Association between vaginal microbiota and vaginal inflammatory immune markers in postmenopausal women.

OBJECTIVE: In premenopausal individuals, vaginal microbiota diversity and lack of Lactobacillus dominance are associated with greater mucosal inflammation, which is linked to a higher risk of cervical dysplasia and infections. It is not known if the association between the vaginal microbiota and inflammation is present after menopause, when the vaginal microbiota is generally higher-diversity and fewer people have Lactobacillus dominance. METHODS: This is a post hoc analysis of a subset of postmenopausal individuals enrolled in a randomized trial for treatment of moderate-severe vulvovaginal discomfort that compared vaginal moisturizer, estradiol, or placebo. Vaginal fluid samples from 0, 4, and 12 weeks were characterized using 16S rRNA gene sequencing (microbiota) and MesoScale Discovery (vaginal fluid immune markers: IL-1b, IL-1a, IL-2, IL-6, IL-18, IL-10, IL-9, IL-13, IL-8, IP10, MIP1a, MIP1b, MIP3a). Global associations between cytokines and microbiota (assessed by relative abundance of individual taxa and Shannon index for alpha, or community, diversity) were explored, adjusting for treatment arm, using linear mixed models, principal component analysis, and Generalized Linear Mixed Model + Microbiome Regression-based Kernel Association Test (GLMM-MiRKAT). RESULTS: A total of 119 individuals with mean age of 61 years were included. At baseline, 29.5% of participants had a Lactobacillus-dominant vaginal microbiota. Across all timepoints, alpha diversity (Shannon index, P = 0.003) was highly associated with immune markers. Individual markers that were associated with Lactobacillus dominance were similar to those observed in premenopausal people: IL-10, IL-1b, IL-6, IL-8 (false discovery rate [FDR] < 0.01), IL-13 (FDR = 0.02), and IL-2 (FDR = 0.09). Over 12 weeks, change in alpha diversity was associated with change in cytokine concentration (Shannon, P = 0.018), with decreased proinflammatory cytokine concentrations observed with decreasing alpha diversity. CONCLUSIONS: In this cohort of postmenopausal individuals, Lactobacillus dominance and lower alpha diversity were associated with lower concentrations of inflammatory immune markers, as has been reported in premenopausal people. This suggests that after menopause lactobacilli continue to have beneficial effects on vaginal immune homeostasis, despite lower prevalence.

Effect of Metronidazole on Concentrations of Vaginal Bacteria Associated with Risk of HIV Acquisition.

Several bacterial vaginosis (BV)-associated bacteria have been associated with elevated risk of HIV acquisition, however susceptibility of these bacteria to antibiotics is poorly understood. Vaginal samples were collected from 22 persons daily for two weeks following BV diagnosis. Metronidazole treatment was prescribed for 5-7 days. Changes in bacterial concentrations were measured with taxon-specific 16S rRNA gene quantitative PCR (qPCR) assays. A culture-based antimicrobial assay confirmed presence of antibiotics in vaginal swab samples. Bacterial DNA concentrations decreased during antibiotic administration for all thirteen bacterial taxa tested. Comparison of bacterial DNA concentrations in samples before administration of antibiotics to samples taken on the last day of antimicrobial assay-confirmed antibiotic presence showed a 2.3-4.5 log10-fold decrease across all taxa. Concentrations were frequently reduced to the qPCR assay's limit of detection, suggesting eradication of bacteria. Mean clearance time varied across taxa (1.2-8.6 days), with several bacteria (e.g., Gemella asaccharolytica, Sneathia spp., Eggerthella-like sp.) taking >7 days to suppress. Metronidazole reduces quantities of bacterial taxa associated with increased HIV acquisition risk. Eradication of high-risk vaginal bacteria using metronidazole is one promising avenue for reducing HIV acquisition risk. A 5-7-day treatment course may not be sufficient to suppress all bacteria.

Identification of fallopian tube microbiota and its association with ovarian cancer.

