Evaluation of Detection and Efficacy of Decontamination of Respiratory Pathogens Including SARS-CoV-2 on Basic Military Trainee Gas Masks.

INTRODUCTION: Basic military trainee (BMT) gas mask training poses a potential mechanism of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) transmission. After training, gas masks are decontaminated. Insufficient decontamination can lead to viral transmission in the next training class. To our knowledge, the decontamination process has not been validated for efficacy in removing respiratory pathogens such as SARS-CoV-2. MATERIALS AND METHODS: Inactivated strains of SARS-CoV-2, influenza A and B, and Bordetella pertussis were separately inoculated onto gas masks in the emitter area (n = 5). Pathogen detection in swabs collected from gas masks was performed by real-time polymerase chain reaction (RT-PCR) using the BioFire® RP2.1 panel and Biomeme Franklin system. For decontamination efficacy experiments, pathogens were inoculated onto gas masks, and contaminated areas were swabbed before and after decontamination, with detection using both PCR platforms. Lastly, 65 gas masks were swabbed after gas mask training, and again after the trainees and guardians decontaminated the masks, to identify the presence of any respiratory pathogen exhaled onto the gas masks. RESULTS: All four pathogens were detected by both PCR platforms. The BioFire® FilmArray® was more sensitive than the Biomeme platform. Decontamination resulted in undetectable levels of all three viruses. B. pertussis was detected on one mask after decontamination. Experiments with live B. pertussis validated that decontamination eliminated all viable bacteria from gas masks. For BMT sampling, all masks were negative for SARS-CoV-2. One mask tested positive for coronavirus 229E. Once decontaminated, all masks tested negative. CONCLUSIONS: BMT gas masks can be monitored for the presence of respiratory pathogens using RT-PCR. The decontamination process removed all viable respiratory pathogens tested from the gas masks. This study demonstrates that RT-PCR can be used to conduct pathogen surveillance on BMT gas masks after training and that the current decontamination process is effective to eliminate respiratory viruses including SARS-CoV-2.

Comparison of next generation diagnostic systems (NGDS) for the detection of SARS-CoV-2.

INTRODUCTION: The World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) a pandemic in March 2020. Initially, supply chain disruptions and increased demand for testing led to shortages of critical laboratory reagents and inadequate testing capacity. Thus, alternative means of biosample collection and testing were essential to overcome these obstacles and reduce viral transmission. This study aimed to 1) compare the sensitivity and specificity of Cepheid GeneXpert® IV and BioFire® FilmArray® 2.0 next generation detection systems to detect SARS-CoV-2, 2) evaluate the performance of both platforms using different biospecimen types, and 3) assess saline as an alternative to viral transport media (VTM) for sample collection. METHODS: A total of 1,080 specimens consisting of nasopharyngeal (NP) swabs in VTM, NP swabs in saline, nasal swabs, oropharyngeal (OP) swabs, and saliva were collected from 216 enrollees. Limit of detection (LoD) assays, NP VTM and NP saline concordance, and saliva testing were performed on the BioFire® FilmArray® 2.0 Respiratory Panel 2.1 and Cepheid GeneXpert® Xpress SARS-CoV-2/Flu/RSV assays. RESULTS: LoD and comparative testing demonstrated increased sensitivity with the Cepheid compared with the BioFire® in detecting SARS-CoV-2 in NP VTM and saline, nasal, and OP swabs. Conversely, saliva testing on the Cepheid showed statistically significant lower sensitivity compared to the BioFire® . Finally, NP swabs in saline showed no significant difference compared with NP swabs in VTM on both platforms. CONCLUSION: The Cepheid and BioFire® NGDS are viable options to address a variety of public health needs providing rapid and reliable, point-of-care testing using a variety of clinical matrices.

Evaluating sensitivity and specificity of the Biomeme Franklin™ three9 real-time PCR device and SARS-CoV-2 go-strips assay using clinical samples.

