Data on autophagy markers and anti-candida cytokines expression in mice in response to vaginal infection of Candida albicans.

The data presented here are related to the research article entitled "Knockout of autophagy gene, ATG5 in mice vaginal cells abrogates cytokine response and pathogen clearance during vaginal infection of Candida albicans" (Shroff et al., 2018) [1]. The cited research article describes the role of autophagy in host immune response against C. albicans infection of mice vagina. In this data report wild-type C57BL/6 mice were infected intravaginally with C. albicans. Vaginal cells were analyzed for the expression of autophagy marker genes LC3 & ATG5 and lysosome marker LAMP1 at the transcript and protein level. Vaginal lavages were also obtained from these infected mice. The levels of pro-inflammatory and T-helper cell related cytokines were determined in these lavages.

Knockout of autophagy gene, ATG5 in mice vaginal cells abrogates cytokine response and pathogen clearance during vaginal infection of Candida albicans.

The female reproductive tract (FRT) presents a unique challenge to the mucosal immune system as it needs to monitor constantly for the presence of opportunistic pathogens amidst its commensal flora. During infection, autophagy plays a critical role in pathogen clearance, presentation of antigens and production of pro-inflammatory cytokines. However, no information is available that describes the role of autophagy in mouse vaginal infection of Candida albicans. The objective of our study is to evaluate the effect of autophagy gene, ATG5 knockout in vaginal cells in response to vaginal C. albicans infection. Mice having knockout of ATG5 in the vaginal cells (PR-ATG5-KO mice) were infected intra-vaginally with the yeast form of Candida albicans. Vaginal lavages were collected once in a week until the infection was cleared. We detected the expression of autophagy marker genes (LC3, ATG5 and LAMP1) in the vaginal cells. We determined the levels of various cytokines (IL-1α, IL-1ß, IL-6, IL-10, IL-17A, IL-22, IL-23p19, TNF-α and G-CSF) involved in anti-candida response. The levels of cytokines in the vaginal lavages were quantified using Aimplex Premixed analyte kit. The vaginal lavages were checked for polymorphonuclear leucocytes (PMNLs) infiltration. The candida clearance rate from the vaginal lumen was determined by Colony Forming Units (CFUs) assay. The results revealed that PR-ATG5-KO mice failed to induce the expression of LC3, ATG5 and LAMP1 indicating an impaired autophagy pathway. The levels of all the cytokines (except IL-10) in C. albicans infected PR-ATG5-KO mice were significantly reduced as compared to the wild type infected C57BL/6 mice. The number of PMNLs infiltrated into the vaginal lavages of infected PR-ATG5-KO mice was reduced. The clearance of C. albicans from the vaginal lumen was also considerably delayed in PR-ATG5-KO mice. In conclusion, the results revealed that impaired autophagy in vaginal cells influences host response during vaginal infection of C. albicans by affecting anti-Candida cytokine levels in the vaginal lavage resulting in reduction of pathogen clearance rate.

Rapamycin (Sirolimus) alters mechanistic target of rapamycin pathway regulation and microRNA expression in mouse meiotic spermatocytes.

