NF-B 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-B activation, an IB kinase (IKK)-dependentprocess. 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 PKC␣, but not PKC␤1, 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 PKC␣A25E induces IKK activity and NF-B-dependent transcription; which is abrogated following the mutations of two aspartates at positions 246 and 248, which are required for Ca 2؉ binding to PKC␣ and cell membrane recruitment. Kinetic studies reveal that an early phase (1 to 5 min) of IKK activation following TCR/CD28 cross-linking is PKC␣ dependent and that a later phase (5 to 25 min) of IKK activation is PKC dependent. Activation of IKK-and NF-B-dependent transcription by PKC␣A25E is abrogated by the PKC inhibitor rottlerin or the expression of the kinase-inactive form of PKC. Taken together, our results suggest that PKC␣ acts upstream of PKC to activate the IKK complex and NF-B in T lymphocytes following TCR activation.Identification of the molecular events regulating T-cell activation is paramount to understanding the regulation of the immune response. The signal transduction pathways triggered by antigen presentation lead to the immediate activation of multiple transcription factors that further amplify the process of lymphocyte activation, ultimately leading to cellular proliferation and division. T-cell receptor (TCR) engagement, together with the second costimulatory signal derived from engagement of the CD28 receptor, results in NF-B activation (13,31,36). When occurring together with NF-AT, AP-1, and octomer, NF-B activation leads to interleukin-2 (IL-2) expression (17,27,30).NF-B is a heterodimer of transcription factors that belong to the Rel family of proteins. The canonical NF-B is a heterodimer of p65 (RelA) with p50 or p52 (35,50,54). This heterodimer is anchored by a group of proteins named IB, which function to retain NF-B in the cytosol by masking its nuclear localization signal (1,4,45,60). IB␣ is the prototype IB molecule known to control the subcellular localization of NF-B (p50/p65). Following activation of certain signal transduction pathways, a site-specific hyperphosphorylation of IB␣ at S32 and S36 renders the inhibitor molecule susceptible to site-specific ubiquitination and subsequent degradation by the proteasome complex (8,9,18,62,68). This releases NF-B, allowing it to undergo nuclear translocation. Two IB␣ kinases, IKK␣ (19,52,70) and IKK␤ (46), which are contained within a high-molecular-weight complex, target the phosphorylation of S32 and S36 of IB␣ following stimulation by various stimuli. While...

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

Summary Human immunodeficiency virus‐1 (HIV‐1) is primarily a sexually transmitted disease. Identification of cell populations within the female reproductive tract that are initially infected, and the events involved in transmission of infection to other cells, remain to be established. In this report, we evaluated expression of HIV receptors and coreceptors on epithelial cells in the uterus and found they express several receptors critical for HIV infection including CD4, CXCR4, CCR5 and galactosylceramide (GalC). Moreover, expression of these receptors varied during the menstrual cycle. Expression of CD4 and CCR5 on uterine epithelial cells is high throughout the proliferative phase of the menstrual cycle when blood levels of oestradiol are high. In contrast, CXCR4 expression increased gradually throughout the proliferative phase. During the secretory phase of the cycle when both oestradiol and progesterone are elevated, CD4 and CCR5 expression decreased whereas CXCR4 expression remained elevated. Expression of GalC on endometrial glands is higher during the secretory phase than during the proliferative phase of the menstrual cycle. Because epithelial cells line the female reproductive tract and express HIV receptors and coreceptors, it is likely that they are one of the first cell types to become infected. The hormonal regulation of HIV receptor expression may affect a woman's susceptibility to HIV infection during her menstrual cycle. Moreover, selective coreceptor expression could account for the preferential transmission of R5‐HIV‐1 strains to women. In addition, these studies provide evidence that the uterus, and potentially the entire upper reproductive tract, are important sites for the initial events involved in HIV infection.

Summary 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.

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.