Antimicrobial host defence peptide, LL-37, as a potential vaginal contraceptive.

STUDY QUESTION: Does antimicrobial peptide, LL-37, inhibit sperm fertilizing ability? SUMMARY ANSWER: Our results indicate that LL-37 inhibits mouse and human sperm fertilizing ability. WHAT IS KNOWN ALREADY: LL-37, a cationic antimicrobial peptide, exerts its microbicidal effects through the disruption of microbial cytoplasmic membranes following its interaction with microbial surface anionic phospholipids. ALL-38 (an LL-37 close analogue: LL-37 + Ala at the N-terminus) is produced in the vagina 2-6 h post-intercourse from its precursor hCAP-18, a seminal plasma component. At this time, motile sperm have already swum into the uterine cavity, thus unexposed to ALL-38. Since sperm contain a substantial amount of acidic sulfogalactosylglycerolipid (SGG) on their surface, treatment of sperm with LL-37 may cause their membrane disruption in an analogous manner to that occurring on microbial membranes. STUDY DESIGN, SIZE AND DURATION: Mouse/human sperm treated (2-30 min) with LL-37 in a physiological concentration range (up to 10.8 µM) were assessed for SGG-dependent LL-37 binding, and parameters relevant to fertilizing ability, namely motility and intactness of the sperm acrosome and plasma membrane. Ability of mouse sperm to fertilize eggs in vitro was also evaluated. Each study was performed with greater than or equal to three different sperm samples. The efficacy of LL-37 to inhibit sperm fertilizing ability in vivo was determined in female mice (n = 26 each for LL-37 treatment and no treatment), using sperm retrieved from 26 males. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm samples were donated by fertile men. LL-37 was chemically synthesized and was biotinylated for sperm binding studies. Sperm motility was assessed by videomicroscopy and the acrosomal status by Coomassie blue staining of acrosome-intact mouse sperm or the exposure of CD46, an inner acrosomal membrane protein, of acrosome reacted human sperm. Sperm membrane permeabilization/disruption was assessed by the loss of hypo-osmotic swelling response, an incorporation of Sytox Green (a membrane impermeable fluorescent DNA dye), and electron microscopy. Mouse IVF was scored by the presence of two pronuclei in eggs 6 h post-insemination. Ability of mouse sperm to fertilize eggs in vivo was determined by the pregnancy outcome of female mice injected transcervically with sperm with or without LL-37. MAIN RESULTS AND THE ROLE OF CHANCE: Biotinylated LL-37 bound to both mouse and human sperm and the binding was partially dependent on sperm surface SGG. Mouse and human sperm became immotile and underwent a premature acrosome reaction upon treatment with LL-37 at 3.6 and 10.8 µM, respectively. The initial action of LL-37 on both mouse and human sperm appeared to be through permeabilization/disruption of sperm surface membranes evidenced by the loss of hypo-osmotic swelling response, Sytox Green staining and electron microscopy revealing ultrastructural damage. Mouse sperm treated with 3.6 µM LL-37 lost the ability to fertilize eggs both in vitro and in vivo. All 26 female mice inseminated with sperm and LL-37 did not become pregnant. No apparent damage to the reproductive tract was observed as revealed by histological characterization in LL-37-inseminated mice and these females resumed fecundity following mating with fertile males. LIMITATIONS, REASONS FOR CAUTION: Direct demonstration that LL-37 treated human sperm fail to fertilize eggs was limited by legal restrictions on obtaining human eggs for such use. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal selective inhibitory effects of LL-37 on sperm fertilizing ability in mice without apparent impairment to the female reproductive tract. LL-37 is therefore a promising candidate to be developed into a vaginal contraceptive with microbicidal activity. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grand Challenges Explorations grant from the Bill & Melinda Gates Foundation (OPP1024509), Canadian Institutes of Health Research (MOP119438 & CCI82413) and International Collaboration and Exchanges NSFC of China (No.30611120525). There are no competing interests to declare.

HIV infection of thymocytes inhibits IL-7 activity without altering CD127 expression.

BACKGROUND: Thymic function is altered in HIV infection and characterized by dysregulation of the thymic epithelial network, reduced thymic output and ultimately an impaired naïve T-cell pool. The IL-7/IL-7 receptor (IL-7R) signalling pathway is critical for the maturation and differentiation of thymocytes. HIV infection is associated with a decrease in IL-7Rα (CD127) expression and impaired CD127 signalling in circulating CD8+ T-cells; however, little is known about the effect of HIV on CD127 expression and IL-7 activity in the thymus. Therefore, the effect of in vitro HIV infection on CD127 expression and IL-7-mediated function in thymocytes was investigated. FINDINGS: In vitro HIV infection of thymocytes did not affect CD127 expression on either total thymocytes or on single positive CD4 or single positive CD8 subsets. However, HIV infection resulted in a decrease in the level of IL-7-induced STAT-5 phosphorylation and Bcl-2 expression in unfractionated thymocytes. CONCLUSION: These findings indicate that HIV infection alters IL-7 responsiveness of thymocytes by a mechanism other than CD127 downregulation and potentially explain the disruption in thymopoiesis observed in HIV infection.

Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells.