We evaluated the sensitivity and specificity of the Biomeme Franklin™ three9 Real-Time PCR Thermocycler and Biomeme SARS-CoV-2 Go-Strips in the detection of SARS-CoV-2. The Biomeme Franklin™ three9 platform is a portable, battery-operated system that could be used in remote settings. We assessed performance of the Biomeme SARS-CoV-2 detection system at a wide range of viral concentrations, examined cross-reactivity of the SARS-CoV-2 Go-Strips against several near-neighbor respiratory pathogens, and evaluated agreement against the BioFire® Respiratory Panel 2.1 in four clinical sample types. Our data indicate the Biomeme Go-Strips can reliably detect SARS-CoV-2 at a concentration of 4.2 × 103 copies/mL. No cross reactivity of the Go-Strips targets was detected against any of the tested near-neighbor respiratory pathogens. Cohen's kappa statistics ranged from 0.68 to 0.92 between results from the Biomeme SARS-CoV-2 Go-Strips and the BioFire® Respiratory Panel 2.1 in all the different sample types. Compared to the BioFire® Respiratory Panel 2.1, the Biomeme SARS-CoV-2 Go-Strips demonstrated statistically significantly lower sensitivity in 3 out of 5 sample types. Overall, our study demonstrates the Biomeme Franklin™ three9 used with the SARS-CoV-2 Go-Strips is an effective system for the detection of SARS-CoV-2 that could potentially be used in a remote or austere environment.

Vitamin D Status Impacts Genital Mucosal Immunity and Markers of HIV-1 Susceptibility in Women.

While vitamin D insufficiency is known to impact a multitude of health outcomes, including HIV-1, little is known about the role of vitamin D-mediated immune regulation in the female reproductive tract (FRT). We performed a pilot clinical study of 20 women with circulating 25(OH)D levels <62.5 nmol/L. Participants were randomized into either weekly or daily high-dose oral vitamin D supplementation groups. In addition to serum vitamin D levels, genital mucosal endpoints, including soluble mediators, immune cell populations, gene expression, and ex vivo HIV-1 infection, were assessed. While systemic vitamin D levels showed a significant increase following supplementation, these changes translated into modest effects on the cervicovaginal factors studied. Paradoxically, post-supplementation vitamin D levels were decreased in cervicovaginal fluids. Given the strong correlation between vitamin D status and HIV-1 infection and the widespread nature of vitamin D deficiency, further understanding of the role of vitamin D immunoregulation in the female reproductive tract is important.

The Effect of Hormonal Contraception on Cervicovaginal Mucosal End Points Associated with HIV Acquisition.

Reproductive age women may choose to concurrently use topical antiretrovirals and hormonal contraceptives (HCs) to simultaneously prevent HIV-1 infection and unintended/mistimed pregnancy. There are conflicting data on the effect of HCs on mucosal susceptibility to HIV-1. The objective of this study was to evaluate cervicovaginal (CV) mucosal data from healthy women before and after initiation of either oral contraceptive pills (OCPs) or depot medroxyprogesterone acetate (DMPA) injection. CONRAD A10-114 was a prospective, open-label, parallel cohort study. We enrolled 74 women and 62 completed the visits (32 and 30 who selected OCPs and DMPA, respectively). Participants provided CV lavage, vaginal biopsies, and CV swabs at baseline in the luteal phase and then ∼6 weeks after initiating HCs. After contraceptive initiation, there were significant increases in vaginal immune cell density among both DMPA and OCP users. Changes for OCP users were concentrated in the subepithelial lamina propria, whereas for DMPA users, they were distributed throughout the vaginal tissue, including the epithelium (CD45+, CD3+, CD4+, and CD1a+). Contraceptive use altered concentrations of soluble CV inflammatory and immune mediators, with significant reductions in some proinflammatory cytokines and secretory leukoprotease inhibitor. Compared with baseline, p24 antigen production after ex vivo HIV-1 infection of vaginal biopsies doubled after DMPA use, but all p-values were >.05. HIV-1 replication was significantly higher in DMPA-exposed tissues compared with those from the OCP group at the end of the tissue culture (p = .01). Although not statistically significant, median in vitro inhibition of HIV-1 by CV fluid (innate antiviral activity), was reduced by ∼50% with HCs (p > .21). Exposure to exogenous contraceptive hormones significantly increased vaginal immune cells and reduced CV proinflammatory cytokines and antimicrobial peptides. DMPA users showed higher susceptibility to HIV-1 ex vivo infection.

Randomized, placebo controlled phase I trial of safety, pharmacokinetics, pharmacodynamics and acceptability of tenofovir and tenofovir plus levonorgestrel vaginal rings in women.