Investigating the human fallopian tube (FT) microbiota has significant implications for understanding the pathogenesis of ovarian cancer (OC). In this large prospective study, we collected swabs intraoperatively from the FT and other surgical sites as controls to profile the microbiota in the FT and to assess its relationship with OC. Eighty-one OC and 106 non-cancer patients were enrolled and 1001 swabs were processed for 16S rRNA gene PCR and sequencing. We identified 84 bacterial species that may represent the FT microbiota and found a clear shift in the microbiota of the OC patients when compared to the non-cancer patients. Of the top 20 species that were most prevalent in the FT of OC patients, 60% were bacteria that predominantly reside in the gastrointestinal tract, while 30% normally reside in the mouth. Serous carcinoma had higher prevalence of almost all 84 FT bacterial species compared to the other OC subtypes. The clear shift in the FT microbiota in OC patients establishes the scientific foundation for future investigation into the role of these bacteria in the pathogenesis of OC.

The Relationship Between Insertive Oral and Anal Sex and Select Measures of the Composition of the Urethral Microbiota Among Men Who Have Sex With Men.

BACKGROUND: Sexual behavior may influence the composition of the male urethral microbiota, but this hypothesis has not been tested in longitudinal studies of men who have sex with men (MSM). METHODS: From December 2014 to July 2018, we enrolled MSM with nongonococcal urethritis (NGU) attending a sexual health clinic. Men attended 5 in-clinic visits at 3-week intervals, collected weekly urine specimens at home, and reported daily antibiotics and sexual activity on weekly diaries. We applied broad-range 16S rRNA gene sequencing to urine. We used generalized estimating equations to estimate the association between urethral sexual exposures in the prior 7 days (insertive oral sex [IOS] only, condomless insertive anal intercourse [CIAI] only, IOS with CIAI [IOS + CIAI], or none) and Shannon index, number of species (observed, oral indicator, and rectal indicator), and specific taxa, adjusting for recent antibiotics, age, race/ethnicity, HIV, and preexposure prophylaxis. RESULTS: Ninety-six of 108 MSM with NGU attended ≥1 follow-up visit. They contributed 1140 person-weeks of behavioral data and 1006 urine specimens. Compared with those with no urethral sexual exposures, those with IOS only had higher Shannon index ( P = 0.03 ) but similar number of species and presence of specific taxa considered, adjusting for confounders; the exception was an association with Haemophilus parainfluenzae . CIAI only was not associated with measured aspects of the urethral microbiota. IOS + CIAI was only associated with presence of H. parainfluenzae and Haemophilus . CONCLUSIONS: Among MSM after NGU, IOS and CIAI did not seem to have a substantial influence on measured aspects of the composition of the urethral microbiota.

Gardnerella Species and their Association with Bacterial Vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a condition marked by high vaginal bacterial diversity. Gardnerella vaginalis has been implicated in BV but is also detected in healthy women. The Gardnerella genus has been expanded to encompass six validly named species and several genomospecies. We hypothesized that particular Gardnerella species may be more associated with BV. METHODS: Quantitative PCR assays were developed targeting the cpn60 gene of species groups including G. vaginalis, G. piotii/pickettii, G. swidsinskii/greenwoodii and G. leopoldii. These assays were applied to vaginal swabs from individuals with (n=101) and without BV (n=150) attending a sexual health clinic in Seattle, Washington. Weekly swabs were collected from 42 participants for up to 12 weeks. RESULTS: Concentrations and prevalence of each Gardnerella species group were significantly higher in participants with BV. 91.1% of BV positive participants had three or more Gardnerella species groups detected compared to 32.0% of BV negative participants (p<0.0001). BV negative participants with three or more species groups detected were more likely to develop BV within 100 days versus those with fewer (60.5% vs 3.7%, p<0.0001). CONCLUSIONS: These results suggest that BV reflects a state of high Gardnerella species diversity. No Gardnerella species group was a specific marker for BV.

IgA coating of vaginal bacteria is reduced in the setting of bacterial vaginosis (BV) and preferentially targets BV-associated species.

