ T cells with Tcm qualities that are amenable to augmentation, HIV-specific CD8+ T-cell clones were derived from HIV-reactive CD28+CD8+ T-cell lines isolated from 7 HIV+ HAART-treated patients, expanded ex vivo, and reinfused into their autologous host. Tracking of the cells in vivo revealed that clones could persist for ≥ 84 days, maintain expression and/or re-express CD28, up-regulate CD62L, secrete IL-2, proliferate on cognate Ag encounter and localize to the rectal mucosa. These results suggest some infused cells exhibited phenotypic and functional characteristics shared with Tcm in vivo, and imply that more effective therapeutic vaccination strategies targeting CD8+ Tcm in patients on HAART might provide hosts with expanded, long-lasting immune responses not only systemically but also in GALT. This study is registered at www.clinicaltrials.gov as NCT00110578.

Leukemia inhibitor factor (LIF) has been shown to potently inhibit HIV-1 replication in vitro and in human organ explant cultures. Furthermore, LIF activates the Jak/Stat signaling pathway with which many viruses, including HIV-1, interfere. We used CXCR4 and the LIF signaling receptor (gp130)-expressing cMAGI cells transfected with CD4, CCR5, and HIV-LTR-beta-galactosidase as a model system to investigate the potential involvement of Stat proteins in the anti-HIV-1 effect of LIF. Pretreatment with recombinant human (rh)LIF resulted in a significantly reduced uptake of HIV-1(BaL) , HIV-1(LAI), and SIVmac251 viral particles without affecting uptake of murine leukemia retroviral particles. HIV-1(BaL), HIV-1(LAI), as well as rhLIF selectively induced phosphorylation of Stat 3 but not Stat 1 or Stat 5. However, treatment of cMAGI cells with rhLIF prior to HIV-1 infection downregulated the HIV-1-mediated Stat 3 phosphorylation. In addition, peripheral blood mononuclear cells (PBMCs) transfected with Stat 3 siRNA prior to HIV-1(LAI) or HIV-1(BaL) infection produced significantly less HIV-1 p24 antigen as compared to nontransfected HIV-1(LAI) and HIV-1(BaL)-infected PBMCs. Thus, the Jak/Stat signaling pathway is important for the HIV-1 replication life cycle and rhLIF excerts its anti-HIV-1 activity by disrupting this signaling cascade.

Objectives Skin biopsies from local sites of herpes simplex virus 2 (HSV-2)-induced ulcers can show infiltrates of inflammatory cells several months after macroscopic healing. We hypothesise that foreskin tissue samples of asymptomatic HSV-2 seropositive men had remaining signs of inflammation at the molecular level. Even in the absence of clinical lesions, genital inflammation may contribute to increased HIV susceptibility on sexual exposure to the virus. Setting Foreskin tissue samples were collected from men undergoing elective circumcision in Kisumu, Kenya. Participants The foreskin tissue samples (n=86) were stratified into study groups based on HSV-2 serology and assessed for mRNA expression of inflammatory markers. Markers of interest were further assessed by immunohistochemical staining within the tissue samples. Results The two study groups had comparable levels of all molecular markers (CD3, CD4, CD8, CD69, CCR5, HLA-DR, Langerin, DC-SIGN, Mannose Receptor 1, IL-1, IL-6, TNF-α, β7, IgA, IFN-α, CCL5, E-cadherin, ZO-1 and occludin), except for lower mRNA levels of the epithelial junction protein claudin-1 in the HSV-2 seropositive group (p=0.008). Although mRNA levels of claudin-1 were lower in HSV-2 seropositive individuals, the corresponding protein could be visualised in the foreskin epithelium of all samples tested. Conclusions Whereas no general inflammation was demonstrated in the foreskin of asymptomatic HSV-2 seropositive individuals, a decreased expression of claudin-1 indicates a less robust genital epithelial barrier. An intact epithelial barrier is essential for blocking mucosal entry of genital infections, including HIV.

