Production and characterization of a human antisperm monoclonal antibody against CD52g for topical contraception in women.

BACKGROUND: Approximately 40% of human pregnancies are unintended, indicating a need for more acceptable effective contraception methods. New antibody production systems make it possible to manufacture reagent-grade human monoclonal antibodies (mAbs) for clinical use. We used the Nicotiana platform to produce a human antisperm mAb and tested its efficacy for on-demand topical contraception. METHODS: Heavy and light chain variable region DNA sequences of a human IgM antisperm antibody derived from an infertile woman were inserted with human IgG1 constant region sequences into an agrobacterium and transfected into Nicotiana benthamiana. The product, an IgG1 mAb ["Human Contraception Antibody" (HCA)], was purified on Protein A columns, and QC was performed using the LabChip GXII Touch protein characterization system and SEC-HPLC. HCA was tested for antigen specificity by immunofluorescence and western blot assays, antisperm activity by sperm agglutination and complement dependent sperm immobilization assays, and safety in a human vaginal tissue (EpiVaginal™) model. FINDINGS: HCA was obtained at concentrations ranging from 0.4 to 4 mg/ml and consisted of > 90% IgG monomers. The mAb specifically reacted with a glycan epitope on CD52g, a glycoprotein produced in the male reproductive tract and found in abundance on sperm. HCA potently agglutinated sperm under a variety of relevant physiological conditions at concentrations ≥ 6.25 µg/ml, and mediated complement-dependent sperm immobilization at concentrations ≥ 1 µg/ml. HCA and its immune complexes did not induce inflammation in EpiVaginal™ tissue. INTERPRETATION: HCA, an IgG1 mAb with potent sperm agglutination and immobilization activity and a good safety profile, is a promising candidate for female contraception. FUNDING: This research was supported by grants R01 HD095630 and P50HD096957 from the National Institutes of Health.

Chlamydia trachomatis Infection, when Treated during Pregnancy, Is Not Associated with Preterm Birth in an Urban Safety-Net Hospital.

Preterm birth is a major public health problem, occurring in more than half a million births per year in the United States. A number of maternal conditions have been recognized as risk factors for preterm birth, but for the majority of cases, the etiology is not completely understood. Chlamydia trachomatis is one of the most prevalent sexually transmitted infections in the world. However, its role in adverse pregnancy outcome in women is still debated. In order to determine if genitourinary tract infection with C. trachomatis during pregnancy was associated with preterm birth, we conducted a case-control study on women who delivered at Boston Medical Center, an urban "safety-net" hospital that serves a socioeconomically disadvantaged and racially diverse population. Women with known risk factors for preterm birth or immune suppression were excluded. Variables collected on enrolled subjects included demographics; diagnosis of C. trachomatis during or prior to pregnancy; tobacco, alcohol, and illicit substance use; gestational age; and birthweight and gender of the newborn. We also collected urine for chlamydia testing at the time of delivery and placental biopsies for nucleic acid amplification and histological studies. A total of 305 subjects were enrolled: 100 who delivered preterm and 205 who delivered full term. Among those subjects, we identified 19 cases of pregnancy-associated C. trachomatis infection: 6/100 preterm and 13/205 full term, a difference which was not statistically significant. Only two cases of untreated chlamydia infection were identified postpartum, and both occurred in women who delivered at term. We conclude that genitourinary tract infection with C. trachomatis during pregnancy, when appropriately treated, is not associated with preterm birth.

Condylomata Acuminata (Anogenital Warts) Contain Accumulations of HIV-1 Target Cells That May Provide Portals for HIV Transmission.

Background: Condylomata acuminata (anogenital warts [AGWs]) are prevalent in human immunodeficiency virus (HIV)-infected individuals and sexually active populations at risk for HIV acquisition and have been associated with HIV transmission. We compared AGW specimens to control tissue specimens for abundance, types, and location of HIV target cells and for susceptibility to HIV infection in vitro, to provide biologic evidence that AGWs facilitate HIV transmission. Methods: We used immunohistologic staining to identify HIV target cells in AGW and control specimens. We also inoculated HIV in vitro into AGW and control specimens from HIV-negative men and assessed infection by means of TZM-bl and p24 assays. Results: CD1a+ dendritic cells, CD4+ T cells, and macrophages were significantly more abundant in the epidermis of AGW specimens than control specimens. These HIV target cells also often appeared in large focal accumulations in the dermis of AGW specimens. Two of 8 AGW specimens versus 0 of 8 control specimens showed robust infection with HIV in vitro. Conclusions: Compared with normal skin, AGWs contain significantly higher concentrations of HIV target cells that may be susceptible to HIV infection. Condylomata may thus promote HIV transmission, especially in the setting of typical lesion vascularity and friability. Prevention or treatment of AGWs may decrease the sexual transmission of HIV.