Immunoglobulin (Ig) bacterial coating has been described in the gastrointestinal tract and linked to inflammatory bowel disease; however, little is known about Ig coating of vaginal bacteria and whether it plays a role in vaginal health including bacterial vaginosis (BV). We examined Ig coating in 18 women with symptomatic BV followed longitudinally before, 1 week, and 1 month after oral metronidazole treatment. Immunoglobulin A (IgA) and/or immunoglobulin G (IgG) coating of vaginal bacteria was assessed by flow cytometry, and Ig coated and uncoated bacteria were sorted and characterized using 16S rRNA sequencing. Despite higher levels of IgG compared to IgA in cervicovaginal fluid, the predominant Ig coating the bacteria was IgA. The majority of bacteria were uncoated at all visits, but IgA coating significantly increased after treatment for BV. Despite similar amounts of uncoated and IgA coated majority taxa ( >1% total) across all visits, there was preferential IgA coating of minority taxa (0.2%-1% total) associated with BV including Sneathia, several Prevotella species, and others. At the time of BV, we identified a principal component (PC) driven by proinflammatory mediators that correlated positively with an uncoated BV-associated bacterial community and negatively with an IgA coated protective Lactobacillus bacterial community. The preferential coating of BV-associated species, increase in coating following metronidazole treatment, and positive correlation between uncoated BV-associated species and inflammation suggest that coating may represent a host mechanism designed to limit bacterial diversity and reduce inflammatory responses. Elucidating the role of Ig coating in vaginal mucosal immunity may promote new strategies to prevent recurrent BV.

Increasing the Motivation to Train Through Haptic Social Interaction - Pilot study.

Motivation is crucial in stroke rehabilitation, as it enhances patient engagement, adherence, and recovery. Robots can be employed to improve motivation through multiplayer rehabilitation games, which allow patients to collaborate and interact in a virtual environment through multimodal sensory cues. This social interaction can provide social support and increase motivation, resulting in better therapy engagement. A hand rehabilitation robot (PLUTO) was used to investigate the potential of social interaction to implement haptic multiplayer games. Twelve unimpaired participants (6 dyads) played in solo, collaborative, and competitive game modes. Surprisingly, no difference was found in self-reported engagement, tension, or competence between solo and multiplayer games. However, the IMI scale indicated that engagement for multiplayer games was rated higher than for solo games. The collaborative game was preferred by 10 out of 12 participants, highlighting its potential for promoting behavioural involvement and engagement. This study indicates that using PLUTO with multiplayer game modes can enhance therapy engagement. This can potentially improve rehabilitation outcomes if translated to the patient population.

Amygdalobacter indicium gen. nov., sp. nov., and Amygdalobacter nucleatus sp. nov., gen. nov.: novel bacteria from the family Oscillospiraceae isolated from the female genital tract.

Four obligately anaerobic Gram-positive bacteria representing one novel genus and two novel species were isolated from the female genital tract. Both novel species, designated UPII 610-JT and KA00274T, and an additional isolate of each species were characterized utilizing biochemical, genotypic and phylogenetic analyses. All strains were non-motile and non-spore forming, asaccharolytic, non-cellulolytic and indole-negative coccobacilli. Fatty acid methyl ester analysis for UPII 610-JT and KA00274T and additional isolates revealed C16 : 0, C18 : 0, C18:1ω9c and C18:2ω6,9c to be the major fatty acids for both species. UPII 610-JT had a 16S rRNA gene sequence similarity of 99.4 % to an uncultured clone sequence (AY724740) designated as Bacterial Vaginosis Associated Bacterium 2 (BVAB2). KA00274T had a 16S rRNA gene sequence similarity of 96.5 % to UPII 610-JT. Whole genomic DNA mol% G+C content was 42.2 and 39.3 % for UPII 610-JT and KA00274T, respectively. Phylogenetic analyses indicate these isolates represent a novel genus and two novel species within the Oscillospiraceae family. We propose the names Amygdalobacter indicium gen. nov., sp. nov., for UPII 610-JT representing the type strain of this species (=DSM 112989T, =ATCC TSD-274T) and Amygdalobacter nucleatus gen. nov., sp. nov., for KA00274T representing the type strain of this species (=DSM 112988T, =ATCC TSD-275T).

Identification of fallopian tube microbiota and its association with ovarian cancer: a prospective study of intraoperative swab collections from 187 patients.