Mechanistic target of rapamycin (mTOR) is a signal transduction pathway that modulates translation initiation in several animals including mammals. Rapamaycin, an allosteric inhibitor of mTOR pathway, is often used as an immunosuppressive drug following kidney transplantation and causes gonadal dysfunction and defects in spermatogenesis. The molecular mechanism behind rapamycin-mediated testicular dysfunction is not known. We have therefore explored the contribution of rapamycin in mTOR regulation and microRNA (miRNA) expression in mouse spermatocytes, the intermediate stage of spermatogenesis, where meiosis takes place. In the present study, we optimized the isolation of highly pure and viable spermatocytes by flow sorting, treated them with rapamycin, and investigated the expression of mTOR and downstream effector molecules. Western blot and immunocytochemical analysis confirm that rapamycin treatment suppresses mTOR and phopsphorylated P70S6 kinase activities in spermatocytes, but not that of phosphorylated 4E-binding protein 1. Also, rapamycin treatment modulates the expression of several spermatocyte-specific miRNAs. To complement these finding an in vivo study was also performed. In silico prediction of target genes of these miRNAs and their functional pathway analysis revealed that, several of them are involved in crucial biological process, cellular process and catalytic activities. miRNA-transcription factor (TF) network analysis enlisted different TFs propelling the transcription machineries of these miRNAs. In silico prediction followed by quatitative real-time PCR revealed two of these TFs namely, PU.1 and CCCTC binding factor (CTCF) are down and upregulated, respectively, which may be the reason of the altered expression of miRNAs following rapamycin treatment. In conclusion, for the first time, the present study provides insight into how rapamycin regulates mTOR pathway and spermatocyte-specific miRNA expression which in turn, regulate expression of target genes post-transcriptionally.

Hemoglobin expression in nonerythroid cells: novel or ubiquitous?

Hemoglobin (Hb) is a major protein involved in transport of oxygen (O2). Red blood cells (RBCs) contain maximum amount of Hb and because of their unique structure and plasticity they transport O2 to various tissues of the body at an optimal concentration. Recently, it has been reported that, apart from RBCs, Hb is also expressed by nonerythroid cells such as epithelial cells of different origin. The cells expressing Hb are from the tissues where maintenance of O2 homeostasis is of paramount importance. Hb expression has been observed in the epithelial cells from human tissues including lungs, neurons, retina, and endometrium. Our group has recently demonstrated that Hb is expressed by the cervicovaginal epithelial cells. We further showed that, apart from maintaining O2 homeostasis, Hb and the peptides derived from it play an indispensable role in the protection of vaginal epithelium by exhibiting antimicrobial activity. In this review, we discuss the significance of Hb expression in vaginal epithelial cells and its role in the recognition of pathogens thereby reducing the risk and/or severity of inflammation and/or infections and the possible mechanism by which Hb exhibits antimicrobial and antioxidative functions.

Regulatory non-coding transcripts in spermatogenesis: shedding light on 'dark matter'.

Global rise in male infertility over the past decades as a result of falling sperm count and quality has been pointed out by different investigations. Therefore, it is important to understand the molecular mechanism of spermatogenesis and its regulation. Mammalian spermatogenesis, a streamlined process through which male germline cells divide and differentiate into mature spermatozoa, is strictly regulated by phase-specific gene expression which, in turn, is controlled by myriads of regulatory non-coding RNAs (ncRNAs). Rapid advancement in genome mining technologies has identified role of ncRNAs including microRNAs, PIWI-interacting RNAs, endogenous small-interfering RNAs and long non-coding RNAs as controller of gene expression at transcriptional as well as post-transcriptional level in different biological context and disease processes. Here, we discuss the recent progress in our understanding about the involvement of these molecules in spermatogenesis. In addition, we describe here the possible roles of long non-coding RNAs in controlling this process which is not delved so far.

Spermicidal activity of Indian seaweeds: an in vitro study.

Contraceptive properties of seaweeds are still stands as lacuna; in this context, the screening of in vitro male contraceptive properties of crude ethanolic extract of Indian seaweeds against normal human sperm is carried out. In total, twelve seaweeds were screened for in vitro spermicidal activity. Among these twelve seaweeds, Halimeda gracilis showed 100% inhibition of human spermatozoa at 10 mg ml(-1) concentration in 20 s and its EC50 value was 2.05 mg ml(-1) in 20 s. Further, dose- and time-dependent spermicidal assay revealed that the sperm was completely immobilised for 20 s. Plasma membrane of sperm was damaged due to the exposure of H. gracilis extract. MTT assay with H. gracilis extract showed 88.5% of cytotoxic incidence. H. gracilis extract tested for cytotoxicity against Artemia salina recorded LC50 value of 34.8 µg ml(-1) . Phytochemical analysis of H. gracilis extract evidenced the presence of alkaloids, flavonoids, proteins and sugars. Results of this study clearly inferred that the synergistic effect of active principles reside within the H. gracilis extract had shown better contraceptive activity.

