Soluble E-cadherin: A marker of genital epithelial disruption.

PROBLEM: The genital epithelial barrier is a crucial first line of defence against HIV, and epithelial disruption may enhance HIV susceptibility. Assessment of genital epithelial integrity requires biopsies, but their collection is not practical in many research settings. A validated biomarker of genital epithelial barrier integrity would therefore be useful. The purpose of this study was to evaluate soluble E-cadherin (sE-cad) as a marker of genital epithelial disruption. METHOD OF STUDY: Using in vitro models of endocervical and foreskin epithelial cells, we assessed changes in sE-cad, IL-6, IL-1ß, and IL-1α levels following mechanical disruption. We also assessed changes in sE-cad levels in vivo in cervicovaginal secretions after epithelial disruption by endocervical cytobrush sampling in Canadian women, and assessed the relationship between levels of sE-cad in coronal sulcus swabs to membrane-bound E-cadherin in the overlying foreskin tissue in Ugandan men. RESULTS: sE-cad levels immediately increased after in vitro epithelial physical disruption with the degree of elevation dependent on the extent of disruption, as did levels of IL-1ß and IL-1α; this was followed by a delayed increase in IL-6 levels. In vivo results confirmed that sE-cad levels in cervicovaginal secretions were elevated 6 h after cytobrush sampling when compared to baseline. Furthermore, levels of sE-cad in the prepuce were inversely correlated with the amount of membrane-bound E-cadherin of overlying tissue. CONCLUSION: Our results validate the use of sE-cad as a marker of epithelial disruption and demonstrate that the processes of physical disruption and inflammation in the genital tract are strongly intertwined.

Altered Innate Immunity and Damaged Epithelial Integrity in Vaginal Microbial Dysbiosis.

The role of neutrophils relative to vaginal dysbiosis is unclear. We hypothesize that bacterial vaginosis (BV)-associated bacteria may induce the activation and accumulation of mucosal neutrophils within the female reproductive tract (FRT), resulting in epithelial barrier damage. We collected endocervical cytobrushes from women with and without BV and assessed bacteria community type and frequency/functional phenotypes of neutrophils. We performed in vitro whole blood co-cultures with BV-associated bacteria and healthy vaginal commensals and assessed their impact on epithelial integrity using transepithelial electrical resistance. We demonstrated increased neutrophil frequency (p < 0.0001), activation (p < 0.0001), and prolonged lifespan (p < 0.0001) in the cytobrushes from women with non-Lactobacillus dominant (nLD) communities. Our in vitro co-cultures confirmed these results and identified significant barrier damage in the presence of neutrophils and G. vaginalis. Here, we demonstrate that BV-associated bacteria induce neutrophil activation and increase lifespan, potentially causing accumulation in the FRT and epithelial barrier damage.

17BIPHE2, an engineered cathelicidin antimicrobial peptide with low susceptibility to proteases, is an effective spermicide and microbicide against Neisseria gonorrhoeae.

