Vaginal Epithelium Transiently Harbours HIV-1 Facilitating Transmission.

Vaginal transmission accounts for majority of newly acquired HIV infections worldwide. Initial events that transpire post-viral binding to vaginal epithelium leading to productive infection in the female reproductive tract are not well elucidated. Here, we examined the interaction of HIV-1 with vaginal epithelial cells (VEC) using Vk2/E6E7, an established cell line exhibiting an HIV-binding receptor phenotype (CD4-CCR5-CD206+) similar to primary cells. We observed rapid viral sequestration, as a metabolically active process that was dose-dependent. Sequestered virus demonstrated monophasic decay after 6 hours with a half-life of 22.435 hours, though residual virus was detectable 48 hours' post-exposure. Viral uptake was not followed by successful reverse transcription and thus productive infection in VEC unlike activated PBMCs. Intraepithelial virus was infectious as evidenced by infection in trans of PHA-p stimulated PBMCs on co-culture. Trans-infection efficiency, however, deteriorated with time, concordant with viral retention kinetics, as peak levels of sequestered virus coincided with maximum viral output of co-cultivated PBMCs. Further, blocking lymphocyte receptor function-associated antigen 1 (LFA-1) expressed on PBMCs significantly inhibited trans-infection suggesting that cell-to-cell spread of HIV from epithelium to target cells was LFA-1 mediated. In addition to stimulated PBMCs, we also demonstrated infection in trans of FACS sorted CD4+ T lymphocyte subsets expressing co-receptors CCR5 and CXCR4. These included, for the first time, potentially gut homing CD4+ T cell subsets co-expressing integrin α4ß7 and CCR5. Our study thus delineates a hitherto unexplored role for the vaginal epithelium as a transient viral reservoir enabling infection of susceptible cell types.

Analysis of sequence diversity and selection pressure in HIV-1 clade C gp41 from India.

Evaluation of viral diversity is critical for the rational design of treatment modalities against Human immunodeficiency virus (HIV). Predominated by HIV-1 clade C (HIV-1C), the epidemic in India represents the third largest population infected with HIV-1 globally. Glycoprotein 41 (gp41) is critical for viral replication and is a target for the design of therapeutic strategies. However, documentation of viral diversity of gp41 gene in infected individuals from India remains limited. Present study employed high throughput sequencing to examine variation in gp41 amplicons generated from blood derived viruses in 24 HIV-1C infected individuals from Mumbai, India. Sequence diversity profiles were documented in different functional domains of gp41. Furthermore, through a meta-analysis approach, all reported gp41 sequences from India (N = 70) were compared with those from South Africa (N = 126), country with the largest HIV epidemic globally, also predominated by HIV-1C. A total of 44 positions displayed statistically significant differential (p < 0.05) Shannon entropy in the two regions. This comparison also identified 11 codon sites undergoing distinct selection, 8 of which remained differentially selected in an extended comparison of data from Asia (N = 137) and Africa(N = 383). Assessment of correlated mutation networks associated with differentially selected residues revealed common as well as distinct interaction networks. Furthermore, codon usage analysis revealed 17 differentially selected codons (Mann-Whitney test, p < 0.001) in Asia and Africa. Dissimilar trends in GC content across codon positions were also observed. In depth understanding of these divergent evolutionary signatures through extended analysis with larger data-sets would assist development of effective interventions being considered for HIV-1C.

Effect of diversity in gp41 membrane proximal external region of primary HIV-1 Indian subtype C sequences on interaction with broadly neutralizing antibodies 4E10 and 10E8.