Effect of Rabbit Epididymal Antimicrobial Peptide, REHbßP, on LPS-Induced Proinflammatory Cytokine Responses in Human Vaginal Cells In Vitro.

Antimicrobial peptides (AMP's) protect epithelial surfaces including epididymis against pathogens and play a key role in orchestrating various defensive responses. Recently, we have identified one such AMP, rabbit epididymal hemoglobin-ß subuit (REHbßP) from the epididymal fluid of rabbit, Oryctologus cuniculus. The demonstration of a protective role of REHbßP in epididymal epithelial cells (EPEC's) led us to investigate: (1) the identification of LPS interactive domain in REHbßP, and (2) whether the REHbßP of rabbit origin mediates vaginal cellular immune responses of another species (human). HeLa-S3, human vaginal epithelial cells (hVECs) were exposed to LPS or the LPS-stimulated cells treated with REHbßP or neutral peptide, nREHbßP. Effect of LPS and cytokines (IL-6 and IL-1α) and chemokines (IL-8, MCP-1) levels was determined in the culture supernatants. In response to the LPS, hVECs synthesized these mediators and the levels were significantly higher than controls. This enhancing effect was ameliorated when the LPS-induced hVECs were treated with REHbßP. Similar results were obtained on NF-κB protein and hBD-1 mRNA expression. Confocal microscopy studies revealed that REHbßP attenuated the LPS-induced internalization of E. coli by macrophages. The chemotaxis studies performed using Boyden chamber Transwell assay, which showed elevated migration of U937 cells when the supernatants of LPS-induced hVECs were used, and the effect was inhibited by REHbßP. REHbßP was found to be localized on the acrosome of rabbit spermatozoa, suggesting its role in sperm protection beside sperm function. In conclusion, REHbßP may have the potential to develop as a therapeutic agent for reproductive tract infections (RTI's).

A rabbit vaginal cell-derived antimicrobial peptide, RVFHbαP, blocks lipopolysaccharide-mediated inflammation in human vaginal cells in vitro.

Antimicrobial peptides (AMPs) constitute a phylogenetically ancient form of innate immunity that provides host defense at various mucosal surfaces, including the vagina. Recently, we have identified one such AMP, rabbit vaginal fluid hemoglobin alpha peptide (RVFHbαP), from the vaginal lavage of rabbits (Oryctolagus cuniculus). The recent demonstration of a protective role of this peptide in erythrocytes and vaginal cells led us to investigate (i) the lipopolysaccharide (LPS) interactive domain in RVFHbαP and (ii) whether RVFHbαP of rabbit origin modulates the cellular immune responses of another species (humans) in vitro. HeLa-S3, a human vaginal epithelial cell line (hVEC), was exposed to LPS alone (10 µg/ml for 6 h), or LPS-induced cells were treated with RVFHbαP (70.45 µM for 1 h) and cultured for 24 h, and the results obtained were compared with the medium control. We show here that RVFHbαP exerts an anti-inflammatory activity in hVECs, as suggested by the prevention of LPS-induced production of extracellular (supernatant) and intracellular (lysate) levels of cytokines (interleukin 6 [IL-6] and IL-1α) and chemokines (IL-8 and monocyte chemoattractant protein 1 [MCP-1]). The demonstration of Toll-like receptor 4 (TLR4) and NF-κB expression in hVECs and the observations of RVFHbαP suppression of human ß-defensin-1 (hBD1) mRNA expression further support the hypothesis of a genomic activity of RVFHbαP. Confocal microscopy and flow cytometry results demonstrate that RVFHbαP inhibits LPS-induced phagocytosis of Escherichia coli by macrophages. The chemotaxis studies performed using the Boyden chamber Transwell method showed the increased migration of U937 cells when supernatants of LPS-induced hVECs were used, and this effect was inhibited by RVFHbαP. In conclusion, our study proposes a novel explanation for the protective role of RVFHbαP in inflammation-associated infections, which not only may provide the new cellular targets for the screening of RVFHbαP ligands acting in the vaginal tissue but also has the potential to develop RVFHbαP as a therapeutic agent for reproductive tract infections.