Differential expression of toll-like receptors in the human placenta across early gestation.

Toll-like receptors (TLRs) are an essential component of the innate immune system. While a number of studies have described TLR expression in the female reproductive tract, few have examined the temporal expression of TLRs within the human placenta. We hypothesized that the pattern of TLR expression in the placenta changes throughout the first and second trimester, coincident with physiological changes in placental function and the demands of innate immunity. We collected first and second trimester placental tissue and conducted quantitative PCR analysis for TLRs 1-10, followed by immunohistochemistry to define the cell specific expression pattern of a subset of these receptors. Except for the very earliest time points, RNA expression for TLRs 1-10 was stable out to 20 weeks gestation. However, the pattern of protein expression evolved over time. Early first trimester placenta demonstrated a strong, uniform pattern predominantly in the inner villous cytotrophoblast layer. As the placenta matured through the second trimester, both the villous cytotrophoblasts and the pattern of TLR expression within them became disorganized and patchy, with putative Hofbauer cells now identifiable in the tissue also staining positive. We conclude from this data that placental TLR expression changes over the course of gestation, with a tight barrier of TLRs forming a wall of defense along the cytotrophoblast layer in the early first trimester that breaks down as pregnancy progresses. These data are relevant to understanding placental immunity against pathogen exposure throughout pregnancy and may aid in our understanding of the vulnerable period for fetal exposure to pathogens.

RNF121 Inhibits Angiogenic Growth Factor Signaling by Restricting Cell Surface Expression of VEGFR-2.

Ligand stimulation promotes downregulation of RTKs, a mechanism by which RTKs, through the ubiquitination pathway are removed from the cell surface, causing a temporary termination of RTK signaling. The molecular mechanisms governing RTK trafficking and maturation in the endoplasmic reticulum (ER)/Golgi compartments are poorly understood. Vascular endothelial growth factor receptor-2 (VEGFR-2) is a prototypic RTK that plays a critical role in physiologic and pathologic angiogenesis. Here we demonstrate that Ring Finger Protein 121 (RNF121), an ER ubiquitin E3 ligase, is expressed in endothelial cells and regulates maturation of VEGFR-2. RNF121 recognizes newly synthesized VEGFR-2 in the ER and controls its trafficking and maturation. Over-expression of RNF121 promoted ubiquitination of VEGFR-2, inhibited its maturation and resulted a significantly reduced VEGFR-2 presence at the cell surface. Conversely, the shRNA-mediated knockdown of RNF121 in primary endothelial cells reduced VEGFR-2 ubiquitination and increased its cell surface level. The RING Finger domain of RNF121 is required for its activity toward VEGFR-2, as its deletion significantly reduced the effect of RNF121 on VEGFR-2. Additionally, RNF121 inhibited VEGF-induced endothelial cell proliferation and angiogenesis. Taken together, these data identify RNF121 as a key determinant of angiogenic signaling that restricts VEGFR-2 cell surface presence and its angiogenic signaling.

Characterization of a Hormone-Responsive Organotypic Human Vaginal Tissue Model: Morphologic and Immunologic Effects.

Estrogen and progesterone regulate proliferation and differentiation of epithelial cells in the female genital tract. We investigated the effects of these hormones on reconstructed human organotypic vaginal epithelial tissue models (EpiVaginal). We ascertained that epithelial cells in the tissue models express estrogen and progesterone receptors. Treatment with estradiol-17ß (E(2)) significantly increased epithelium thickness and transepithelial electrical resistance (TEER), whereas progesterone (P) treatment resulted in thinning of the epithelium and decreased TEER when compared with untreated controls. Exposure to E(2) increased (1) the expression of the progesterone receptor B (PR-B), (2) accumulation of glycogen in suprabasal cells, (3) epithelial differentiation, and (4) the expression of a number of gene pathways associated with innate immunity, epithelial differentiation, wound healing, and antiviral responses. These findings indicate that EpiVaginal tissues are hormone responsive and can be used to study the role of female reproductive hormones in innate immune responses, microbial infection, and drug delivery in the vaginal mucosa.