Human Immunodeficiency Virus-1 Clade C (HIV-1C) dominates the AIDS epidemic in India, afflicting 2.1 million individuals within the country and more than 15 million people worldwide. Membrane proximal external region (MPER) is an attractive target for broadly neutralizing antibody (bNAb) based therapies. However, information on MPER sequence diversity from India is meagre due to limited sampling of primary viral sequences. In the present study, we examined the variation in MPER of HIV-1C from 24 individuals in Mumbai, India by high throughput sequencing of uncultured viral sequences. Deep sequencing of MPER (662-683; HXB2 envelope amino acid numbering) allowed quantification of intra-individual variation up to 65% at positions 662, 665, 668, 674 and 677 within this region. These variable positions included contact sites targeted by bNAbs 2F5, Z13e1, 4E10 as well as 10E8. Both major and minor epitope variants i.e. 'haplotypes' were generated for each sample dataset. A total of 23, 34 and 25 unique epitope haplotypes could be identified for bNAbs 2F5, Z13e1 and 4E10/10E8 respectively. Further analysis of 4E10 and 10E8 epitopes from our dataset and meta-analysis of previously reported HIV-1 sequences from India revealed 26 epitopes (7 India-specific), heretofore untested for neutralization sensitivity. Peptide-Ab docking predicted 13 of these to be non-binding to 10E8. ELISA, Surface Plasmon Resonance and peptide inhibition of HIV-1 neutralization assays were then performed which validated predicted weak/non-binding interactions for peptides corresponding to six of these epitopes. These results highlight the under-representation of 10E8 non-binding HIV-1C MPER sequences from India. Our study thus underscores the need for increased surveillance of primary circulating envelope sequences for development of efficacious bNAb-based interventions in India.

Membrane-initiated estrogen signaling in prostate cancer: A route to epithelial-to-mesenchymal transition.

The plasma membrane (PM) is considered as a major druggable site. More than 50% of the existing drugs target PM proteins. In the wake of emerging data indicating a key role of estrogens in prostate cancer (PCa) pathogenesis, the study was undertaken to explore whether the estrogen binding sites exist on the PM and if such sites are functionally relevant in PCa. Estradiol (E2) binding to the PM was detected in androgen-dependent (LNCaP), androgen-independent (PC3, DU145) PCa cell lines, nontumorigenic (RWPE1) prostate epithelial cell line, and rat prostate cells. Conventional estrogen receptors (nuclear estrogen receptors), known for their nuclear localization, were detected in the PM enriched extracts. This was indirectly confirmed by reduced localization of ERs on the PM of cells, silenced for the expression of their cognate genes. Further, unlike cell-permeable E2, stimulation with cell-impermeable estradiol (E2-BSA) did not induce proliferation in LNCaP cells. However, stimulation with E2-BSA led to alterations in the phosphorylation status of several kinases including GSK3 and AKT, along with the hyperphosphorylation of cytoskeletal proteins such as ß-actin and cytokeratin 8 in LNCaP. This was accompanied by epithelial-to-mesenchymal (EMT) features such as increased migration and invasion; higher vimentin expression, and a concomitant decrease in the E-cadherin expression. These effects were not observed in RWPE1 cells. Interestingly, cell-permeable E2 failed to induce EMT in PCa cells. This in vitro study is the first to suggest that the PM-initiated estrogen signaling contributes to higher invasiveness in PCa cells. Plasma membrane ERs may act as novel targets for PCa therapeutics.

Evaluation of Phytopolyphenols for their gp120-CD4 Binding Inhibitory Properties by In Silico Molecular Modelling & In Vitro Cell Line Studies.

BACKGROUND: Lack of effective early-stage HIV-1 inhibitor instigated the need for screening of novel gp120-CD4 binding inhibitor. Polyphenols, a secondary metabolite derived from natural sources are reported to have broad spectrum HIV-1 inhibitory activity. However, the gp120-CD4 binding inhibitory activity of polyphenols has not been analysed in silico yet. OBJECTIVES: To establish the usage of phytopolyphenols (Theaflavin, Epigallocatechin (EGCG), Ellagic acid and Gallic acid) as early stage HIV-1 inhibitor by investigating their binding mode in reported homology of gp120-CD4 receptor complex using in silico screening studies and in vitro cell line studies. METHODS: The in silico molecular docking and molecular simulation studies were performed using Schrödinger 2013-2 suite installed on Fujitsu Celsius Workstation. The in vitro cell line studies were performed in the TZM-bl cell line using MTT assay and ß-galactosidase assay. RESULTS: The results of molecular docking indicated that Theaflavin and EGCG exhibited high XP dock score with binding pose exhibiting Van der Waals interaction and hydrophobic interaction at the deeper site in the Phe43 cavity with Asp368 and Trp427. Both Theaflavin and EGCG form a stable complex with the prepared HIV-1 receptor and their binding mode interaction is within the vicinity 4 Å. Further, in vitro cell line studies also confirmed that Theaflavin (SI = 252) and EGCG (SI = 138) exert better HIV-1 inhibitory activity as compared to Ellagic acid (SI = 30) and Gallic acid (SI = 34). CONCLUSION: The results elucidate a possible binding mode of phytopolyphenols, which pinpoints their plausible mechanism and directs their usage as early stage HIV-1 inhibitor.