BACKGROUND Leukemia inhibitor factor (LIF), a member of the interleukin-6 cytokine family, has recently been shown to inhibit HIV-1 replication both in vivo and in vitro. OBJECTIVE LIF and its corresponding receptors gp130 and LIF receptor-alpha (LIFR-alpha) were studied in lymphoid tissue (LT) to reveal potential systemic immunoregulatory effects during the course of HIV-1 infection. METHODS LIF, gp130, LIFR-alpha and HIV-1 replicating cells were identified at the single cell level by immunohistochemistry and quantified by computerized in situ imaging in tonsil and lymph nodes biopsies (LT) from patients with primary HIV-1 infection (PHI), chronic HIV-1 infection (cHI), long-term non-progressors (LTNP) and HIV-1 seronegative controls. RESULTS LIF and its receptors, gp130 and LIFR-alpha were significantly (P < 0.005) upregulated in LT from PHI patients as compared with HIV-1 seronegative controls. Expression of LIF in cHI was comparable to LIF levels in HIV-1 seronegative controls whereas LTNP showed significantly reduced LIF expression (P < 0.05). LIF receptors, gp130 and LIFR-alpha were significantly upregulated in cHI (P < 0.005) but downregulated in LTNP (P < 0.05 and P < 0.005, respectively). LIF expressing cells could be demonstrated in LT 2 days after onset of acute retroviral syndrome. LIF expression was evident in CD3, CD4 and CD8 cells. Furthermore, high plasma viral load was associated with high expression of LIF in LT. Finally, no HIV-1 replication could be found in CD4 gp130-positive cells in PHI. CONCLUSIONS LIF, gp130 and LIFR-alpha showed increased expression in LT from patients with PHI. Furthermore, HIV-1 replication did not occur in cells expressing the LIF signaling receptor, gp130, indicating that LIF may be associated with control of HIV-1 replicating cells in vivo.

Dendritic cells (DC) are antigen-presenting cells (APC) that most efficiently initiate and control immune responses. Migration processes of blood DC are crucial to exert their professional antigen-presenting functions. Matrix-degrading metalloproteinases (MMP) are proteolytic enzymes, which are considered to be key enzymes in extracellular matrix (ECM) turnover and mediators of cell migration. Tissue inhibitors of metalloproteinases (TIMP) are important regulators of MMP activity. Here we investigate whether blood monocyte-derived immature DC (iDC) and mature DC (mDC) express, produce and secrete functionally active MMP-1, -2, -3 and -9 and their inhibitors TIMP-1 and -2, and examine their involvement in multiple sclerosis (MS). On mRNA level, we observed high numbers of MMP-2 and TIMP-2 mRNA expressing iDC in MS. On protein level, high percentages of MMP-1, -2 and -9 expressing iDC by flow cytometry, and high MMP-1 secretion by Western blot together with high MMP-2 and -9 activities in iDC supernatants as studied with zymography were observed. Similarly, MS is associated with high percentages of MMP-2 and -3 and of TIMP-1 expressing mDC by flow cytometry together with high MMP-3 secretion and high MMP-9 activity in culture supernatants. Spontaneous migratory capacity of both iDC and mDC over ECM-coated filters was higher in MS compared to healthy controls (HC). In conclusion, blood monocyte-derived iDC and mDC express, produce and secrete several MMP and TIMP. Alterations in these molecules as observed in MS may be functionally important for DC functioning.

We have examined the role of alpha and beta chemokines in the promotion of the ontogenetic development of the brain. RANTES was expressed preferentially in human fetal astrocytes in an age-dependent manner. Astrocytes from 5-week-old brains showed high proliferation and reduced survival, whereas 10-week-old astrocytes exhibited opposite effects. These effects were suppressed by anti-RANTES or anti-RANTES receptor antibodies and were enhanced by recombinant RANTES. RANTES induced tyrosine phosphorylation of several cellular proteins and nuclear translocation of STAT-1 in astrocytes. Interferon-gamma (IFN-gamma) was required for RANTES effects because RANTES induced IFN-gamma and only 10-week-old astrocytes expressed the IFN-gamma receptor. Blocking of IFN-gamma with antibody reversed the effects of RANTES, indicating that cytokine/chemokine networks are critically involved in brain development.

We addressed the host-parasite interplay and the immunopathogenetic events occurring in the central nervous system (CNS) during human African trypanosomiasis. Human first trimester forebrain cells were stimulated with a trypanosome lymphocyte-triggering factor (TLTF) and studied for their immune response as exemplified by cell proliferation and IFN-gamma production. TLTF induced proliferation of human first trimester forebrain cells and IFN-gamma production at the mRNA and protein levels. Astrocytes are the major producers of IFN-gamma in response toTLTE These data illustrated for the first time a direct effect of a parasite factor on human brain cells. TargetingTLTF during the course of the disease may be considered in preventing the deadly neurological complications of human African trypanosomiasis. NeuroReport