The structure of the human vaginal stratum corneum and its role in immune defense.

The superficial layers of the human vaginal epithelium, which form an interface between host and environment, are comprised of dead flattened cells that have undergone a terminal cell differentiation program called cornification. This entails extrusion of nuclei and intercellular organelles, and the depletion of functional DNA and RNA precluding the synthesis of new proteins. As a consequence, the terminally differentiated cells do not maintain robust intercellular junctions and have a diminished capacity to actively respond to microbial exposure, yet the vaginal stratum corneum (SC) mounts an effective defense against invasive microbial infections. The vaginal SC in reproductive-aged women is comprised of loosely connected glycogen-filled cells, which are permeable to bacterial and viral microbes as well as molecular and cellular mediators of immune defense. We propose here that the vaginal SC provides a unique microenvironment that maintains vaginal health by fostering endogenous lactobacilli and retaining critical mediators of acquired and innate immunity. A better understanding of the molecular and physicochemical properties of the vaginal SC could promote the design of more effective topical drugs and microbicides.

The Neonatal Fc receptor (FcRn) enhances human immunodeficiency virus type 1 (HIV-1) transcytosis across epithelial cells.

The mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses mucosal surfaces to establish infection are unknown. Acidic genital secretions of HIV-1-infected women contain HIV-1 likely coated by antibody. We found that the combination of acidic pH and Env-specific IgG, including that from cervicovaginal and seminal fluids of HIV-1-infected individuals, augmented transcytosis across epithelial cells as much as 20-fold compared with Env-specific IgG at neutral pH or non-specific IgG at either pH. Enhanced transcytosis was observed with clinical HIV-1 isolates, including transmitted/founder strains, and was eliminated in Fc neonatal receptor (FcRn)-knockdown epithelial cells. Non-neutralizing antibodies allowed similar or less transcytosis than neutralizing antibodies. However, the ratio of total:infectious virus was higher for neutralizing antibodies, indicating that they allowed transcytosis while blocking infectivity of transcytosed virus. Immunocytochemistry revealed abundant FcRn expression in columnar epithelia lining the human endocervix and penile urethra. Acidity and Env-specific IgG enhance transcytosis of virus across epithelial cells via FcRn and could facilitate translocation of virus to susceptible target cells following sexual exposure.

A quantitative glycogen assay to verify use of self-administered vaginal swabs.

BACKGROUND: Self-administered swabs are used to sample vaginal contents for a variety of clinical purposes including detection of sexually transmitted infections, condom breakage, and vaginal product use. The goal of this study was to determine whether a quantitative glycogen assay can be used to assess whether a swab has been exposed to the vagina to assure study compliance. STUDY DESIGN: Buccal, skin, or vaginal samples were tested to determine whether a commercial quantitative glycogen assay can differentiate vaginal specimens. In addition, archived remnant de-identified vaginal swabs from clinical trials were tested. Periodic acid-Schiff stain was used to identify glycogen-positive cells as a confirmation test. RESULTS: Glycogen concentrations in eluates of vaginal swabs from reproductive-aged women were significantly higher than those from unused swabs (mean ± SE, 964 ± 135 µg/mL vs. 14.7 ± 2.5 µg/mL, P < 0.001) and swabs exposed to buccal and finger/hand epithelia (40.3 ± 4.8 and 18.5 ± 5.4 µg/mL, P < 0.001). Glycogen concentrations were lower and more variable in vaginal swabs from older perimenopausal/menopausal women (mean ± SE, 235 ± 123, P < 0.01). Semen and sample storage longer than 1 year did not affect glycogen detection. Using a cutoff of 100 µg/mL of glycogen, 30 of 30 vaginal swabs from reproductive-aged women versus 0 of 28 control swabs were positive, for an assay sensitivity of 1 (95% confidence interval, 0.86-1) and specificity of 1 (95% confidence interval, 0.85-1). Periodic acid-Schiff stain correlated with soluble glycogen results but was less specific. CONCLUSIONS: The quantitative glycogen assay provides a simple and inexpensive method to validate the use of self-administered swabs for sampling vaginal contents in clinical studies.