STUDY QUESTION: Is 17BIPHE2, an engineered cathelicidin antimicrobial peptide with low susceptibility to proteases, a better spermicide in cervicovaginal fluid (CVF) than its parental peptides, LL-37 and GF-17? SUMMARY ANSWER: At the same mass concentration, 17BIPHE2 exhibited the highest spermicidal activity on human sperm resuspended in CVF-containing medium. WHAT IS KNOWN ALREADY: LL-37 and its truncated peptide GF-17 exert both spermicidal and microbicidal activities, although they are prone to proteolytic degradation in body fluids. STUDY DESIGN, SIZE, DURATION: Spermicidal activities of 17BIPHE2 were evaluated in vitro in mouse and human sperm, both resuspended in medium, and then on human sperm incubated in CVF-containing medium; in the latter condition, the spermicidal activity and peptide stability in CVF of 17BIPHE2 were compared with that of LL-37 and GF-17. The in vivo contraceptive effects of 17BIPHE2 and the reversibility thereof were then assessed in mice. Finally, in vitro microbicidal effects of 17BIPHE2 on Neisseria gonorrhoeae were determined. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm motility and plasma membrane integrity were assessed by videomicroscopy and exclusion of Sytox Green, a membrane-impermeable fluorescent dye, respectively. Successful in vitro fertilization (IVF) was determined by the presence of two pronuclei in oocytes following their coincubation with capacitated untreated or 17BIPHE2-treated sperm. Sperm alone or with 17BIPHE2 were transcervically injected into female mice and successful in vivo fertilization was indicated by the formation of two-cell embryos 42-h postinjection, and by pregnancy through pup delivery 21-25 days afterwards. Peptide intactness was assessed by immunoblotting and HPLC. Reversibility of the contraceptive effects of 17BIPHE2 was evaluated by resumption of pregnancy of the female mice, pretranscervically injected with 17BIPHE2, following natural mating with fertile males. Minimum inhibitory/bactericidal concentrations of 17BIPHE2 on N. gonorrhoeae were obtained through microdilution broth assay. MAIN RESULTS AND THE ROLE OF CHANCE: At the same mass concentration, 17BIPHE2 was a more effective spermicide than LL-37 or GF-17 on human sperm resuspended in CVF-containing medium, with the spermicidal concentration of 32.4 µM. This was mainly due to lower susceptibility of 17BIPHE2 to CVF proteases. Importantly, the reproductive tract of mouse females treated three times with 32.4 µM 17BIPHE2 remained normal and their fecundity resumed after stopping 17BIPHE2 treatment. LIMITATIONS, REASONS FOR CAUTION: For ethical reasons, the inhibitory effects of 17BIPHE2 on fertilization and pregnancy cannot presently be performed in women. Also, while our study has proven the effectiveness of 17BIPHE2 as a spermicide for mouse and human sperm in vitro, dosage formulation (e.g. in hydrogel) of 17BIPHE2 still needs to be developed to allow 17BIPHE2 to remain in the vagina/uterine cavity with controlled release for its spermicidal action. WIDER IMPLICATIONS OF THE FINDINGS: Since 17BIPHE2 also exerted bactericidal activity against N. gonorrhoeae at its spermicidal concentration, it is a promising candidate to be developed into a vaginal multipurpose prevention technology agent, thus empowering women against unplanned pregnancies and sexually transmitted infections. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Canadian Institutes of Health Research (PJT 173268 to N.T.). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Longitudinal Assessment of SARS-CoV-2-Specific T Cell Cytokine-Producing Responses for 1 Year Reveals Persistence of Multicytokine Proliferative Responses, with Greater Immunity Associated with Disease Severity.

Cell-mediated immunity is critical for long-term protection against most viral infections, including coronaviruses. We studied 23 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected survivors over a 1-year post-symptom onset (PSO) interval by ex vivo cytokine enzyme-linked immunosorbent spot assay (ELISpot) assay. All subjects demonstrated SARS-CoV-2-specific gamma interferon (IFN-γ), interleukin 2 (IL-2), and granzyme B (GzmB) T cell responses at presentation, with greater frequencies in severe disease. Cytokines, mainly produced by CD4+ T cells, targeted all structural proteins (nucleocapsid, membrane, and spike) except envelope, with GzmB and IL-2 greater than IFN-γ. Mathematical modeling predicted that (i) cytokine responses peaked at 6 days for IFN-γ, 36 days for IL-2, and 7 days for GzmB, (ii) severe illness was associated with reduced IFN-γ and GzmB but increased IL-2 production rates, and (iii) males displayed greater production of IFN-γ, whereas females produced more GzmB. Ex vivo responses declined over time, with persistence of IL-2 in 86% and of IFN-γ and GzmB in 70% of subjects at a median of 336 days PSO. The average half-life of SARS-CoV-2-specific cytokine-producing cells was modeled to be 139 days (~4.6 months). Potent T cell proliferative responses persisted throughout observation, were CD4 dominant, and were capable of producing all 3 cytokines. Several immunodominant CD4 and CD8 epitopes identified in this study were shared by seasonal coronaviruses or SARS-CoV-1 in the nucleocapsid and membrane regions. Both SARS-CoV-2-specific CD4+ and CD8+ T cell clones were able to kill target cells, though CD8 tended to be more potent. IMPORTANCE Our findings highlight the relative importance of SARS-CoV-2-specific GzmB-producing T cell responses in SARS-CoV-2 control and shared CD4 and CD8 immunodominant epitopes in seasonal coronaviruses or SARS-CoV-1, and they indicate robust persistence of T cell memory at least 1 year after infection. Our findings should inform future strategies to induce T cell vaccines against SARS-CoV-2 and other coronaviruses.