Investigating the human fallopian tube (FT) microbiota has significant implications for understanding the pathogenesis of ovarian cancer (OC). In this large prospective study, we collected swabs intraoperatively from the FT and other surgical sites as controls to profile the microbiota in the FT and to assess its relationship with OC. 81 OC and 106 non-cancer patients were enrolled and 1001 swabs were processed for 16S rRNA gene PCR and sequencing. We identified 84 bacterial species that may represent the FT microbiota and found a clear shift in the microbiota of the OC patients when compared to the non-cancer patients. Of the top 20 species that were most prevalent in the FT of OC patients, 60% were bacteria that predominantly reside in the gastrointestinal tract, while 30% normally reside in the mouth. Serous carcinoma had higher prevalence of almost all 84 FT bacterial species compared to the other OC subtypes. The clear shift in the FT microbiota in OC patients establishes the scientific foundation for future investigation into the role of these bacteria in the pathogenesis of ovarian cancer. .

Intestinal microbiota controls graft-versus-host disease independent of donor-host genetic disparity.

Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic stem cell transplantation (SCT), and severe intestinal manifestation is the major cause of early mortality. Intestinal microbiota control MHC class II (MHC-II) expression by ileal intestinal epithelial cells (IECs) that promote GVHD. Here, we demonstrated that genetically identical mice of differing vendor origins had markedly different intestinal microbiota and ileal MHC-II expression, resulting in discordant GVHD severity. We utilized cohousing and antibiotic treatment to characterize the bacterial taxa positively and negatively associated with MHC-II expression. A large proportion of bacterial MHC-II inducers were vancomycin sensitive, and peri-transplant oral vancomycin administration attenuated CD4+ T cell-mediated GVHD. We identified a similar relationship between pre-transplant microbes, HLA class II expression, and both GVHD and mortality in a large clinical SCT cohort. These data highlight therapeutically tractable mechanisms by which pre-transplant microbial taxa contribute to GVHD independently of genetic disparity.

Optimizing metaproteomics database construction: lessons from a study of the vaginal microbiome.

Metaproteomics, a method for untargeted, high-throughput identification of proteins in complex samples, provides functional information about microbial communities and can tie functions to specific taxa. Metaproteomics often generates less data than other omics techniques, but analytical workflows can be improved to increase usable data in metaproteomic outputs. Identification of peptides in the metaproteomic analysis is performed by comparing mass spectra of sample peptides to a reference database of protein sequences. Although these protein databases are an integral part of the metaproteomic analysis, few studies have explored how database composition impacts peptide identification. Here, we used cervicovaginal lavage (CVL) samples from a study of bacterial vaginosis (BV) to compare the performance of databases built using six different strategies. We evaluated broad versus sample-matched databases, as well as databases populated with proteins translated from metagenomic sequencing of the same samples versus sequences from public repositories. Smaller sample-matched databases performed significantly better, driven by the statistical constraints on large databases. Additionally, large databases attributed up to 34% of significant bacterial hits to taxa absent from the sample, as determined orthogonally by 16S rRNA gene sequencing. We also tested a set of hybrid databases which included bacterial proteins from NCBI RefSeq and translated bacterial genes from the samples. These hybrid databases had the best overall performance, identifying 1,068 unique human and 1,418 unique bacterial proteins, ~30% more than a database populated with proteins from typical vaginal bacteria and fungi. Our findings can help guide the optimal identification of proteins while maintaining statistical power for reaching biological conclusions. IMPORTANCE Metaproteomic analysis can provide valuable insights into the functions of microbial and cellular communities by identifying a broad, untargeted set of proteins. The databases used in the analysis of metaproteomic data influence results by defining what proteins can be identified. Moreover, the size of the database impacts the number of identifications after accounting for false discovery rates (FDRs). Few studies have tested the performance of different strategies for building a protein database to identify proteins from metaproteomic data and those that have largely focused on highly diverse microbial communities. We tested a range of databases on CVL samples and found that a hybrid sample-matched approach, using publicly available proteins from organisms present in the samples, as well as proteins translated from metagenomic sequencing of the samples, had the best performance. However, our results also suggest that public sequence databases will continue to improve as more bacterial genomes are published.