Structure and function of intercellular junctions in human cervical and vaginal mucosal epithelia.

The mucosal epithelium is a major portal for microbial invasion. Mucosal barrier integrity is maintained by the physical interactions of intercellular junctional molecules on opposing epithelial cells. The epithelial mucosa in the female reproductive tract provides the first line of defense against sexually transmitted pathogenic bacteria and viruses, but little is known concerning the structure and molecular composition of epithelial junctions at this site. In the present study, the distribution of tight, adherens, and desmosomal junctions were imaged in the human endocervix (columnar epithelium) and ectocervix (stratified squamous epithelium) by electron microscopy, and permeability was assessed by tracking the penetration of fluorescent immunoglobulin G (IgG). To further define the molecular structure of the intercellular junctions, select junctional molecules were localized in the endocervical, ectocervical, and vaginal epithelium by fluorescent immunohistology. The columnar epithelial cells of the endocervix were joined by tight junctions that excluded apically applied fluorescent IgG. In contrast, the most apical layers of the ectocervical stratified squamous epithelium did not contain classical cell-cell adhesions and were permeable to IgG. The suprabasal and basal epithelial layers in ectocervical and vaginal tissue contained the most robust adhesions; molecules characteristic of exclusionary junctions were detected three to four cellular layers below the luminal surface and extended to the basement membrane. These data indicate that the uppermost epithelial layers of the ectocervix and vagina constitute a unique microenvironment; their lack of tight junctions and permeability to large-molecular-weight immunological mediators suggest that this region is an important battlefront in host defense against microbial pathogens.

Innate and acquired immunity in the human penile urethra.

In men, the penile urethra is a primary infection site for sexually transmitted pathogens. Research on the immunology of this mucosal site has been limited in part due to sampling challenges, but available evidence indicates that the urethra contains a rich contingent of immunological mediators that can mount vigorous innate and adaptive immune responses against infectious organisms. Further research is needed to define approaches to stimulate immunity at this mucosal site to prevent the transmission of HIV-1 and other sexually transmitted pathogens.

HIV infection and immune defense of the penis.

Recent evidence that circumcision decreases HIV infection in heterosexual men by 50-60% has focused research on the foreskin as a target of HIV infection. In this review article, we discuss potential mechanisms underlying the circumcision effect and re-examine the assumption that the foreskin is the principle penile HIV infection site. HIV target cells are present in the foreskin epithelium, but are also found in the epithelia of the penile shaft, glans/corona, meatus and urethral introitus. Sexually transmitted infections (STIs) can affect any of these sites and increase susceptibility to HIV acquisition by eroding the protective epithelial layer and by attracting and activating HIV target cells in the epithelium. The moist subpreputial cavity, which encompasses the entire penile tip in most uncircumcised men including the glans, meatus and urethral introitus, plays an important role in STI acquisition. Circumcised men have a lower rate of STIs that infect not only the foreskin but also other distal penile sites, especially the urethra. Likewise, the foreskin may trap HIV and HIV-infected cells after intercourse thereby increasing the risk of HIV acquisition not only through the inner foreskin but also other sites covered by the foreskin. The subpreputial cavity also hosts a unique microbiome that may also play a role in HIV infection. We hypothesize that the penile urethra may be the primary HIV acquisition site in circumcised men and possibly also in non-circumcised men because of the presence of superficial HIV target cells and a high incidence of STIs at this site. Both innate and adaptive immune defense mechanisms are operative in the lower male genital region. The penile urethral mucosa contains accumulations of IgA(+) plasma cells and T lymphocytes and may provide a responsive target for future mucosal vaccines to prevent HIV sexual transmission.

Development of an in vitro alternative assay method for vaginal irritation.