Antibacterial activity of a synthetic peptide that mimics the LPS binding domain of Indian mud crab, Scylla serrata anti-lipopolysaccharide factor (SsALF) also involved in the modulation of vaginal immune functions through NF-kB signaling.

Recently the cDNA coding for anti-lipopolysaccharide factor (ALF) has been identified from the Indian mud crab, Scylla serrata and has been named S. serrata anti-lipopolysaccharide factor (SsALF). SsALF protein sequence demonstrated the presence of two highly conserved cystine residues between which the putative lipopolysaccharide (LPS) binding domain is known to be located. In this study, we have designed and synthesized a 24 amino acid linear (lSsALF24) and a cyclic (cSsALF24) peptides based on this putative LPS binding domain and demonstrated the ability of these peptides to bind to LPS. The peptides were active against vaginal pathogens demonstrated by MIC, CFU and phagocytosis assays. cSsALF24 did not show toxicity to human vaginal epithelial cells (HeLa-S3), macrophages and rabbit erythrocytes even at high concentration (64.64 µM). Flow cytometry results demonstrated that cSsALF24 peptide suppressed LPS induced phagocytosis of FITC labeled E. coli. HeLa cells were stimulated with LPS (10 µg/ml) alone for 6 h or after two washings with PBS, treated for 1 h with cSsALF24 (64.64 µM). After washing, the cells were cultured for 24 h in fresh media. The spent media as well as cells were collected for the determination of cytokine/chemokine levels such as interleukin-6 (IL-6) interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and interleukin-1α (IL-1α) using ELISA and RT-PCR respectively. Similar results were obtained with LPS stimulated cells treated with c/nSsALF24 or unstimulated cells treated with c/nSsALF24. The expression of cytokine/chemokines and mRNA's coding these proteins were unaffected in c/nSsALF24 treated cells. In contrast, in LPS stimulated cells, the expression levels of these molecules were up-regulated via the induction of nuclear factor kappa-B (NF-kB) levels. However, the expression of these pro-inflammatory markers was decreased in LPS stimulated cells following the treatment with cSsALF24, attributing anti-inflammatory potential of the peptide. Collectively, these findings suggest that cSsALF24 might regulate the vaginal epithelial cell immune responses indirectly through modulation of LPS-TLR4 binding in NF-kB pathway.

Identification and characterization of anti-microbial peptides from rabbit vaginal fluid.

Antimicrobial peptides (AMPs) serve as a first line of host defense and represent an important, though poorly understood components of the innate immune system. The present study was an attempt to identify and characterize the major molecules having anti-bacterial activities from the vaginal fluid of rabbit, Oryctologus cuniculus. AMPs from the rabbit vaginal fluid (RVF) were identified in the acid extracts of pooled RVF samples after RP-HPLC purification. The protein, RVFAMP was effective against gram negative (Escherichia coli and Pseudomonas aeruginosa) and gram positive (Staphylococcus aureus and Streptococcus pyogenes) bacteria. The results of acid urea-PAGE-gel overlay assay (AU-PAGE-GOA) demonstrated clear zone of growth inhibition of E. coli corresponding to 6 and 15 kDa protein bands. LC-MS data of these proteins indicated that 15 kDa protein consists of lysozyme, lipopolysaccharide binding protein (LBP), hemoglobin-α and ß subunits (Hb-α/ß), whereas 9 kDa protein band consists of transthyretin and calcyclin while uteroglobulin and neutrophil antibacterial peptide-5 (NAMP-5) are present in the 6 kDa protein band. Of the eight proteins, Hb-α derived protein was further characterized, as it showed the highest Probability Based Mowse Score (PBMS) of 288. A 25mer peptide, RVFHbαp was active against several clinical pathogens as demonstrated by minimum inhibitory concentration (MIC) and radial diffusion assays (RDA). The interaction of RVFHbαP with bacterial liposome membrane was assessed by calcein dye leakage assay. RVFHbαP did not show cytotoxicity against human endocervical cells (End1/E6E7) or erythrocytes. RT-PCR and immunofluorescence results revealed the expression of RVFHbαP mRNA and protein in rabbit vaginal tissue. To the best our knowledge, this is the first report describing the detection of AMPs in RVFs. In conclusion, these studies indicated that vaginal epithelial cells (VEC) derived RVFHbαP may have therapeutic potential in the management of reproductive well being of rabbits. The major reason for undertaking this study in rabbits is that, it forms an excellent in vivo model system for human's studies.