Changes in Vaginal Bacteria and Inflammatory Mediators From Periconception Through the Early Postpartum Period in a Cohort of Kenyan Women Without HIV.

BACKGROUND: Women's increased risk of HIV acquisition during pregnancy and postpartum may be mediated by changes in vaginal microbiota and/or cytokines. METHODS: A cohort of 80 Kenyan women who were HIV-1 seronegative contributed 409 vaginal samples at 6 pregnancy time points: periconception, positive pregnancy test result, first trimester, second trimester, third trimester, and postpartum. Concentrations of vaginal bacteria linked with HIV risk and Lactobacillus spp were measured using quantitative polymerase chain reaction. Cytokines were measured by immunoassay. RESULTS: Based on Tobit regression, later pregnancy time points were associated with lower concentrations of Sneathia spp (P = .01), Eggerthella sp type 1 (P = .002), and Parvimonas sp type 2 (P = .02) and higher concentrations of Lactobacillus iners (P < .001), Lactobacillus crispatus (P < .001), Lactobacillus vaginalis (P < .001), interleukin 6 (P < .001), TNF (P = .004), C-X-C motif chemokine ligand 10 (CXCL10; P < .001), C-C motif ligand 3 (P = .009), C-C motif ligand 4 (P < .001), C-C motif ligand 5 (P = .002), interleukin 1ß (P = .02), and interleukin 8 (P = .002). Most cervicovaginal cytokines and vaginal bacteria clustered separately in principal component analysis, except for CXCL10, which did not group with either cytokines or bacteria. The shift toward a Lactobacillus-dominated microbiota during pregnancy mediated the relationship between pregnancy time point and CXCL10. CONCLUSIONS: Increases in proinflammatory cytokines, but not vaginal bacterial taxa linked with higher HIV risk, could provide an explanation for increased HIV susceptibility during pregnancy and postpartum.

Unwinding the mechanism of macrophage repolarization potential of Oceanimonas sp. BPMS22-derived protein protease inhibitor through Toll-like receptor 4 against experimental visceral leishmaniasis.

The Oceanimonas sp. BPMS22-derived protein protease inhibitor (PPI) has been proven to shift macrophages towards an inflammatory state and reduce Leishmania donovani infection in vitro and in vivo. The current study explored and validated the mechanistic aspects of the PPI and Toll-like receptor (TLR) interaction. The PPI exhibited the upregulation of TLR2, TLR4, and TLR6 during treatment which was proven to orchestrate parasite clearance effectively. An in silico study confirmed the high interaction with TLR4 and PPI. Immune blotting confirmed the significant upregulation of TLR4 in macrophages irrespective of L. donovani infection. Pharmacological inhibition and immune blot study confirmed the involvement of the PPI in TLR4-mediated phosphorylation of p38 MAPK and dephosphorylation of ERK1/2, repolarizing to pro-inflammatory macrophage state against experimental visceral leishmaniasis. In addition, in TLR4 knockdown condition, PPI treatment failed to diminish M2 phenotypical markers (CD68, Fizz1, Ym1, CD206, and MSR-2) and anti-inflammatory cytokines (IL-4, IL-10, and TGF-ß). Simultaneously, the PPI failed to upregulate the M1 phenotypical markers and pro-inflammatory cytokines (IL-1ß, IL-6, IL-12, and IFN-γ) (p < 0.001) during the TLR4 knockdown condition. In the absence of TLR4, the PPI also failed to reduce the parasite load and T-cell proliferation and impaired the delayed-type hypersensitivity response. The absence of pro-inflammatory cytokines was observed during a co-culture study with PPI-treated macrophages (in the TLR4 knockdown condition) with day 10 T-cell obtained from L. donovani-infected mice. This study supports the immunotherapeutic potential of the PPI as it interacted with TLR4 and promoted macrophage repolarization (M2-M1) to restrict the L. donovani parasite burden and helps in the mounting immune response against experimental visceral leishmaniasis.

