PlmCas12e (CasX2) cleavage of CCR5: impact of guide RNA spacer length and PAM sequence on cleavage activity.

Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from Streptococcus pyogenes and Staphylococcus aureus, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5-7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in CCR5 (CCR5-[Formula: see text]32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR/Cas. We determined that CCR5 cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the CCR5-[Formula: see text]32 mutation in haematopoietic stem cells.

CAS12e (CASX2) CLEAVAGE OF CCR5: IMPACT OF GUIDE RNA LENGTH AND PAM SEQUENCE ON CLEAVAGE ACTIVITY.

CRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs. We investigated varying guide RNA (spacer) lengths and alternative PAM sequences to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation of CCR5 (delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined that CCR5 cleavage activity varied with the target site, guide RNA length, and the terminal nucleotide in the PAM sequence. Our analyses demonstrated a PlmCas12e PAM preference for purines (A, G) over pyrimidines (T, C) in the fourth position of the CasX2 PAM (TTCN). These analyses have contributed to a better understanding of CasX2 cleavage requirements and will position us more favorably to develop a therapeutic that creates the delta-32 mutation in the CCR5 gene in hematopoietic stem cells.

Human uterine natural killer cells but not blood natural killer cells inhibit human immunodeficiency virus type 1 infection by secretion of CXCL12.

Natural killer (NK) cells derived from the human female reproductive tract (FRT) are phenotypically and functionally distinct from those obtained from peripheral blood. Because the FRT is a primary site of human immunodeficiency virus type 1 (HIV-1) infection in women, we determined whether soluble factors secreted by uterine-derived NK (uNK) cells inhibit HIV-1 infection. Clonal populations of uNK cells were activated with interleukin-12 (IL-12) and IL-15, and conditioned media (CM) from these cultures evaluated for their ability to inhibit infection of cells by HIV-1(IIIB), HIV-1(NL4.3), and HIV-1(HC4) (X4-tropic) or HIV-1(BaL) (R5-tropic) viruses. We found that soluble factors secreted by activated uNK cells significantly inhibited X4-tropic virus infection of TZM-bl cells, peripheral blood mononuclear cells, and primary human endometrial cells, but not infection by HIV-1(BaL). In contrast, CM from peripheral blood NK (bNK) cells did not inhibit HIV-1 infection of cells. Analysis of factors secreted from uNK clones with anti-HIV-1 activity demonstrated significantly higher levels of CXCL12 compared to uNK clones without this activity, and the HIV inhibitory activity was neutralized by antibodies to CXCL12. Collectively, these data demonstrate that human uNK cells release chemokines with anti-HIV-1 activity for X4-tropic strains and this suggest that these chemokines may contribute to the inhibition of X4-tropic strain transmission across mucosal tissues.

A mathematical model of HIV dynamics treated with a population of gene-edited haematopoietic progenitor cells exhibiting threshold phenomenon.

The use of gene-editing technology has the potential to excise the CCR5 gene from haematopoietic progenitor cells, rendering their differentiated CD4-positive (CD4+) T cell descendants HIV resistant. In this manuscript, we describe the development of a mathematical model to mimic the therapeutic potential of gene editing of haematopoietic progenitor cells to produce a class of HIV-resistant CD4+ T cells. We define the requirements for the permanent suppression of viral infection using gene editing as a novel therapeutic approach. We develop non-linear ordinary differential equation models to replicate HIV production in an infected host, incorporating the most appropriate aspects found in the many existing clinical models of HIV infection, and extend this model to include compartments representing HIV-resistant immune cells. Through an analysis of model equilibria and stability and computation of $R_0$ for both treated and untreated infections, we show that the proposed therapy has the potential to suppress HIV infection indefinitely and return CD4+ T cell counts to normal levels. A computational study for this treatment shows the potential for a successful 'functional cure' of HIV. A sensitivity analysis illustrates the consistency of numerical results with theoretical results and highlights the parameters requiring better biological justification. Simulations of varying level production of HIV-resistant CD4+ T cells and varying immune enhancements as the result of these indicate a clear threshold response of the model and a range of treatment parameters resulting in a return to normal CD4+ T cell counts.

Analysis of CRISPR/Cas9 Guide RNA Efficiency and Specificity Against Genetically Diverse HIV-1 Isolates.