To prevent the global health burdens of human immunodeficiency virus [HIV] and unintended/mistimed pregnancies, we developed an intravaginal ring [IVR] that delivers tenofovir [TFV] at ~10mg/day alone or with levonorgestrel [LNG] at ~20µg/day for 90 days. We present safety, pharmacokinetics, pharmacodynamics, acceptability and drug release data in healthy women. CONRAD A13-128 was a randomized, placebo controlled phase I study. We screened 86 women; 51 were randomized to TFV, TFV/LNG or placebo IVR [2:2:1] and 50 completed all visits, using the IVR for approximately 15 days. We assessed safety by adverse events, colposcopy, vaginal microbiota, epithelial integrity, mucosal histology and immune cell numbers and phenotype, cervicovaginal [CV] cytokines and antimicrobial proteins and changes in systemic laboratory measurements, and LNG and TFV pharmacokinetics in multiple compartments. TFV pharmacodynamic activity was measured by evaluating CV fluid [CVF] and tissue for antiviral activity using in vitro models. LNG pharmacodynamic assessments were timed based on peak urinary luteinizing hormone levels. All IVRs were safe with no significant colposcopic, mucosal, immune and microbiota changes and were acceptable. Among TFV containing IVR users, median and mean CV aspirate TFV concentrations remained above 100,000 ng/mL 4 hours post IVR insertion and mean TFV-diphosphate [DP] concentrations in vaginal tissue remained above 1,000 fmol/mg even 3 days post IVR removal. CVF of women using TFV-containing IVRs completely inhibited [94-100%] HIV infection in vitro. TFV/LNG IVR users had mean serum LNG concentrations exceeding 300 pg/mL within 1 hour, remaining high throughout IVR use. All LNG IVR users had a cervical mucus Insler score <10 and the majority [95%] were anovulatory or had abnormal cervical mucus sperm penetration. Estimated in vivo TFV and LNG release rates were within expected ranges. All IVRs were safe with the active ones delivering sustained high concentrations of TFV locally. LNG caused changes in cervical mucus, sperm penetration, and ovulation compatible with contraceptive efficacy. The TFV and TFV/LNG rings are ready for expanded 90 day clinical testing. Trial registration ClinicalTrials.gov #NCT02235662.

Comparison of Mucosal Markers of Human Immunodeficiency Virus Susceptibility in Healthy Premenopausal Versus Postmenopausal Women.

The objective of this study was to characterize cervicovaginal (CV) mucosal factors modulating susceptibility to human immunodeficiency virus (HIV) acquisition in healthy premenopausal (PRE) and postmenopausal (POST) women before and after treatment with estradiol (E2). We compared CV mucosal epithelial histology and immune cells, vaginal microbiota, antimicrobial activity of and soluble mucosal protein concentrations in the CV fluid lavage (CVL), and p24 antigen production after ex vivo infection of ectocervical tissues with HIV-1BaL among PRE women (n = 20) in the follicular and luteal phases of the menstrual cycle and POST women (n = 17) at baseline and after ∼1 month of treatment with 0.01% vaginal E2 cream. Compared to PRE women, we measured higher levels of p24 antigen after ex vivo infection in tissues from POST women. POST women had a significantly thinner vaginal epithelium with decreased tight junction proteins and a higher density of mucosal immune T cells and lower levels of CD1a antigen-presenting cells, antimicrobial peptides, and inflammatory cytokines in the CVL (p values <.05). POST women had higher vaginal pH and lower vaginal Lactobacilli (p values <.05) than PRE women. After vaginal E2 therapy, CV endpoints and ex vivo HIV replication in POST tissues were similar to those observed in PRE tissues. The CV mucosa in POST women is thinned and compromised, with increased HIV-target immune cells and decreased antimicrobial factors, being more susceptible to HIV infection. After POST women receive topical E2 treatment, mucosal endpoints are similar to PRE levels.

Comparison of Follicular and Luteal Phase Mucosal Markers of HIV Susceptibility in Healthy Women.