Genital Herpes Simplex Virus Type 2 Suppression With Valacyclovir Is Not Associated With Changes in Nugent Score or Absolute Abundance of Key Vaginal Bacteria.

Background: In women, genital herpes simplex virus type 2 (HSV-2) infection is associated with increased risk for recurrent bacterial vaginosis (BV), but causal relationships are unclear. Methods: Women with a self-reported history of BV and HSV-2 seropositivity self-collected vaginal and anogenital swabs for 2 nonconsecutive 28-day periods, in the absence or presence of valacyclovir suppressive therapy (500 mg daily). HSV polymerase chain reaction was performed on anogenital swabs; vaginal swabs were used for assessment of BV by Nugent score and quantification of vaginal microbiota. Days with BV, defined by Nugent score ≥7, were compared during the observational period and valacyclovir treatment. Results: Forty-one women collected swabs for a median of 28 days (range, 20-32 days) each study period. The HSV-2 shedding rate decreased from 109 of 1126 days (9.7%) presuppression to 6 of 1125 days (0.05%) during valacyclovir (rate ratio [RR], 0.06 [95% confidence interval {CI}, .02-.13]). BV occurred on 343 of 1103 days (31.1%) during observation and 302 of 1091 days (27.7%) during valacyclovir (RR, 0.90 [95% CI, .68-1.20]). The median per-person Nugent score was 3.8 during observation and 4.0 during valacyclovir. Average log10 concentrations of vaginal bacterial species did not change significantly during valacyclovir treatment. Conclusions: Short-term HSV-2 suppression with valacyclovir did not significantly affect the Nugent score or the vaginal microbiome despite potent suppression of HSV-2 shedding.

Next generation sequencing to examine associations between vaginal washing and vaginal microbiota: A cohort study.

BACKGROUND: The association between vaginal washing and HIV risk may be mediated by vaginal washing-associated changes in vaginal microbiota. METHODS: Data from a cohort of HIV-negative US and Kenyan women enrolled in the Preventing Vaginal Infections trial were analyzed. Vaginal fluid samples and vaginal washing data were collected every 2 months for 12 months. Bacterial relative abundances were measured by broad-range 16S rRNA gene polymerase chain reaction with next generation sequencing. Generalized estimating equations were used to evaluate the association between vaginal washing and i) the Shannon Diversity Index (SDI); and ii) mean change in percent bacterial relative abundances, with application of a 10% false discovery rate (FDR). RESULTS: Participants (N = 111) contributed 93/630 (14.8%) vaginal washing visits. Mean SDI was 0.74 points higher (95% CI 0.35, 1.14; p < 0.001) at washing visits among US participants (N = 26). Vaginal washing was not associated with SDI in Kenyan participants (N = 85). There were no associations between vaginal washing and vaginal bacterial relative abundances after applying the FDR. CONCLUSIONS: The discordant results in Kenyan versus US women suggests the link between vaginal washing and sub-optimal vaginal microbiota may be context specific. Vaginal microbial shifts may not fully explain the association between vaginal washing and HIV acquisition.

Economic Evaluation of a Point-of-Care Test for Bacterial Vaginosis Among Women With Vaginal Symptoms.

BACKGROUND: There is an unmet need for a clinical diagnostic technology to detect bacterial vaginosis (BV) rapidly and accurately. Novel point-of-care (POC) tests have the potential to fulfill this gap. Our objective was to determine the cost-effectiveness of a hypothetical clinician-administered POC test for diagnosing BV in the United States. METHODS: We developed a state-transition microsimulation model to evaluate the cost-effectiveness of using the POC test versus usual care among women of reproductive age with vaginal symptoms. We adopted a healthcare sector perspective that included relevant healthcare costs and a societal perspective that further incorporated productivity costs. Model parameters were empirically estimated based on commercial insurance claims data or derived from published literature. The primary model outcome was incremental cost-effectiveness ratio. We started with analyzing a hypothetical POC test with a sensitivity and specificity of 0.9 and a cost of $40, followed by extensive sensitivity analyses. RESULTS: Using the hypothetical POC test to diagnose BV increased costs by $16 and quality-adjusted life-years by 0.0005 per person compared with the usual care, leading to an incremental cost-effectiveness ratio of $31,108 per quality-adjusted life-year gained. When also capturing the productivity costs, the POC test resulted in an average cost savings of $57. The sensitivity analyses showed that the POC test's sensitivity was more influential on its cost-effectiveness than specificity. CONCLUSIONS: Using the POC test to diagnose BV is likely to be cost-effective relative to usual care, especially with a high sensitivity or a substantial positive effect on productivity.