Tetrahydrocurcumin-loaded vaginal nanomicrobicide for prophylaxis of HIV/AIDS: in silico study, formulation development, and in vitro evaluation.

A vaginal microbicide is a front-line women-dependent approach and an alternative to a condom for prevention of unprotected sexual intercourse-associated HIV. The microbicide research is still in its infancy with several products in the clinical studies being reported to have good efficacy, safe, but with poor adherence. One such molecule reported with an excellent efficacy when tested preclinically is curcumin, a natural polyphenol derived from Curcuma longa. Despite its potential HIV-1 inhibitory activity, it has intense yellow color staining properties, which would result in poor consumer compliance and adherence for vaginal application. To address this issue, tetrahydrocurcumin (THC), a colorless derivative of curcumin, was subjected to in silico screening (molecular docking and dynamics simulation studies) using homology model of gp120-CD4 binding. It was found that THC exhibited equivalent gp120-CD4 binding inhibitory activity as compared with curcumin due to its stable hydrophobic interactions with residues Asp368 and Trp427 deeper in the Phe43 cavity of CD4 receptor. Hence, it can be effectively used as a potential microbicide candidate. THC, a BCS Class II molecule exhibits poor solubility, spreadability, and intracellular uptake when used in the conventional form. Thus, it was decided to develop a lipid-based nanomicrobicide gel for delivery of THC. The developed THC-loaded o/w microemulsion gel was characterized for physicochemical properties (globule size, drug content, drug release, and permeation) and further used for in vitro cell line studies (cell viability, cellular uptake, and anti-HIV activity). The developed formulation was found to be stable with coitus-independent release profile and exhibited a rapid time-independent intracellular uptake. In addition, it exhibited a fourfold increase in efficacy as compared with conventional THC. Thus, the novel THC-loaded o/w microemulsion gel exhibited the potential for prevention of HIV-1 infection associated with unprotected sexual intercourse.

Immunogenicity of virus-like Semliki Forest virus replicon particles expressing Indian HIV-1C gag, env and polRT genes.

Development of a vaccine targeting human immunodeficiency virus-1 subtype C (HIV-1C) is an important public health priority in regions with a high prevalence of the clade C virus. The present study demonstrates the immunogenicity of recombinant Semliki Forest virus (SFV)-based virus-like replicon particles (VRPs) expressing Indian HIV-1C env/gag/polRT genes. Immunization of mice with recombinant VRPs in a homologous prime-boost protocol, either individually or in combination, elicited significant antigen-specific IFN-γ T cell responses as detected by the ELISPOT assay. Additionally, Gag-specific TNF-α secreting CD8+ and CD4+ T cells and Env-specific IL-2 secreting T cells were also elicited by mice immunized with Gag and Env constructs, respectively, as estimated by intracellular cytokine staining assay. Moreover, an HIV Pol-specific TNF-α response was elicited in mice immunized with a combination of the three VRP constructs. Furthermore, HIV-1C Gag and Env-specific binding antibodies were elicited as verified by gp120 ELISA and p24 Gag ELISA, respectively. The immunogenicity of VRPs was found to be higher as compared to that of RNA replicons and VRPs may therefore be promising preventive and therapeutic candidate vaccines for the control and management of HIV/AIDS.

Current status of a unique vaccine preventing pregnancy.

The ability of a vaccine linking beta hCG to a carrier to generate antibodies against hCG, its reversibility and safety was established by Phase I clinical trials conducted in India, Finland, Sweden, Chile and Brazil. Employing a hetero-species dimer (beta hCG-αoLH) linked to tetanus toxoid further improved the immunogenicity of the vaccine. Phase II clinical trials showed that anti-hCG titres above 50 ng/ml prevented pregnancy of sexually active fertile women without derangement of ovulation and menstrual regularity. On decline of antibodies, women conceived again to give birth to normal progeny. A genetically engineered vaccine consisting of beta hCG linked to B subunit of heat labile enterotoxin of E. coli has been made. It is expressed as DNA as well as protein. Priming with DNA followed by protein version of the vaccine generates very high titres against hCG in mice. Extensive toxicology studies in 2 species of rodents, and marmosets have shown complete safety of the vaccine. The vaccine is cleared for Clinical trials by the National Review committee on Genetic Manipulation and Drugs Controller General of India.