The purpose of this study was to evaluate differences in vaginal immune cell populations, vaginal tissue gene expression, antimicrobial activity of the cervicovaginal (CV) lavage (CVL), vaginal flora, and p24 antigen production from CV tissues after ex vivo human immunodeficiency virus (HIV) infection between follicular (FOL) and luteal (LUT) phases of the menstrual cycle. CV tissue biopsies, CV secretions, and blood samples were obtained as part of two longitudinal clinical trials of healthy women (CONRAD D11-119 and A12-124 studies). Participants (n = 39) were HIV-seronegative women not using exogenous hormone supplementation, with normal menstrual cycles, who were screened to exclude sexually transmitted and reproductive tract infections. Serum levels of estradiol and progesterone were significantly higher in the LUT versus the FOL phase of the menstrual cycle. Controlling for race, reported contraceptive use/sexual practices, and clinical trial, we found no differences in vaginal tissue immune cell populations and activation status, transcriptomes, inhibition of HIV, herpes simplex virus type 2 and Escherichia coli by the CVL, vaginal pH or Nugent score, or production of p24 antigen after ex vivo infection by HIV-1BaL between CV samples obtained in the FOL phase versus the LUT phase of the menstrual cycle. There were no significant correlations between serum estradiol and progesterone levels and CV endpoints. The hypothesis that the LUT phase of the menstrual cycle represents a more vulnerable stage for mucosal infection with HIV was not supported by data from samples obtained from the lower genital tract (ectocervix and vagina) from these two clinical trials.

Blocking CXCL9 Decreases HIV-1 Replication and Enhances the Activity of Prophylactic Antiretrovirals in Human Cervical Tissues.

OBJECTIVES: The interferon-gamma-induced chemokine CXCL9 is expressed in a wide range of inflammatory conditions including those affecting the female genital tract. CXCL9 promotes immune cell recruitment, activation, and proliferation. The role of CXCL9 in modulating HIV-1 infection of cervicovaginal tissues, a main portal of viral entry, however, has not been established. We report a link between CXCL9 and HIV-1 replication in human cervical tissues and propose CXCL9 as a potential target to enhance the anti-HIV-1 activity of prophylactic antiretrovirals. DESIGN: Using ex vivo infection of human cervical tissues as a model of mucosal HIV-1 acquisition, we described the effect of CXCL9 neutralization on HIV-1 gene expression and mucosal CD4 T-cell activation. The anti-HIV-1 activity of tenofovir, the leading mucosal pre-exposure prophylactic microbicide, alone or in combination with CXCL9 neutralization was also studied. METHODS: HIV-1 replication was evaluated by p24 ELISA. HIV-1 DNA and RNA, and CD4, CCR5, and CD38 transcription were evaluated by quantitative real-time polymerase chain reaction. Frequency of activated cervical CD4 T cells was quantified using fluorescence-activated cell sorting. RESULTS: Antibody blocking of CXCL9 reduced HIV-1 replication by decreasing mucosal CD4 T-cell activation. CXCL9 neutralization in combination with suboptimal concentrations of tenofovir, possibly present in the cervicovaginal tissues of women using the drug inconsistently, demonstrated an earlier and greater decrease in HIV-1 replication compared with tissues treated with tenofovir alone. CONCLUSIONS: CXCL9 neutralization reduces HIV-1 replication and may be an effective target to enhance the efficacy of prophylactic antiretrovirals.

Enhancing Interferon Regulatory Factor 7 Mediated Antiviral Responses and Decreasing Nuclear Factor Kappa B Expression Limit HIV-1 Replication in Cervical Tissues.

Establishment of a productive HIV-1 infection in the female reproductive tract likely depends on the balance between anti-viral and pro-inflammatory responses leading to activation and proliferation of HIV target cells. Immune modulators that boost anti-viral and depress pro-inflammatory immune responses may decrease HIV-1 infection or replication. Polyinosinic:polycytidylic [Poly (I:C)] has been reported to down-regulate HIV-1 replication in immune cell subsets and lymphoid tissues, yet the scope and mechanisms of poly (I:C) regulation of HIV-1 replication in the cervicovaginal mucosa, the main portal of viral entry in women remain unknown. Using a relevant, underexplored ex vivo cervical tissue model, we demonstrated that poly (I:C) enhanced Interferon Regulatory Factor (IRF)7 mediated antiviral responses and decreased tissue Nuclear Factor Kappa B (NFκB) RNA expression. This pattern of cellular transcription factor expression correlated with decreased HIV-1 transcription and viral release. Reducing IRF7 expression up-regulated HIV-1 and NFκB transcription, providing proof of concept for the critical involvement of IRF7 in cervical tissues. By combining poly (I:C) with a suboptimal concentration of tenofovir, the leading anti-HIV prophylactic microbicide candidate, we demonstrated an earlier and greater decrease in HIV replication in poly (I:C)/tenofovir treated tissues compared with tissues treated with tenofovir alone, indicating overall improved efficacy. Poly (I:C) decreases HIV-1 replication by stimulating IRF7 mediated antiviral responses while reducing NFκB expression. Early during the infection, poly (I:C) improved the anti-HIV-1 activity of suboptimal concentrations of tenofovir likely to be present during periods of poor adherence i.e. inconsistent or inadequate drug use. Understanding interactions between anti-viral and pro-inflammatory immune responses in the genital mucosa will provide crucial insights for the identification of targets that can be harnessed to develop preventative combination strategies to improve the efficacy of topical or systemic antiviral prophylactic agents and protect women from HIV-1 and other sexually transmitted infections.