Partial characterization of purified glycoprotein from nutshell of Arachis hypogea L. towards macrophage activation and leishmaniacidal activity.

Arachis hypogea L. protein fraction-2 (AHP-F2) from the Peanut shell was extracted and characterized and its potent immunomodulatory and anti-leishmanial role was determined in this present study. AHP-F2 was found to be a glycoprotein as the presence of carbohydrates were confirmed by the analysis of high-performance liquid chromatography (HPLC) yielded glucose, galactose, mannose, and xylose. AHP-F2 molecular mass was found to be ∼28 kDa as indicated in MALDI-TOF and peptide mass fingerprinting analysis followed by Mascot search. The peptide matches revealed the similarity of the mannose/glucose binding lectin with 71.07% in the BLAST analysis. After that, the 3D structure of the AHP-F2 model was designed and validated by the Ramachandran plot. The immunomodulatory role of AHP-F2 was established in murine peritoneal macrophages as induction of nitric oxide (NO), and stimulation of proinflammatory cytokines (IL-12 and IFN-γ) in a dose-dependent manner was observed. Interestingly, it was also found that AHP-F2 has interacted with the innate immune receptor, toll-like receptors (TLRs) as established in molecular docking as well as mRNA expression. The anti-leishmanial potential of AHP-F2 was revealed with a prominent inhibition of amastigote growth within the murine macrophages with prompt induction of nitrite release. Altogether, the isolated AHP-F2 from Arachis hypogea L. has strong immunomodulatory and anti-leishmanial potential which may disclose a new path to treat leishmaniasis.

Bacterial vaginosis-associated bacteria in cisgender men who have sex with women: prevalence, association with non-gonococcal urethritis and natural history.

OBJECTIVES: Bacterial vaginosis-associated bacterium 2 (BVAB2), Mageeibacillus indolicus and Sneathia spp are highly predictive of bacterial vaginosis (BV) in cisgender women. They have been associated with non-gonococcal urethritis (NGU) in cisgender men in some but not all populations. We evaluated this association in a cross-sectional study of cisgender men who have sex with women only (MSW). METHODS: MSW without gonorrhoea attending a sexual health clinic (SHC) from 2014 to 2018 completed a computer-assisted self-interview, clinical interview and examination. NGU was defined as ≥5 polymorphonuclear leucocytes/high-power field in urethral exudates plus either urethral symptoms or visible discharge. Urine was tested for Chlamydia trachomatis and Mycoplasma genitalium using Aptima (Hologic) and for BVAB2, M. indolicus, Sneathia spp, Trichomonas vaginalis, Ureaplasma urealyticum, Haemophilus influenzae, herpes simplex virus and adenovirus using quantitative PCR. RESULTS: Of 317 MSW age 17-71, 67 (21.1%) had Sneathia spp, 36 (11.4%) had BVAB2, and 17 (5.4%) had M. indolicus at enrolment. Having ≥3 partners in the past 2 months was the only characteristic that was more common among MSW with than those without these bacteria (BVAB2: 47% vs 23%, M. indolicus: 53% vs 24%, Sneathia spp: 42% vs 22%; p≤0.03 for all). One-hundred seventeen men (37%) were diagnosed with NGU at enrolment. There was no significant association of BVAB2, M. indolicus or Sneathia spp with NGU (adjusted OR=0.59, 95% CI 0.14 to 2.43; aOR=3.40, 95% CI 0.68 to 17.06; aOR=0.46, 95% CI 0.16 to 1.27). Of 109 MSW with monthly samples, 34 (31.2%) had one of the bacteria at one or more follow-up visits, 22 of which were co-colonised with >1. Median persistence over 6 months did not differ significantly (BVAB2=30.5 days, IQR=28-87; M. indolicus=87 days, IQR=60-126; Sneathia spp=70 days, IQR=30-135; p≥0.20 for each comparison). CONCLUSIONS: Neither BVAB2, M. indolicus nor Sneathia spp were associated with increased risk of prevalent NGU in MSW attending an SHC.