Nevirapine Loaded Core Shell Gold Nanoparticles by Double Emulsion Solvent Evaporation: In vitro and In vivo Evaluation.

BACKGROUND: HIV/AIDS is a macrophage resident infection localized in the reticuloendothelial system and remote locations of brain and bone marrow. We present core shell nanoparticles of gold(AuNPs) and nevirapine(NVP) for targeted delivery to the multiple HIV reservoirs. The aim of the study was to design core shell NVP loaded AuNPs with high drug loading and to evaluate biodistribution of the nanoparticles in possible HIV reservoirs in vivo. A specific objective was to assess the possible synergy of AuNPs with NVP on anti-HIV activity in vitro. METHOD: Core shell nanoparticles were prepared by double emulsion solvent evaporation method and characterized. RESULTS: Glyceryl monostearate-nevirapine-gold nanoparticles(GMS-NVP-AuNPs) revealed high entrapment efficiency (>70%), high loading (~40%), particle size <250 nm and zeta potential -35.9± 1.41mv and exhibited sustained release with good stability. Surface plasmon resonance indicated shell formation while SEM coupled EDAX confirmed the presence of Au. TEM confirmed formation of spherical core shell nanoparticles. GMS-NVP-AuNPs revealed low hemolysis (<10 %) and serum stability upto 6 h. GMS-NVP-AuNPs exhibited rapid, high and sustained accumulation in the possible HIV reservoir organs, including the major organs of liver, spleen, lymph nodes, thymus and also remote locations of brain, ovary and bone marrow. High cell viability and enhanced uptake in PBMC's and TZM-bl cells were observed. While uptake in PBMC's proposed monocytes/macrophages enabled brain delivery. GMS-NVP-AuNPs demonstrated synergistic anti-HIV activity. CONCLUSION: The superior anti-HIV activity in vitro coupled with extensive localization of the nanoparticles in multiple HIV reservoirs suggests great promise of the core shell GMS-NVP-AuNPs for improved therapy of HIV.

Immunogenicity of Semliki Forest virus based self-amplifying RNA expressing Indian HIV-1C genes in mice.

Development of recombinant vaccines is considered as a promising approach to prevent transmission and eradication of HIV/AIDS. Candidate vaccines tested so far have shown poor to modest efficacy. Self-amplifying RNAs of positive strand alphaviruses are reported to be promising vectors for development of recombinant vaccines. This study describes the construction, in vitro expression and in vivo immunogenicity of recombinant RNA vaccines developed by individually cloning gag, env and polRT genes of primary HIV-1C Indian isolates using Semliki Forest virus (SFV) vector. HIV-1C specific T cell responses were detected in mice immunized with rSFV2gen/gag RNA by IFN-γ ELISPOT assay. Furthermore, using flow cytometry based intracellular cytokine staining (ICCS) assay HIV-1C specific IL-2 responses were detected in immunized mice that were mediated by both CD4(+) and CD8(+) T cells. Mice immunized with rSFV2gen/env RNA elicited HIV-1C Env-specific antibodies as detected by gp120 ELISA. The Env, Gag and Pol (RT) RNA constructs in combination elicited better HIV-1C Env-specific humoral responses compared to mice immunized with Env RNA alone. In conclusion, rSFV2gen RNA constructs encoding HIV-1C antigens elicited clear cell mediated and humoral immune responses in mice, thus demonstrating the potential of self-amplifying rSFV2gen RNA as a promising candidate for anti-HIV vaccine development.

Advances in development of a contraceptive vaccine against human chorionic gonadotropin.