Gene editing approaches using CRISPR/Cas9 are being developed as a means for targeting the integrated HIV-1 provirus. Enthusiasm for the use of gene editing as an anti-HIV-1 therapeutic has been tempered by concerns about the specificity and efficacy of this approach. Guide RNAs (gRNAs) that target conserved sequences across a wide range of genetically diverse HIV-1 isolates will have greater clinical utility. However, on-target efficacy should be considered in the context of off-target cleavage events as these may comprise an essential safety parameter for CRISPR-based therapeutics. We analyzed a panel of Streptococcus pyogenes Cas9 (SpCas9) gRNAs directed to the 5' and 3' long terminal repeat (LTR) regions of HIV-1. We used in vitro cleavage assays with genetically diverse HIV-1 LTR sequences to determine gRNA activity across HIV-1 clades. Lipid-based transfection of gRNA/Cas9 ribonucleoproteins was used to assess targeting of the integrated HIV-1 proviral sequence in cells (in vivo). For both the in vitro and in vivo experiments, we observed increased efficiency of sequence disruption through the simultaneous use of two distinct gRNAs. Next, CIRCLE-Seq was utilized to identify off-target cleavage events using genomic DNA from cells with integrated HIV-1 proviral DNA. We identified a gRNA targeting the U3 region of the LTR (termed SpCas9-127HBX2) with broad cleavage efficiency against sequences from genetically diverse HIV-1 strains. Based on these results, we propose a workflow for identification and development of anti-HIV CRISPR therapeutics.

Toward a VA Women's Health Research Agenda: setting evidence-based priorities to improve the health and health care of women veterans.

The expansion of women in the military is reshaping the veteran population, with women now constituting the fastest growing segment of eligible VA health care users. In recognition of the changing demographics and special health care needs of women, the VA Office of Research & Development recently sponsored the first national VA Women's Health Research Agenda-setting conference to map research priorities to the needs of women veterans and position VA as a national leader in Women's Health Research. This paper summarizes the process and outcomes of this effort, outlining VA's research priorities for biomedical, clinical, rehabilitation, and health services research.

Toll-like receptor agonists are potent inhibitors of human immunodeficiency virus-type 1 replication in peripheral blood mononuclear cells.

Innate immune responses to microbial pathogens are initiated following the binding of ligand to specific pattern recognition receptors. Each pattern recognition receptor, which includes members of the Toll-like receptor (TLR) family, is specific for a particular type of pathogen associated molecular pattern ensuring that the organism can respond rapidly to a wide range of pathogens including bacteria, viruses, and fungi. We studied the extent to which agonists to endosomal TLR could induce anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs). When agonists to TLR3, TLR7, TLR8 and TLR9 were added prior to infection with HIV-1, they significantly reduced infection of peripheral blood mononuclear cells. Interestingly, agonists to TLR8 and TLR9 were highly effective at blocking HIV replication even when added as late as 48 h or 72 h, respectively, after HIV-1 infection, indicating that the anti-viral effect was durable and long lasting. Analysis of the induction of anti-viral genes after agonist activation of TLR indicated that all of the agonists induced expression of the type I interferons and interferon stimulated genes, although to variable levels that depended on the agonist used. Interestingly, only the agonist to TLR9, ODN2395 DNA, induced expression of type II interferon and the anti-HIV proteins Apobec3G and SAMHD1. By blocking TLR activity using an inhibitor to the MyD88 adaptor protein, we demonstrated that, at least for TLR8 and TLR9, the anti-HIV activity was not entirely mediated by TLR activation, but likely by the activation of additional anti-viral sensors in HIV target cells. These findings suggest that agonists to the endosomal TLR function to induce expression of anti-HIV molecules by both TLR-mediated and non-TLR-mediated mechanisms. Moreover, the non-TLR-mediated mechanisms induced by these agonists could potentially be exploited to block HIV-1 replication in recently HIV-exposed individuals.

Gardiquimod: a Toll-like receptor-7 agonist that inhibits HIV type 1 infection of human macrophages and activated T cells.

Immune response modifiers are being studied as therapeutic agents for viral infections and cancer. These molecules include agonists for the Toll-like receptors (TLR), a family of innate immune receptors. TLR7 and 8, located in cellular endosomes, bind single-stranded RNA characteristic of viral genomes, and trigger intracellular signaling pathways that induce inflammatory cytokines and antiviral innate immune factors. We studied the anti-HIV-1 effects of gardiquimod, a specific TLR7 agonist when used at concentrations below 10 µM, in macrophages and activated peripheral blood mononuclear cells (PBMCs). Gardiquimod, added prior to or within 2 days after infection with X4, R5, or dual-tropic (R5/X4) strains of HIV-1, significantly reduced infection in these cells. Cocultures of activated PBMCs added to gardiquimod-treated and HIV-1-exposed macrophages demonstrated minimal HIV-1 replication for up to 10 days, suggesting that gardiquimod inhibited activated PBMCs viral amplification from HIV-1-exposed macrophages. Gardiquimod treatment of both activated PBMCs and macrophages induced interferon-alpha (IFN-α) transcription within hours of addition, and sustained IFN-α protein secretion for several days. Treatment of cells with a peptide inhibitor to the MyD88 adaptor protein blocked the induction of IFN-α by gardiquimod, and partially reversed the anti-HIV effects in activated PBMCs. Blocking the IFN-α receptor with a neutralizing antibody also reduced the anti-HIV effect of gardiquimod. Gardiquimod inhibited HIV-1 reverse transcriptase, an early step in the life cycle of HIV-1. These findings suggest that gardiquimod, functioning as both an immune system modifier and a reverse transcriptase inhibitor, could be developed as a novel therapeutic agent to block systemic and mucosal transmission of HIV-1.