IL-8 decreases HIV-1 transcription in peripheral blood lymphocytes and ectocervical tissue explants.

IL-8 is enhanced in the peripheral blood and lymphoid tissue of HIV-infected individuals, suggesting that IL-8 is important in the pathogenesis of HIV-1 infection and progression to AIDS. Characterizing the mechanisms of IL-8 regulation of HIV-1 replication may be relevant in addressing the role of IL-8 as a therapeutic target in HIV-1 infection. We evaluated replication of primary R5-tropic HIV-1 in peripheral blood lymphocytes and ectocervical tissue explants infected in vitro in the presence of physiological concentrations of IL-8 found in the serum and genital tract secretions of HIV-infected individuals. To identify the specific stages of the viral life cycle targeted by IL-8, we performed real-time polymerase chain reaction to detect HIV-1 reverse transcription, integration, and transcription. Early during the infection, IL-8 decreased HIV-1 reverse transcription and viral integration. This effect was transient, as on day 4 after infection, we detected no differences on HIV-1 DNA or proviral DNA in peripheral blood lymphocyte. IL-8 decreased HIV-1 transcription in both lymphocytes and ectocervical tissue explants. The decrease in viral RNA expression was associated with reduced HIV-1 replication, as measured by viral p24 release in the culture supernatants. This is the first report to suggest that IL-8 decreases replication of primary R5-tropic HIV-1 by transcriptional mechanisms.

HIV type 1 infection in women: increased transcription of HIV type 1 in ectocervical tissue explants.

BACKGROUND: Mucosal surfaces of the female reproductive tract are the main routes of heterosexual transmission of human immunodeficiency virus type 1 (HIV-1), but the contribution of each of the reproductive sites to mucosal transmission is unknown. METHODS: We compared levels of HIV-1 transcription between ectocervical and endometrial tissue explants infected ex vivo with HIV-1. RESULTS: We detected higher levels of HIV-1 transcription in the ectocervix. Although CD45 expression was also increased at this site, higher levels of HIV-1 transcription could not be accounted for exclusively by differences in CD45 expression. This suggests that factors other than CD45 levels regulate HIV-1 transcription within the ectocervix. We detected higher levels of interleukin (IL)-6 at this site. Furthermore, addition of recombinant IL-6 to tissue explants enhanced HIV-1 transcription to a much greater degree in the ectocervix than in the endometrium. CONCLUSIONS: This is, to our knowledge, the first study to compare ectocervix and endometrium in a tissue explant model of HIV-1 infection and to demonstrate greater HIV-1 transcription in the ectocervix. Our results suggest that the ectocervix is more conducive to HIV-1 replication than is the endometrium and that IL-6 enhances HIV-1 transcription at this site. Thus, the ectocervix is an important site to be considered in heterosexual transmission of HIV-1.

Estradiol and progesterone regulate HIV type 1 replication in peripheral blood cells.

Endogenous levels of estradiol and progesterone fluctuate in the peripheral blood of premenopausal women during the reproductive cycle. We studied the effects of these sex hormones on HIV-1 replication in peripheral blood mononuclear cells (PBMCs). We compared HIV-1 replication in PBMCs infected in the presence of mid-secretory (high concentrations) and mid-proliferative (low concentrations) or in the absence of sex hormones. With PBMCs from men, we used concentrations of estradiol and progesterone that are normally present in their plasma. Our findings demonstrate that mid-proliferative phase conditions increased, and mid-secretory phase conditions decreased, HIV-1 replication. To determine if sex hormones affect specific stages of the viral life cycle we performed real-time PCR assays and found decreased levels of HIV-1 integration in the mid-secretory phase and increased levels viral transcription in the mid-proliferative phase. No significant effects on HIV-1 reverse transcription or on CCR5 expression were found. In addition, we assessed hormonal regulation of the HIV-1 LTR in the absence of the viral regulatory protein Tat. We observed that mid-proliferative hormone levels enhanced, whereas mid-secretory hormone concentrations reduced, the activity of the LTR. These findings demonstrate that in HIV-1-infected cells, estradiol and progesterone regulate HIV-1 replication most likely by directly altering HIV-1 transcriptional activation. An additional indirect mechanism of sex hormone regulation of cytokine and chemokine secretion cannot be excluded.