The vaginal mucosa is commonly exposed to chemicals and therapeutic agents that may result in irritation and/or inflammation. In addition to acute effects, vaginal irritation and inflammation can make women more susceptible to infections such as HIV-1 and herpes simplex virus 2 (HSV-2). Hence, the vaginal irritation potential of feminine care formulations and vaginally administered therapeutic agents is a significant public health concern. Traditionally, testing of such materials has been performed using the rabbit vaginal irritation (RVI) assay. In the current study, we investigated whether the organotypic, highly differentiated EpiVaginal™ tissue could be used as a non-animal alternative to the RVI test. The EpiVaginal tissue was exposed to a single application of ingredients commonly found in feminine hygiene products and the effects on tissue viability (MTT assay), barrier disruption (measured by transepithelial electrical resistance, TEER and sodium fluorescein (NaFl) leakage), and inflammatory cytokine release (interleukin (IL)-1α, IL-1ß, IL-6, and IL-8) patterns were examined. When compared to untreated controls, two irritating ingredients, nonoxynol 9 and benzalkonium chloride, reduced tissue viability to <40% and TEER to <60% while increasing NaFl leakage by 11-24% and IL-1α and IL-1ß release by >100%. Four other non-irritating materials had minimal effects on these parameters. Assay reproducibility was confirmed by testing the chemicals using three different tissue production lots and by using tissues reconstructed from cells obtained from three different donors. Coefficients of variation between tissue lots reconstructed with cells obtained from the same donor or lots reconstructed with cells obtained from different donors were less than 10% and 12%, respectively. In conclusion, decreases in tissue viability and barrier function and increases in IL-1α and IL-1ß release appear to be useful endpoints for preclinical screening of topically applied chemicals and formulations for their vaginal irritation potential.

Expression of toll-like receptors in genital tract tissues from normal and HIV-infected men.

PROBLEM: cells of the innate immune system use Toll-like receptors (TLRs) to recognize and respond to invading pathogens. This study was carried out to characterize TLR expression in the human male genital tract, an initial infection site for several sexually transmitted pathogens. METHOD OF STUDY: immunohistochemistry was used to detect expression of TLRs 1-9 in genital tract tissues from HIV(-) and HIV(+) men. RESULTS: in HIV(-) men, TLR1(+) leukocytes were detected throughout the genital tract. Leukocytes in the penile urethra also expressed TLRs2, 3, 5, 7 and 9. Epithelial cells in most tissues did not express TLRs; exceptions were the prostate, where TLRs3 and 8 were observed on the apical surface of luminal epithelial cells, and the penile urethra, where epithelial cells expressed TLR9. In genital tissues from HIV(+) men with AIDS, few TLR(+) cells were detected. CONCLUSION: cells in the male genital tract can express a variety of TLRs. The penile urethra contained the highest number of TLR(+) cells, indicating that this tissue plays a major role in the innate immune defense of the male genital tract. Overall, TLR expression was reduced in genital tissues from HIV(+) men.

Acipimox, an inhibitor of lipolysis, attenuates atherogenesis in LDLR-null mice treated with HIV protease inhibitor ritonavir.

OBJECTIVE: The advent of HIV protease inhibitors has greatly extended the life span of AIDS patients. With an aging HIV(+) population, the cardiometabolic side effects of these drugs are becoming increasingly important clinical concerns. The purpose of this study was to test the hypothesis that inhibition of adipose lipolysis will retard atherogenic lesion development induced by the antiviral protease inhibitors. METHODS AND RESULTS: LDLR-null mice receiving ritonavir were compared with those receiving ritonavir plus lipolysis inhibitor acipimox or vehicle alone to determine how acipimox would affect ritonavir-induced atherogenesis. Intermittent high-fat high-cholesterol diet was used to facilitate optimal atheromatous lesion development. Drug effects were assessed as changes in aortic lesion score, plasma lipid and lipoprotein profile, body fat mass, and insulin-induced suppression of plasma fatty acid concentrations. Ritonavir increased aortic lesions, in association with decreased body fat mass, impaired antilipolysis action of insulin, and increased proatherogenic plasma lipoproteins. All these adverse effects were attenuated by cotreatment with acipimox. CONCLUSIONS: Our results provide the first direct evidence that supports the hypothesis that dysregulation of adipose lipolysis is an important contributor to the proatherogenic role of selected HIV protease inhibitors.

Mucin gene expression in human male urogenital tract epithelia.