Breast milk from Tanzanian women has divergent effects on cell-free and cell-associated HIV-1 infection in vitro.

Transmission of HIV-1 during breastfeeding is a significant source of new pediatric infections in sub-Saharan Africa. Breast milk from HIV-positive mothers contains both cell-free and cell-associated virus; however, the impact of breast milk on HIV-1 infectivity remains poorly understood. In the present study, breast milk was collected from HIV-positive and HIV-negative Tanzanian women attending antenatal clinics in Dar es Salaam. Milk was analyzed for activity in vitro against both cell-free and cell-associated HIV-1. Potent inhibition of cell-free R5 and X4 HIV-1 occurred in the presence of milk from all donors regardless of HIV-1 serostatus. Inhibition of cell-free HIV-1 infection positively correlated with milk levels of sialyl-Lewis(X) from HIV-positive donors. In contrast, milk from 8 of 16 subjects enhanced infection with cell-associated HIV-1 regardless of donor serostatus. Milk from two of these subjects contained high levels of multiple pro-inflammatory cytokines including TNFα, IL-1ß, IL-6, IL-8, MIP-1α, MIP-1ß, MCP-1 and IP-10, and enhanced cell-associated HIV-1 infection at dilutions as high as 1∶500. These findings indicate that breast milk contains innate factors with divergent activity against cell-free and cell-associated HIV-1 in vitro. Enhancement of cell-associated HIV-1 infection by breast milk may be associated with inflammatory conditions in the mother and may contribute to infant infection during breastfeeding.

Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants.

Human Immunodeficiency Virus-type 1 (HIV-1) binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA) transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV-1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA) sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α), a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

Innate factors in human breast milk inhibit cell-free HIV-1 but not cell-associated HIV-1 infection of CD4+ cells.

BACKGROUND: Transmission of HIV from mother to child through breast-feeding remains a global health challenge, particularly in developing countries. Breast milk from an HIV-infected women may contain both cell-free HIV-1 and cell-associated virus; however, the impact of human breast milk on HIV infection and replication in CD4 cells remain poorly understood. OBJECTIVES: In the present study, we evaluated the effects of breast milk in vitro on infection of CD4 cells with cell-free HIV-1, including effects on HIV-1 receptor expression, reverse transcription, integration, and viral transcription. Additionally, we evaluated the ability of breast milk to inhibit cell-associated transmission of HIV-1 from infected CD4 T lymphocytes. RESULTS: Our results demonstrate that breast milk potently inhibits infection with cell-free HIV-1 in vitro independently of viral tropism and significantly decreases HIV-1 reverse transcription and integration in CD4 cells. However, the inhibitory effect of breast milk on HIV-1 infection of CD4 cells was lost during extended culture, and direct coculture of HIV-infected CD4 T lymphocytes with susceptible target cells revealed that breast milk was ineffective at blocking cell-associated HIV-1 infection. CONCLUSIONS: Our findings suggest that breast milk may provide a protective function against cell-free HIV-1 but may be less effective at blocking infection by cell-associated virus.

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 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.

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.

Intravenous fentanyl increases natural killer cell cytotoxicity and circulating CD16(+) lymphocytes in humans.

UNLABELLED: Opioids, including fentanyl, are often administered to patients who may be at risk for the consequences of impaired immune function. We performed a clinical study to test the effects of the synthetic opioid fentanyl on human immune function. Participants received an IV fentanyl initial dose of 3 microg/kg followed by a 2-h IV infusion of 1.2 microg x kg(-1) x h(-1). Peripheral blood was drawn before and after fentanyl administration to test for neutrophil phagocytic function, neutrophil antibody-dependent cell cytotoxicity, natural killer cell cytotoxicity, percentage of lymphocyte populations, T-lymphocyte proliferative response, and in vivo antibody response to a pneumococcal vaccine inoculation given at the end of the fentanyl infusion. Fentanyl exposure under the conditions of this study caused a rapid and significant increase in natural killer cell cytotoxicity, which was coincident with an increase in the percentage of CD16(+) and CD8(+) cells in peripheral blood. Fentanyl did not significantly affect any of the other immune measurements. IMPLICATIONS: Many previous studies have suggested that opioid drugs can impair immune resistance in patients who may be at risk for infection. This study suggests that the opioid fentanyl, when given to healthy humans without coexisting diseases, does not suppress immune resistance. On the basis of these results, the use of fentanyl should not be restricted because of concerns that it may suppress immune function.

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.

Human immunodeficiency virus receptor and coreceptor expression on human uterine epithelial cells: regulation of expression during the menstrual cycle and implications for human immunodeficiency virus infection.

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.