INTRODUCTION: There is continuing need for contraceptives. According to World Health Organization, 210 million pregnancies occur each year, out of which some 80 million are unintended. A vaccine offering privacy and periodic intake would be an attractive proposition. AREAS COVERED: The article is a brief review of three vaccines developed against human chorionic gonadotropin (hCG) with progressively better attributes. Clinical trials have proven in more than one country the complete safety and reversibility of the anti-hCG vaccine(s) in women. Vaccination does not entail any disturbance in levels of reproductive tract hormones of the woman or any disturbance in menstrual regularity and bleeding profiles. Phase II clinical trials show the effective prevention of pregnancy in sexually active women of proven fertility. A recombinant vaccine amenable to industrial production has been developed; it induces substantially higher antibody titers in mice of four different genetic strains than those required to prevent pregnancy in women. Rigorous toxicology studies have been completed on this vaccine in rodents and marmosets. EXPERT OPINION: This unique vaccine, requiring periodic intake and demonstrating no impairment of ovulation, hormonal profiles and menstrual regularity, is on the verge of final clinical trials under the aegis of the Indian Council of Medical Research and should be a valuable addition to the available contraceptives.

Evaluation of cystatin C activities against HIV.

BACKGROUND & OBJECTIVES: Several host defense proteins known to possess antimicrobial activities are present on mucosal surfaces and are consequently found in body fluids of vertebrates. Naturally occurring protease inhibitors like cystatins, especially cystatin C (cys C), are abundantly present in human seminal plasma. Although its antiviral activity against herpes simplex virus (HSV) has been demonstrated, the role of this protein against HIV is not well studied. Therefore, the aim of the present study was to evaluate the anti-HIV activities of cys C, which is present innately in the male reproductive tract. METHODS: Protein-protein interaction of cys C with various HIV proteins was studied using a commercially available HIV blot and specific interaction with HIV protease was studied by dot-blot technique using commercially available cys C. To purify biologically active cys C from human seminal plasma to be used for subsequent experiments, gel-permeation chromatography followed by affinity chromatography was used. The HIV infectivity inhibition activity of the purified cystatin C was tested in TZM-bl cells. To study its activity on HIV protease, time-course enzyme kinetics studies were performed using spectrometric assay. RESULTS: Cystatin C reacted with some HIV proteins including HIV protease. Biologically active cys C was purified using gel permeation chromatography followed by affinity chromatography. When tested in TZM-bl cells, purified cystatin C demonstrated HIV-infectivity inhibitory activity (IC 50: 0.28 µM). Enzyme kinetic studies demonstrated that it abrogated the action of HIV protease on its substrate. INTERPRETATION & CONCLUSIONS: The present data demonstrate that cystatin C possesses anti-HIV activities. Molecular models need to be designed with this protein which would assist towards prevention/ therapeutics against HIV.

Evaluation of a lactogenic activity of an aqueous extract of Cyperus rotundus Linn.

ETHNOPHARMACOLOGICAL RELEVANCE: In Ayurveda, the rhizome of Cyperus rotundus Linn has been reported for wide spectrum of biological activities including lactational therapy for increasing milk quantity. However, not a single report is available on validation of its herbal galactagogue potentiality in literature. Thus, the present study is aimed to assess the lactogenic property of aqueous extract of Cyperus rotundus (CRE). MATERIALS AND METHODS: The effect of aqueous extract of Cyperus rotundus rhizome was evaluated by measuring weight of the pups during suckling period. Quantitatively, total protein and carbohydrate contents of mammary tissue and serum prolactin and cortisol level were calculated. Histopathological analysis of mammary gland, pituitary gland, heart, liver, spleen, kidney, and ovary tissues was carried out. Acute toxicity of CRE against rat was assessed by the Hippocratic test and biochemical profile of blood serum. RESULTS: Oral administration of 300 and 600mg of CRE induced about 23% and 40% more milk in experimental group of animals as compared to the control group of animals. Weight gain by pups and mother rats of treated groups were significantly higher following administration of CRE as compared to that of control group. Moreover protein and carbohydrate content of mammary gland tissue were also significantly more than control group of animals. The CRE was found to stimulate the synthesis of prolactin significantly. In addition, the mammary gland tissues of experimental group showed obvious lobulo-alveolar development with milk secretion. Administration of CRE did not cause any signs or symptoms of toxicity which implied that Cyperus rotundus is toxicologically safe. CONCLUSION: This study demonstrates that the aqueous extract of Cyperus rotundus can stimulate milk production in the female rats which may be consequently effective in increasing the lactation of human too.