Effect of antimicrobial Peptide, nisin, on the reproductive functions of rats.

Our previous studies have demonstrated that naturally occurring peptide, Nisin possess antibacterial activity and did not interfere with rabbit vaginal mucosa. In this study, the reproductive toxicity of the Nisin in male rats was evaluated. Rats were fed orally with Nisin (10, 25, and 50 mg/kg/day) for 13 weeks. No treatment related mortality was observed. The body weight gain, food consumption and serum biochemical parameters were at par with the control group. Histomorphology of the selected reproductive (testis, epididymis, ventral prostate, and seminal vesicle) and nonreproductive (liver and kidney) tissues was observed to be normal. There was no treatment-related increase or decrease in the expression of testis-specific genes (c-Kit, GATA-1, and HILS-1) and the activity levels of epididymal α-glucosidase, ventral prostate alkaline phosphatase (AlP), liver alanine aminotransferase (AlAT) and aspartate aminotransferase (AAT). Fructose and lactic acid levels in the seminal vesicles also remained unchanged. These studies suggest that Nisin did not affect the normal physiology of these organs. In addition, no adverse effects were observed on the reproductive performance of Nisin-treated male rats and their offspring. In conclusion, the current studies support our earlier studies, which demonstrated suitability of Nisin as a safe and effective microbicide.

Interaction of contraceptive antimicrobial peptide nisin with target cell membranes: implications for use as vaginal microbicide.

BACKGROUND: Nisin, a naturally occurring antimicrobial peptide (AMP), is currently the focus of clinical trials as an intravaginal microbicide. Therefore its mechanism of interaction with various cell membranes was studied. STUDY DESIGN: Flow cytometry was used for quantitative estimation of membrane damage by nisin which was further determined by scanning electron microscopy (SEM). Affinity of nisin for different unilamellar liposome vesicles was determined spectroflurometrically and confirmed using laser scanning confocal microscopy (LSCM). RESULTS: Propidium iodide (PI) staining by flow cytometry exhibited selective membrane permeabilizing effect of nisin on sperm and bacterial membranes which correlated with ultrastructural changes. In vitro interaction of nisin with liposome model vesicles revealed significant leakage of calcein from liposomes composed of phosphatidylcholine/phosphatidylglycerol (POPC/POPG) (e.g., bacteria) and phosphatidylcholine/phosphatidylserine (POPC/POPS) (e.g., spermatozoa) as compared to phosphatidylcholine/phosphatidylethanolamine (POPC/POPE) vesicles (e.g., red blood corpuscles). LSCM results were in complete agreement with cell membrane affinity studies. CONCLUSION: This unique property of nisin can be exploited in the development of a safe and effective vaginal microbicide for the prevention of sexually transmitted infections/acquired immunodeficiency syndrome (STIs/AIDS) and unplanned pregnancies.

siRNA-mediated silencing of c-kit in mouse primary spermatogonial cells induces cell cycle arrest.