HIV-1 infection of the female reproductive tract.

The human immunodeficiency virus type 1 (HIV-1) infects cells within mucosal tissues, including those of the female reproductive tract (FRT). The mechanism of viral transmission within the FRT and the mode of viral spread to the periphery are not completely understood. We performed phenotypical analyses and infectivity studies of primary FRT cells to identify potential targets of infection within the FRT. Our findings indicate that expression of HIV-1 receptors and coreceptors in the FRT varies as a function of menstrual-cycle stage, suggesting that sex hormone levels may influence a woman's susceptibility to infection by HIV-1. Moreover, HIV-1 strains that utilize the CXCR4 chemokine receptor for infectivity are able to undergo reverse transcription, integration, viral DNA transcription, and viral release, whereas viral strains that utilize CCR5 do not undergo these early replicative events, and are only released unmodified from these cells. This indicates that several mechanisms for viral infection and transmission are present throughout the FRT.

Human immunodeficiency virus reactivation by phorbol esters or T-cell receptor ligation requires both PKCalpha and PKCtheta.

Latently human immunodeficiency virus (HIV)-infected memory CD4(+) T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKCtheta, PKCthetaA148E, and PKCalpha, PKCalphaA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4(+) T cells and that both PKCthetaA148E and PKCalphaA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKCalpha and PKCtheta with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKCalpha or PKCtheta. Since suppression of PKCalpha and PKCtheta together has no greater inhibitory effect on HIV reactivation than inhibition of PKCalpha alone, our data confirm that PKCalpha and PKCtheta act in sequence. The requirement for PKCalpha and PKCtheta for prostratin-induced HIV reactivation and the ability of selective PKCalpha or PKCtheta agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.

Chemokine receptor expression in the human ectocervix: implications for infection by the human immunodeficiency virus-type I.

Human immunodeficiency virus-type 1 (HIV-1) is a sexually transmitted pathogen that can infect cells in the female reproductive tract (FRT). The mechanism of viral transmission within the FRT and the mode of viral spread to the periphery are not well understood. To characterize the frequency of potential targets of HIV infection within the FRT, we performed a systematic study of the expression of HIV receptors (CD4, galactosyl ceramide (GalCer)) and coreceptors (CXCR4 and CCR5) on epithelial cells and leucocytes from the ectocervix. The ectocervix is a likely first site of contact with HIV-1 following heterosexual transmission, and expression of these receptors is likely to correlate with susceptibility to viral infection. We obtained ectocervical tissue specimens from women undergoing hysterectomy, and compared expression of these receptors among patients who were classified as being in the proliferative or secretory phases of their menstrual cycle at the time of hysterectomy, as well as from postmenopausal tissues. Epithelial cells from tissues at early and mid-proliferative stages of the menstrual cycle express CD4, although by late proliferative and secretory phases, CD4 expression was absent or weak. In contrast, GalCer expression was uniform in all stages of the menstrual cycle. CXCR4 expression was not detected on ectocervical epithelial cells and positive staining was only evident on individual leucocytes. In contrast, CCR5 expression was detected on ectocervical epithelial cells from tissues at all stages of the menstrual cycle. Overall, our results suggest that HIV infection of cells in the ectocervix could most likely occur through GalCer and CCR5. These findings are important to define potential targets of HIV-1 infection within the FRT, and for the future design of approaches to reduce the susceptibility of women to infection by HIV-1.

Synergy between IL-8 and GM-CSF in reproductive tract epithelial cell secretions promotes enhanced neutrophil chemotaxis.