BACKGROUND: Mucins are large, hydrophilic glycoproteins that protect wet-surfaced epithelia from pathogen invasion as well as provide lubrication. At least 17 mucin genes have been cloned to date. This study sought to determine the mucin gene expression profile of the human male urogenital tract epithelia, to determine if mucins are present in seminal fluid and to assess the effect of androgens on mucin expression. METHODS AND RESULTS: Testis, epididymis, vas deferens, seminal vesicle, prostate, bladder, urethra and foreskin were assessed for mucin expression by RT-PCR (for 14 mucin genes) and immunohistochemistry (nine antibodies for five mucins). Epithelia of the vas deferens, prostate and urethra expressed the greatest number of mucins, each with mRNA for between 5 and 8 mucins. Except for MUC20 in epididymis, mRNA for MUC1 and MUC20, both membrane-associated mucins, was detected in all tissues analysed. By comparison, MUC6 was more restricted in expression, being primarily detected in seminal vesicle. MUC1, MUC5B and MUC6 were detected in seminal fluid samples by immunoblot analysis. Androgens had no effect on mucin expression in cultured human prostatic epithelial cells. CONCLUSIONS: Each region of urogenital tract epithelium expressed a unique mucin gene repertoire. Secretory mucins are present in seminal fluid, and androgens do not appear to regulate mucin gene expression in prostatic epithelial cells in culture.

Organotypic human vaginal-ectocervical tissue model for irritation studies of spermicides, microbicides, and feminine-care products.

A three-dimensional organotypic vaginal-ectocervical (VEC) tissue model has been developed to test the irritation of topically applied spermicides, microbicides, and vaginal-care products. The in vitro tissue model was reconstructed using normal VEC epithelial cells and is well stratified, containing differentiated basal, suprabasal, intermediate, and superficial cell layers similar to in vivo tissue. The intermediate and superficial cell layers contain glycogen, and the expression of cytokeratins 13 and 14 in the tissue also parallels that of native tissue. The MTT viability assay and histological assessment were used to test inter-lot and intra-lot reproducibility. The MTT average intra-lot coefficient of variation (CV) was less than 10% and the time required to reduce tissue viability by 50% (ET-50) following application of 1% Triton X-100 averaged 1.25+/-0.24h (n=23) upon completion of the 11-day culture period and 1.30 h+/- 0.19 for the same tissues stored overnight at 4 degrees C on agarose gels. The utility of the VEC model for irritation studies was examined by testing commercially available products using the MTT assay and histological assessment. The average ET-50 values ranged between 1.8 and 2.7h for feminine washes, 3.9-6.7 h for spermicides, 6.8-18 h for anti-itch creams, and >18 h for douches, lubricants, and anti-fungal creams. Studies of cytokines released from VEC cultures following product application showed that elevated concentrations of IL-1alpha and IL-1beta were associated with toxicity of test materials. In conclusion, the VEC tissue model is a highly reproducible, non-animal means to assess the irritation of contraceptives, microbicides, and vaginal-care products.

Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone.

Cell-mediated immunity (CMI) is key to defense against intracellular pathogens such as Chlamydia trachomatis and viruses that infect the lower female genital tract, but little is known about CMI at this site. Recent studies indicate that there are immunological microenvironments within the female genital tract, and that immune functions are affected by hormones as well as infections and inflammatory processes. To determine the distribution of mediators of CMI within the lower female genital tract, we have enumerated and characterized T-lymphocyte subsets and natural killer and antigen presenting cells (APCs; macrophages and dendritic cells) in the introitus, vagina, ectocervix, endocervix and cervical transformation zone (TZ) from healthy women, and have examined the effects of the menstrual cycle, menopause and inflammation on these parameters. In women without inflammation, T cells and APCs were most prevalent in the cervical TZ and surrounding tissue. Intraepithelial lymphocytes were predominantly CD8+ T cell+; most CD8+ cells in the TZ and endocervix, and a proportion of cells in the ectocervix, expressed T-cell internal antigen-1, a marker of cytotoxic potential. In contrast, the normal vaginal mucosa contained few T cells and APCs. Cervicitis and vaginitis cases had increased numbers of intraepithelial CD8+ and CD4+ lymphocytes and APCs. The menstrual cycle and menopause had no apparent effect on cellular localization or abundance in any of the lower genital tract tissues. These data indicate that the cervix, especially the TZ, is the major inductive and effector site for CMI in the lower female genital tract. Because CD4+ T cells and APCs are primary host cells for human immunodeficiency virus type 1 (HIV-1), these data also provide further evidence that the cervix is a primary infection site of HIV-1, and that inflammation increases the risk of HIV transmission.