Several genes/gene products are known to act in a concert to regulate the process of spermatogenesis. One such gene is c-kit, a transmembrane tyrosine kinase receptor which plays an indispensable role in the maturation and differentiation of spermatogonial germ cells (SGCs). In the present study, siRNA approach was used to assess the role of c-kit in survival and proliferation of murine primary SGCs. The effect of different concentrations of anti-c-kit siRNA-1 and siRNA-2 (0.15, 0.315, 0.625, 1.25, 2.50, 5, and 10 nM) on c-kit protein and mRNA expression at post-transfection time (0, 6, 12, 24, 48, and 72 hours) was assessed using an array of techniques such as flow cytometry, ELISA, Western blot, and RT-PCR. Transfection of cells with anti-c-kit siRNAs (0.15-10 nM) at various time points after (0-72 hours) showed significant knockdown c-kit mRNA and protein expression. MTT, Alamar blue assays, and RT-PCR were used to investigate the effects of c-kit silencing on survival, proliferation, distribution, and apoptosis of cells. Experiments were also conducted to determine the effects of c-kit knockdown on cell cycle distribution, DNA laddering, and apoptosis. The results indicated that the transfection with anti-c-kit siRNA induces DNA fragmentation and cell cycle arrest at G(2)/M phase leading to significant reduction in cell viability and proliferation. In addition, enhanced suppression of c-kit protein in P815 cells was observed after transfection as compared to ES-E14TG2alpha cells, suggesting early onset of c-kit protein repression in P815 cells leading to prolongation in cell doubling time. In conclusion, our data provide the first evidence of specific knockdown of c-kit expression in mouse primary SGCs, which emphasizes the critical role played by c-kit in germ cell survival, proliferation, and apoptosis.

Assessment of cervicovaginal cytokine levels following exposure to microbicide Nisin gel in rabbits.

Topical microbicides is an emerging female controlled strategy for preventing the acquisition and transmission of STIs/HIV infections. Since they are intended for repeated vaginal and/or rectal use it is essential to validate their safety. Nisin, a naturally occurring contraceptive antimicrobial peptide (AMP) is currently the focus of clinical trials. The present in vitro vaginal tissue explants culture studies revealed that Nisin did not effect vaginal cell viability analyzed at 15, 30, 45 and 60min following treatment with different concentrations of Nisin gel prepared in 1% polycarbophil gel (30.3, 60.6, 121.2, 242.4 and 484.8 microM/g tissue) and SDS (0.35, 0.70, 1.4, 2.8 and 5.6 microM/g tissue) gels compared to placebo gel treated groups. The levels of various pro-inflammatory (IL-6, IL-8 and TNF-alpha,) and immuno-regulatory cytokines (IL-10 and GM-CSF) in the explant culture supernatants of the Nisin treated cells were unaffected. Repeated intravaginal application of high dose of Nisin gel (15,150 microM/day/14 days) on cervicovaginal epithelium was evaluated in rabbits and the results were compared with SDS treated (56 microM) and 1% polycarbophil gel (placebo) groups. We examined vaginal cell morphology, structural integrity of vaginal epithelium and local production of cytokines (PICs) in the cervicovaginal lavage (CVL) of Nisin treated animals and compared with placebo and SDS treated groups. The results demonstrated no treatment related abnormalities either in the vaginal cell morphology or structural abnormalities in the mucosal epithelium. There was no change in the cytokine levels in cervicovaginal lavage (CVL) compared to SDS gel treated animals indicating Nisin gel did not induce irritation and/or inflammation in the vaginal epithelium. CVL cytokine levels were in accordance with immunohistochemical (IHC) localization of cytokines and flow cytometric evaluation of CD45 immune cell population in cervicovaginal epithelium. The levels of cytokines in the CVLs appear to be sensitive indicators in identifying and/or screening out suitable candidate microbicides before they enter phase-1 trials. In conclusion, the lack of vaginal toxicity of Nisin gel means that it has clinical potential as a safe, prophylactic contraceptive in addition to its antimicrobial activities to curb sexual transmission of HIV in human.