Neutrophils occur in tissues of the female reproductive tract (FRT) under non-infected conditions. These cells generally enter tissues under the influence of chemoattractants called chemokines. Primary epithelial cells (EC) from FRT were a potent source of chemokines, IL-8 being the chief neutrophil chemoattractant secreted. Blocking with neutralizing anti-IL-8 showed that IL-8 did not account for all of the chemoattraction observed. A mixture of 25 ng/mL rIL-8 and 1 ng/mL rGM-CSF mediated 2.7-fold more chemotaxis than that expected if the two agents were additive. We then found that GM-CSF was produced by EC in amounts that synergised strongly with IL-8 to enhance chemotaxis. Treatment of uterine EC conditioned medium with saturating doses of anti-IL-8 plus anti-GM-CSF antibodies produced an 84% inhibition of chemotaxis. These findings demonstrate that the majority of neutrophil chemoattractant activity produced by FRT EC results from the synergistic effects of IL-8 and GM-CSF.

Transmission of HIV-1 by primary human uterine epithelial cells and stromal fibroblasts.

Women can become infected with human immunodeficiency virus type 1 (HIV-1) after the heterosexual transmission of virus from an infected male partner. To understand the events that result in transmission of HIV-1 across the female reproductive tract, we characterized the life-cycle events of HIV-1 in primary cultures of human uterine epithelial cells and stromal fibroblasts. Epithelial cells and stromal fibroblasts released virus particles after exposure to either X4- or R5-tropic strains of HIV-1. Virus released by these cells was able to infect CD4(+) T cells. When exposed to an X4-tropic strain of HIV-1, these cells supported HIV-1 reverse transcription, integration, and viral DNA transcription. When exposed to an R5-tropic strain, however, these cells released unmodified virus. These data suggest that uterine cells are targets for productive infection with X4-tropic strains and release unmodified R5-tropic viruses that would then be able to infect submucosal target cells, including T cells and macrophages.

Protein kinase Calpha (PKCalpha) acts upstream of PKCtheta to activate IkappaB kinase and NF-kappaB in T lymphocytes.

NF-kappaB is an ubiquitous transcription factor that is a key in the regulation of the immune response and inflammation. T-cell receptor (TCR) cross-linking leads to NF-kappaB activation, an IkappaB kinase (IKK)-dependent process. However, the upstream kinases that regulate IKK activity following TCR activation remain to be fully characterized. Herein, we demonstrate using genetic analysis, pharmacological inhibition, and RNA interference (RNAi) that the conventional protein kinase C (PKC) isoform PKCalpha, but not PKCbeta1, is required for the activation of the IKK complex following T-cell activation triggered by CD3/CD28 cross-linking. We find that in the presence of Ca(2+) influx, the catalytically active PKCalphaA25E induces IKK activity and NF-kappaB-dependent transcription; which is abrogated following the mutations of two aspartates at positions 246 and 248, which are required for Ca(2+) binding to PKCalpha and cell membrane recruitment. Kinetic studies reveal that an early phase (1 to 5 min) of IKK activation following TCR/CD28 cross-linking is PKCalpha dependent and that a later phase (5 to 25 min) of IKK activation is PKCtheta dependent. Activation of IKK- and NF-kappaB-dependent transcription by PKCalphaA25E is abrogated by the PKCtheta inhibitor rottlerin or the expression of the kinase-inactive form of PKCtheta. Taken together, our results suggest that PKCalpha acts upstream of PKCtheta to activate the IKK complex and NF-kappaB in T lymphocytes following TCR activation.

Human immunodeficiency virus type 1 infection of human uterine epithelial cells: viral shedding and cell contact-mediated infectivity.

We examined the mechanism of human immunodeficiency virus (HIV) type 1 infection of human uterine epithelial cells to gain a clearer understanding of the events by which HIV-1 infects cells within the female reproductive tract. We demonstrated that these cells can be productively infected by HIV-1 and that infection is associated with viral RNA reverse transcription, DNA transcription, and secretion of infectious virus. Levels of viral DNA and secreted virus decreased gradually after infection. Moreover, virus released by the uterine epithelial cells shortly after infection was able to infect human T cell lines, but virus released later did not. In contrast, human CD4(+) T cell lines were infected after cocultivation with epithelial cells at both early and late stages of infection. These data demonstrated that HIV-1 infects human epithelial cells of upper reproductive tract origin and that productive viral infection of epithelial cells may be an important mechanism of transmission of HIV-1 infection in women.