Immune parameters of HIV susceptibility in the female genital tract before and after penile-vaginal sex.

Background: In women, most HIV infections are acquired through penile-vaginal sex. Inflammation in the female genital tract (FGT) increases the risk of HIV acquisition and transmission, likely through recruitment of HIV target cells and disruption of epithelial barrier integrity. Although sex may have important immune and epithelial effects, the impact of receptive penile-vaginal sex on the immune correlates of HIV susceptibility in the female genital tract is not well described. Methods: STI-free heterosexual couples were recruited to the Sex, Couples and Science (SECS) Study, with the serial collection of cervical secretions (CVS), endocervical cytobrushes, blood and semen before and up to 72 h after either condomless (n = 29) or condom-protected (n = 8) penile-vaginal sex. Immune cells were characterized by flow cytometry, and immune factors including cytokines and soluble E-cadherin (sE-cad; a marker of epithelial disruption) were quantified by multiplex immunoassay. Co-primary endpoints were defined as levels of IP-10 and IL-1α, cytokines previously associated with increased HIV susceptibility. Results: Here we show that cervicovaginal levels of vaginal IP-10, sE-cad and several other cytokines increase rapidly after sex, regardless of condom use. The proportion of endocervical HIV target cells, including Th17 cells, activated T cells, and activated or mature dendritic cells (DCs) also increase, particularly after condomless sex. Although most of these immune changes resolve within 72 h, increases in activated cervical CD4 + T cells and Tcm persist beyond this time. Conclusions: Penile-vaginal sex induces multiple genital immune changes that may enhance HIV susceptibility during the 72 h post-sex window that is critical for virus acquisition. This has important implications for the mucosal immunopathogenesis of HIV transmission.

Insertive condom-protected and condomless vaginal sex both have a profound impact on the penile immune correlates of HIV susceptibility.

The penis is the primary site of HIV acquisition in heterosexual men. Elevated penile inflammatory cytokines increase sexual acquisition risk, and topically applied cytokines enhance foreskin HIV susceptibility in an explant model. However, the impact of penile-vaginal sex on these immune parameters is undefined. Heterosexual couples were recruited to the Sex, Couples and Science (SECS) Study, with the collection of penile swabs, semen, cervico-vaginal secretions, and blood after a period of abstinence, and repeated sampling up to 72 hours after either condomless (n = 30) or condom-protected (n = 8) penile-vaginal sex. Soluble immune parameters were quantified by multiplex immunoassay. Co-primary immune endpoints were penile levels of IL-8 and MIG, cytokines previously linked to penile HIV acquisition. One hour after sex there were dramatic increases in penile IL-8 and MIG levels, regardless of condom use, with a gradual return to baseline by 72 hours; similar patterns were observed for other chemoattractant chemokines. Penile cytokine changes were similar in circumcised and uncircumcised men, and repeated measures ANOVA and ANCOVA models demonstrated that the degree of change after condomless sex was explained by cytokine levels in their partners' cervico-vaginal secretions. This may have important implications for the biology of penile HIV acquisition.

Low-Level Anorectal HIV Shedding despite Effective Antiretroviral Therapy Is Not Driven by Mucosal Inflammation.

Although antiretroviral treatment (ART) suppresses HIV RNA in blood and prevents transmission, low-level anorectal HIV RNA shedding persists in some ART-treated men who have sex with men. We collected anorectal biopsies and swabs from 55 men who have sex with men on effective ART, hypothesizing that anorectal shedding would be linked to microbiota-driven mucosal T cell activation. Lymphocytes were assessed by flow cytometry, soluble immune factors by multiplex immunoassay, neutrophils and epithelial integrity by immunofluorescence microscopy, and the anorectal microbiome by quantitative PCR and 16S rRNA gene sequencing. Unexpectedly, we found no evidence that anorectal HIV shedding was associated with the parameters of mucosal inflammation, including T cell activation, inflammatory cytokines, the density of neutrophils, or epithelial integrity. Moreover, the anorectal bacterial load was actually lower in the shedding group, with no major differences in bacterial composition. Instead, the strongest mucosal immune correlates of HIV shedding were an increase in central memory cell frequency and Ki67 expression as well as higher concentrations of the cytokine IL-7 in anorectal secretions. Anorectal HIV RNA shedding during effective ART was not driven by local inflammation; the associations seen with local homeostatic T cell proliferation will require further confirmation.