Oceanimonas sp. BPMS22-derived protein protease inhibitor induces anti-leishmanial immune responses through macrophage M2 to M1 repolarization.

The present study aimed to validate the potential of a novel serine protein protease inhibitor (PPI), purified from marine Oceanimonas sp. BPMS22, induced M2 to M1 repolarization of the macrophages to treat visceral leishmaniasis (VL). Peptide mass fingerprint of the purified trypsin digested PPI peptide was obtained using matrix-assisted laser desorption ionization-time of flight combined with tandem mass spectrometry (MALDI-TOF MS/MS) and the sequence was used to construct a 3D protein model by homology modelling. The IC50 of PPI were 25.28 ± 1.675 µg/mL and 0.415 ± 0.015 µg/mL against promastigotes and intracellular amastigotes, respectively, indicating the host-directed therapy using PPI. The PPI enhanced the effector molecule i.e., nitric oxide (NO), and dampened the arginase activity in a dose-dependent manner. In vitro studies revealed that the BPMS22-derived PPI significantly (p < 0.05) decreased the mRNA expressions of M2 markers (FIZZ-1, YM-1, CD206, Arg-1) and increased the mRNA expressions of M1 markers (iNOS, IL-1ß, IL-12) in rIL-4 + rIL-10 induced M2 macrophages. Interestingly, the BPMS22-derived PPI also significantly (p < 0.05) decreased the FIZZ-1, YM-1, CD206, and Arg-1; significantly (p < 0.05) increased iNOS, IL-12, and IFN-γ mRNA expression in L. donovani -infected murine macrophages, alongside the decreased parasite load in it. Hence, PPI has the potential to repolarize the cytokines (rIL-4 + rIL-10) pre-stimulated and L. donovani-infected M2 macrophages to M1 phenotype in vitro. A decrease in parasite burden after treatment with PPI indicated the acceleration of the parasite killing by enhancing the macrophage effector functions. Further, in vivo PPI treatment reduced hepatic and splenic Leishman donovan units (LDU) up to 93.34 % and 87.63 %, respectively. This was followed by a surge in pro-inflammatory cytokines and dampening anti-inflammatory cytokines (p < 0.01), which exhibited anti-VL immunity. These observations might open new perspectives on PPI in macrophage repolarization to treat VL.

Associations Between Vaginal Bacteria and Bacterial Vaginosis Signs and Symptoms: A Comparative Study of Kenyan and American Women.

Background: Bacterial colonization and associations with bacterial vaginosis (BV) signs and symptoms (Amsel criteria) may vary between populations. We assessed relationships between vaginal bacteria and Amsel criteria among two populations. Methods: Kenyan participants from the placebo arm of the Preventing Vaginal Infections (PVI) trial and participants from a Seattle-based cross-sectional BV study were included. Amsel criteria were recorded at study visits, and the vaginal microbiota was characterized using 16S rRNA gene sequencing. Logistic regression models, accounting for repeat visits as appropriate, were fit to evaluate associations between bacterial relative abundance and each Amsel criterion. Results: Among 84 PVI participants (496 observations) and 220 Seattle participants, the prevalence of amine odor was 25% and 40%, clue cells 16% and 37%, vaginal discharge 10% and 52%, elevated vaginal pH 69% and 67%, and BV 13% and 44%, respectively. BV-associated bacterium 1 (BVAB1) was positively associated with all Amsel criteria in both populations. Eggerthella type 1, Fannyhessea (Atopobium) vaginae, Gardnerella spp., Sneathia amnii, and Sneathia sanguinegens were positively associated with all Amsel criteria in the Seattle study, and all but discharge in the PVI trial. Conclusions: Core vaginal bacteria are consistently associated with BV signs and symptoms across two distinct populations of women.