Increased activity of goat liver plasma membrane alkaline phosphatase upon release by phosphatidylinositol-specific phospholipase C.

Mammalian alkaline phosphatase (ALP) is attached to the plasma membrane by a unique glycosylphosphatidylinositol (GPI) anchor. The influence of such a complex anchoring device on the enzyme function is not fully understood. Here, we report the effect of cleavage of the GPI anchor on the activity of goat liver plasma membrane ALP (GLPM-ALP). Phosphatidylinositol-specific phospholipase C (PI-PLC) purified from Bacillus cereus was used for the cleavage of the GPI anchor (delipidation) and hence for release of ALP from the membrane. Detergents--octyl-beta-D-glucopyranoside (OG) and triton X100 (TX100) were also used for solubilization of ALP from the membrane. Resistance to solubilization by TX100 suggested the association of GPI-ALP with lipid rafts. Solubilization of GLPM-ALP with OG had no effect on the enzyme activity; however, delipidation with PI-PLC resulted in enhanced ALP activity. Kinetic analysis showed catalytic activation of PI-PLC-treated GLPM-ALP with an increase in V(max) (35%) without a significant change in K(m). Moreover, this change in Vmax was observed to be independent of pH and buffer. The results suggested the implication of GPI anchor in modulating the catalytic property of GLPM-ALP, thus indicating the role of this special anchoring structure in the enzyme regulation.

Foeniculum vulgare Mill: a review of its botany, phytochemistry, pharmacology, contemporary application, and toxicology.

Foeniculum vulgare Mill commonly called fennel has been used in traditional medicine for a wide range of ailments related to digestive, endocrine, reproductive, and respiratory systems. Additionally, it is also used as a galactagogue agent for lactating mothers. The review aims to gather the fragmented information available in the literature regarding morphology, ethnomedicinal applications, phytochemistry, pharmacology, and toxicology of Foeniculum vulgare. It also compiles available scientific evidence for the ethnobotanical claims and to identify gaps required to be filled by future research. Findings based on their traditional uses and scientific evaluation indicates that Foeniculum vulgare remains to be the most widely used herbal plant. It has been used for more than forty types of disorders. Phytochemical studies have shown the presence of numerous valuable compounds, such as volatile compounds, flavonoids, phenolic compounds, fatty acids, and amino acids. Compiled data indicate their efficacy in several in vitro and in vivo pharmacological properties such as antimicrobial, antiviral, anti-inflammatory, antimutagenic, antinociceptive, antipyretic, antispasmodic, antithrombotic, apoptotic, cardiovascular, chemomodulatory, antitumor, hepatoprotective, hypoglycemic, hypolipidemic, and memory enhancing property. Foeniculum vulgare has emerged as a good source of traditional medicine and it provides a noteworthy basis in pharmaceutical biology for the development/formulation of new drugs and future clinical uses.

Traditional uses, phytochemistry and pharmacology of Ficus carica: a review.

CONTEXT: Ficus carica Linn (Moraceae) has been used in traditional medicine for a wide range of ailments related to digestive, endocrine, reproductive, and respiratory systems. Additionally, it is also used in gastrointestinal tract and urinary tract infection. OBJECTIVE: This review gathers the fragmented information available in the literature regarding morphology, ethnomedicinal applications, phytochemistry, pharmacology, and toxicology of Ficus carica. It also explores the therapeutic potential of Ficus carica in the field of ethnophytopharmacology. MATERIALS AND METHODS: All the available information on Ficus carica was compiled from electronic databases such as Academic Journals, Ethnobotany, Google Scholar, PubMed, Science Direct, Web of Science, and library search. RESULTS: Worldwide ethnomedical uses of Ficus carica have been recorded which have been used traditionally for more than 40 types of disorders. Phytochemical research has led to the isolation of primary as well as secondary metabolites, plant pigment, and enzymes (protease, oxidase, and amylase). Fresh plant materials, crude extracts, and isolated components of Ficus carica have shown a wide spectrum of biological (pharmacological) activities. CONCLUSION: Ficus carica has emerged as a good source of traditional medicine for the treatment of various ailments such as anemia, cancer, diabetes, leprosy, liver diseases, paralysis, skin diseases, and ulcers. It is a promising candidate in pharmaceutical biology for the development/formulation of new drugs and future clinical uses.