The Effect of Aqueous Extract of Glycyrrhiza glabra on Herpes Simplex Virus 1.

BACKGROUND: Herpes Simplex Virus 1 (HSV-1) resistance to drugs and the side effects of drugs have drawn the attention of investigators to herbal plants. OBJECTIVES: The main aim of the current research was to investigate the effects of Glycyrrhiza glabra (liquorice root) on HSV-1. One of the objectives of the current research was to determine the efficacy and the effect of the elapsed incubation time of treating the Vero cells infected with HSV-1 by G. glabra. In addition, the effect of cells pretreatment with licorice root extract, preincubation of virus with licorice root extract, and the antiviral activity were assessed. PATIENTS AND METHODS: Vero cells were incubated after adding different concentrations of aqueous extracts of G. glabra. The cells were incubated during various time courses. Cytotoxicity assay, determining the 50% tissue culture infectious dose (TCID50), and incubation of HSV-1 with licorice root extract prior to viral infection were performed. RESULTS: Internal association among different experiment groups showed the significant difference in the efficacy of the extract with regard to incubation period between one and four hours, one and eight hours, four and 12 hours, and eight and 12 hours. Moreover, there was a significant difference with regard to efficacy among the pretreatment of cells with extract for two hours, incubation of virus with extract for one hour, incubation of virus with extract for two hours. CONCLUSIONS: G. glabra showed the characteristics of a novel antiviral medication; however, more in vitro experiments are needed to determine the antiherpetic activities of the G. glabra.

The Effect of GBV-C Infection on CD4 Count and Viral Loads in Patients Infected With HIV.

BACKGROUND: The picture that has emerged from studies investigating HIV infected people with GBV-C viremia is that they have lower plasma HIV viral loads in comparison with HIV-positive people who did not have the GBV-C viremia. OBJECTIVES: Since GBV-C HIV coinfection has not been studied in Iran, we have designed a survey to study the outcomes of GBV-C infection on HIV infected individuals. PATIENTS AND METHODS: We analyzed 78 serum samples from HIV-positive patients in Tehran. The HIV positive statue was confirmed by Western blot in our laboratory. Next we detected GBV-C RNA by RT nested-PCR and divided our patient into GBV-C positive and GBV-C negative groups. The final step was measuring the CD4 count and HIV viral load and comparing the means of the CD4 count and HIV viral load in HIV-infected individuals in the GBV-C positive and GBV-C negative groups. RESULTS: We detected GBV-C RNA in 15 patients out of 78. The mean CD4 count was 607.13 compared to 415.87 in the GBV-C negative group and the difference was significant (P = 0.005). In contrast to the CD4 count there was no significant difference in HIV viral loads between HIV infected individuals in the GBV-C positive and GBV-C negative groups. CONCLUSION: Although there was no significant difference in the mean of the HIV viral load between the GBV-C positive and GBV-C negative groups, the significantly higher CD4 mean in the GBV-C positive group compared with the GBV-C negative group suggests a beneficial effect of this coinfection.

Bacteriophage: time to re-evaluate the potential of phage therapy as a promising agent to control multidrug-resistant bacteria.

Nowadays the most difficult problem in treatment of bacterial infections is the appearance of resistant bacteria to the antimicrobial agents so that the attention is being drawn to other potential targets. In view of the positive findings of phage therapy, many advantages have been mentioned which utilizes phage therapy over chemotherapy and it seems to be a promising agent to replace the antibiotics. This review focuses on an understanding of phages for the treatment of bacterial infectious diseases as a new alternative treatment of infections caused by multiple antibiotic resistant bacteria. Therefore, utilizing bacteriophage may be accounted as an alternative therapy. It is appropriate time to re-evaluate the potential of phage therapy as an effective bactericidal and a promising agent to control multidrug-resistant bacteria.