A novel polyherbal microbicide with inhibitory effect on bacterial, fungal and viral genital pathogens.

A polyherbal cream (Basant) has been formulated using diferuloylmethane (curcumin), purified extracts of Emblica officinalis (Amla), purified saponins from Sapindus mukorossi, Aloe vera and rose water along with pharmacopoeially approved excipients and preservatives. Basant inhibits the growth of WHO strains and clinical isolates of Neisseria gonorrhoeae, including those resistant to penicillin, tetracycline, nalidixic acid and ciprofloxacin. It has pronounced inhibitory action against Candida glabrata, Candida albicans and Candida tropicalis isolated from women with vulvovaginal candidiasis, including three isolates resistant to azole drugs and amphotericin B. Basant displayed a high virucidal action against human immunodeficiency virus HIV-1NL4.3 in CEM-GFP reporter T and P4 (Hela-CD4-LTR-betaGal) cell lines with a 50% effective concentration (EC50) of 1:20000 dilution and nearly complete (98-99%) inhibition at 1:1000 dilution. It also prevented the entry of HIV-1(IIIB) virus into P4-CCR5 cells (EC50 approximately 1:2492). Two ingredients, Aloe and Amla, inhibited the transduction of human papillomavirus type 16 (HPV-16) pseudovirus in HeLa cells at concentrations far below those that are cytotoxic and those used in the formulation. Basant was found to be totally safe according to pre-clinical toxicology carried out on rabbit vagina after application for 7 consecutive days or twice daily for 3 weeks. Basant has the potential of regressing vulvovaginal candidiasis and preventing N. gonorrhoeae, HIV and HPV infections.

Identification of genes regulated by an interaction between alphavbeta3 integrin and vitronectin in murine decidua.

The delicate balance between embryo invasion and suppression of maternal immune rejection requires a fully functional decidua in species with haemochorial placenta. Our understanding of the decidual function is very limited due to the molecular and cellular complexity involved in decidualisation. The cell adhesion molecule alpha(v)beta(3) integrin and its ligand vitronectin are upregulated in the mouse decidua during mid-pregnancy. The implications of interactions between alpha(v)beta(3) and vitronectin in regulating decidual function are not known. In the present study, interactions between alpha(v)beta(3) and vitronectin in the decidual cells of the mouse were blocked in vitro and effects on cell fate were evaluated by studying the differentially regulated genes by cDNA array and real-time polymerase chain reaction (PCR). The results indicate that expression of various genes involved in apoptotic and cell cycle pathways, as well as cytokine receptors, was deranged. Signalling through alpha(v)beta(3) seems to be important to maintain a balance between cell proliferation and apoptosis, along with the modulation of inflammatory responses of decidual cells.

Toll-like receptors and cytokines as surrogate biomarkers for evaluating vaginal immune response following microbicide administration.

Topical microbicides are intended for frequent use by women in reproductive age. Hence, it is essential to evaluate their impact on mucosal immune function in the vagina. In the present study, we evaluated nisin, a naturally occurring antimicrobial peptide (AMP), for its efficacy as an intravaginal microbicide. Its effect on the vaginal immune function was determined by localizing Toll-like receptors (TLRs-3, 9) and cytokines (IL-4, 6 , 10 and TNF-alpha) in the rabbit cervicovaginal epithelium following intravaginal administration of high dose of nisin gel for 14 consecutive days. The results revealed no alteration in the expression of TLRs and cytokines at both protein and mRNA levels. However, in SDS gel-treated group, the levels were significantly upregulated with the induction of NF-kappaB signalling cascade. Thus, TLRs and cytokines appear as sensitive indicators for screening immunotoxic potential of candidate microbicides.