Echography of the cervix and uterus during the proliferative and secretory phases of the menstrual cycle in bonnet monkeys (Macaca radiata).

We undertook the present study to investigate the echographic characteristics of the uterus and cervix of female bonnet monkeys ( Macaca radiata ) during the proliferative and secretory phases of the menstrual cycle. The cervix was tortuous in shape and measured 2.74 ± 0.30 cm (mean ± SD) in width by 3.10 ± 0.32 cm in length. The cervical lumen contained 2 or 3 colliculi, which projected from the cervical canal. The echogenicity of cervix varied during proliferative and secretory phases. The uterus was pyriform in shape (2.46 ± 0.28 cm × 1.45 ± 0.19 cm) and consisted of serosa, myometrium, and endometrium. The endometrium generated a triple-line pattern; the outer and central lines were hyperechogenic, whereas the inner line was hypoechogenic. The endometrium was significantly thicker during the secretory phase (0.69 ± 0.12 cm) than during the proliferative phase (0.43 ± 0.15 cm). Knowledge of the echogenic changes in the female reproductive organs of bonnet monkeys during a regular menstrual cycle may facilitate understanding of other physiologic and pathophysiologic changes.

HIV gp120 induced gene expression signatures in vaginal epithelial cells.

Gp120 is the envelope protein of HIV which binds to CD4 independent proteins on vaginal epithelial cells. HIV-gp120 has been reported to modulate gene expression in several cell types. How this interaction may alter the physiologic vaginal milieu during the earliest stages of vaginal transmission of HIV, is currently unknown. Vaginal epithelial cells were treated with HIV-gp120, and a global snapshot of changes in gene expression profiles, were unraveled by microarray analysis. The differentially expressed genes were involved in diverse cellular functions. Genes of immunomodulatory processes and induction of proteases were highly enriched. We propose that the induction of inflammation and proteases may act in concert to weaken the vaginal epithelium, making it more permeable to viral entry. Identification of the gene signatures involved in vaginal-HIV dialogue would aid in understanding the environ induced by HIV itself, as the virus invades and gains entry into its host.

Association of human mannose receptor in sexual transmission of human immunodeficiency virus in serodiscordant couples.

HIV binds specifically to the human mannose receptor (hMR) on vaginal epithelial cells that are devoid of a conventional CD4 receptor. HIV binding to hMR on vaginal epithelial cells induces the production of matrix metalloproteinase 9 (MMP9) leading to degradation of the extracellular matrix, which may increase the risk of HIV entry into vaginal epithelial cells and further transmission into distal cells. Immunofluorescent localization of hMR on vaginal epithelial cells of seronegative females from the general population included the control group (n=52) and seronegative females from serodiscordant couples. There was PCR amplification of DNA from peripheral blood mononuclear cells (PBMCs) of the serodiscordant females for the CCR5 gene flanking the CCR5-Δ32 region; PCR amplification and sequencing of the C2-V3 region of HIV variants in PBMCs and sperm of the infected male partners of the serodiscordant couples; and the presence of hMR on 0-11% of the vaginal epithelial cells of seronegative females (n=39) from serodiscordant couples and 90-95% that of a control group of females (n=52). Nine of these serodiscordant females did not show a CCR5-Δ32 deletion. The translated amino acid sequence of the C2-V3 region of the env gene of HIV-1C in PBMCs (n=9) and sperm (n=5) of the male partners showed the presence of distinct variants and the variation in PBMCs and sperm of serodiscordant males was almost similar to that of infected males from concordant couples. The presence of hMR in a smaller number of vaginal epithelial cells of serodiscordant females prevented binding and HIV entry into these cells and therefore prevented sexual transmission of HIV.

HIV gp120 binds to mannose receptor on vaginal epithelial cells and induces production of matrix metalloproteinases.

BACKGROUND: During sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR) as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs) downstream of HIV gp120 binding to hMR. PRINCIPAL FINDINGS: Human vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line) and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody. CONCLUSION: hMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the vaginal epithelium.