Differential regulation of gene expression in mouse spermatogonial cells after blocking c-kit-SCF interaction with RNAi.

c-Kit, the gene product of the W locus is a receptor tyrosine kinase that regulates the survival, growth and differentiation of spermatogonial cells (SGCs). Stem cell factor (SCF), the gene product of the steel (Sl) locus is the ligand for c-kit. Normal function of SGCs requires cross-talk between c-kit and SCF through which the receptor-ligand pair regulates the functions of SGCs. The implications of cross-talk between c-kit and SCF in regulating SGC function remains unclear due to the molecular complexity of this interaction. In the present study, we analyzed the interactions between c-kit and SCF in mouse primary SGCs after blocking the c-kit expression by c-kit siRNA and its effect on cell fate were determined using cDNA Expression Array and Real-time PCR. Immunofluorescence (IF) and western blot studies revealed that c-kit protein was detected in SGCs and knocked down to undetectable levels at 24 hr post transfection with 10 nM concentration of c-kit siRNA. We further demonstrated that expression of various genes involved in cell signaling, cell differentiation, apoptosis and cell cycle pathways was altered. SGC functions are affected by SCF signaling through c-kit receptor and this signaling appears to be important to maintain balance between cell proliferation and apoptosis along with the modulation of inflammatory responses of SGCs. To the best of our knowledge, this is the first report that identifies the putative molecular pathways in murine SGCs in response to specific blocking of c-kit-SCF interactions by siRNA. In conclusion, the present study may provide useful insights into siRNA function and hopefully aid in understanding the involvement of c-kit in the early events of SGC activities and spermatogenesis in mice.

Evaluation of developmental toxicity of microbicide Nisin in rats.

In the present study, we have investigated the developmental toxicity of a naturally occurring peptide, Nisin in rats in order to determine its suitability as a safe vaginal microbicide. Our earlier studies indicated that, Nisin is a dual function microbicide having contraceptive and antibacterial activities. However, as part of the safety evaluation of any vaginal microbicide, it is essential to determine its teratogenic potential in a suitable animal model before it is found suitable to enter clinical trials. Sixty pregnant rats allocated into four groups were orally administered with 10, 25 and 50 mg Nisin/kg/day from day 6 to day 15 of gestation. Individual food/water consumption and body weight changes were measured daily. Nisin did not cause maternal mortality nor did the treated animals show any clinical signs of toxicity when compared to the control animals. There were no biologically significant differences in maternal liver, kidney, thymus, ovary, gravid and empty uterine weights. Mean number of corpora lutea and implantation sites also did not differ in the treated groups when compared to their respective controls. All the fetuses were weighed, sexed and examined carefully for externally visible malformations. No gross external fetal alterations were observed at any dose tested. When stained by the double staining method, no skeletal malformations and visceral defects were observed in the fetuses. The growth and reproductive performance of the F1 progeny was also unaffected. In conclusion, Nisin shows unique clinical potential as a safe prophylactic microbicide to curb the transmission of STIs/HIV and unintended pregnancies.

Expression pattern of integrins and their ligands in mouse feto-maternal tissues during pregnancy.

The role of integrins, the cell-surface glycoproteins involved in various cellular functions, is well documented. However, information about their role and expression profile during pregnancy is still scant. In the present study, the expression of the integrin subunits beta(3), alpha(6) and alpha(5), along with their ligands vitronectin, osteopontin, laminin and fibronectin, was investigated in mouse uterus during different stages of pregnancy, namely 6.5, 8.5 and 13.5 days post coitus (d.p.c.) by immunohistochemical localisation. Integrins beta(3), alpha(6) and alpha(5) and the extracellular matrix molecules vitronectin and osteopontin exhibited dynamic spatiotemporal changes in their expression pattern in gestational endometrium, whereas fibronectin and laminin demonstrated more-or-less ubiquitous expression. The inter-implantation sites showed localisation of most of these molecules predominantly in the luminal epithelium, whereas their expression was negligible in the stroma. The present study explores the possible role and relevance of the spatiotemporal expression of integrins and their ligands in endometrial/decidual function and the maintenance of pregnancy.