PROBLEM: Sperm are the major cells in semen. Human sperm possess a number of HIV-1 gp120 binding ligands including sulfogalactosylglycerolipid (SGG). However, the mechanisms of how sperm capture HIV-1 onto their surface are unclear. Furthermore, the ability of sperm to deliver HIV-1 to vaginal/cervical epithelial cells lining the lower female reproductive tract, as a first step in HIV-1 transmission, needs to be determined. METHOD OF STUDY: Sperm from healthy donors were incubated with dual-tropic HIV-1CS204 (clinical isolate), and virus capture was determined by p24 antigen ELISA. The involvement of SGG in HIV-1 capture was assessed by determining Kd values of HIV-1 gp120-SGG binding as well as computational docking of SGG to the gp120 V3 loop. The ability of sperm-associated HIV-1 to infect peripheral blood mononuclear cells (PBMCs) and TZM-bl indicator cells was determined. Lastly, infection of vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cells mediated by HIV-1-associated sperm was evaluated. RESULTS: Sperm were able to capture HIV-1 in a dose-dependent manner, and the capture reached a maximum within 5 minutes. Captured HIV-1, however, could be removed from sperm by Percoll-gradient centrifugation. Affinity of gp120 for SGG was substantial, implicating sperm SGG in HIV-1 capture. Sperm-associated HIV-1 could productively infect PBMCs and TZM-bl cells, and was capable of being transmitted into vaginal/cervical epithelial cells. CONCLUSION: Sperm are able to capture HIV-1, which remains infectious and is able to be transmitted into vaginal/cervical epithelial cells, a result indicating the importance of sperm in HIV transmission.

CpG increases vaccine antigen-specific cell-mediated immunity when administered with hepatitis B vaccine in HIV infection.

BACKGROUND: Lack of adequate adjuvancy is a possible explanation for lack of vaccine immunogenecity. Immunostimulatory CpGs are potent vaccine adjuvants and may be an important component of the development vaccines, particularly those for which a cellular immune response is required for protection. We have previously demonstrated that CpG ODN co-administration with hepatitis B vaccine results in earlier, stronger and more sustained antibody responses to hepatitis B surface antigen in HIV infected individuals, and wished to determine if, in this population, helper T-cell responses were also enhanced. METHODS: We conducted a double-blind, placebo-controlled trial in hepatitis B susceptible, effectively treated HIV-seropositive individuals. Participants received hepatitis B vaccine, with either placebo or CPG 7909 1.0 mg at week 0, 4 and 8. To determine the impact of CpG on cellular immune responses, lymphoproliferative responses (LPR) were evaluated by [3H]-thymidine incorporation at baseline and weeks 4, 8, 12, 24, and 48. Immunophenotyping of lymphocyte subsets was also determined at these time points. RESULTS: Of 36 patients enrolled, 18 received hepatitis B vaccine alone, and 18 received hepatitis B vaccine with CpG. Inclusion of CPG 7909 was associated with a greater proliferative response to HBsAg at all time points following initial vaccination. This increase was statistically significant at 8 weeks (p = 0.042) and 48 weeks (p = 0.024). Similar results were observed when LPR were evaluated as stimulation indices (SI). No differences in proliferative responses to HIV p24 Ag were observed, nor were there any differences in lymphocyte subsets. CONCLUSION: In addition to enhancing humoral responses to vaccination, we describe for the first time that CPG 7909 enhances cellular immunity to vaccine antigen in a typically hyporesponsive population. This adjuvancy may be important in the development of an effective vaccine for which a cellular immune response is required for protection.

Optimization of culture and storage conditions for an in vitro system to evaluate thymocyte phenotype and function.

Studies on thymopoiesis are critical to the understanding of T-cell homeostasis as well as the host response to T-cell depletion. Various in vitro culture systems have been used in the study of thymocyte development; however it is unclear if current co-culture methods have been fully optimized. In this study in vitro suspension cultures have been re-evaluated and the optimal storage conditions for thymocytes have been established by evaluating various methods of storing/isolating thymic tissue and isolated thymocytes as well as the source of thymic epithelial cells (TEC). It was determined that thymocytes must be freshly isolated from whole thymic tissue and ideally stored at 4 degrees C prior to co-culture. Co-culture with either autologous or allogeneic TEC results in similar thymocyte subset distribution as well as interleukin-7 receptor-alpha (CD127) expression on these subsets. To evaluate the influence of the source of TEC on one aspect of thymocyte function the effect of IL-7 stimulation on the expression of CD127 was evaluated. IL-7 stimulation resulted in a downregulation of the expression of CD127 on all thymic subsets similar to that observed in circulating CD8+ T-cells. The effect of this was the same whether TEC were autologous or allogeneic. Optimizing culture techniques and facilitating the study of individual thymocyte subsets will lead to a better understanding of thymic function and development. It could also lead to therapeutic approaches that enhance immune recovery after T-cell depletion in HIV infection, bone marrow transplantation or following chemotherapy.

Long-term follow-up of a cohort of HIV-infected patients who discontinued maintenance therapy for cytomegalovirus retinitis.

PURPOSE: To determine the long-term safety of discontinuation of maintenance therapy for cytomegalovirus retinitis (CMVR) and to identify predictors for relapse. METHOD: This was a prospective cohort study. Patients with treated CMVR who responded to HAART were followed by ophthalmologic assessment, markers for CMV replication (blood and urine cultures, CMV antigenemia, CMV DNA by PCR), and in vitro lymphoproliferative responses to CMV and other antigens after discontinuation of CMVR maintenance therapy. RESULTS: 23 patients were followed a median of 34 (range, 5-61) months. Median CD4 count was 321/mm3 at enrollment and 395/mm3 at last follow-up. HIV RNA was <50 copies/mL in 78% of patients at enrollment and 65% at last follow-up. One CMVR reactivation occurred at 12 months at a CD4 count of 395/mm3 (21%) and HIV RNA <50 copies/mL. Urine cultures were a poor predictive marker for reactivation. Other CMV replication markers had good negative predictive value. 96% of patients had a good lymphoproliferative response to CMV antigen in vitro. CONCLUSION: Maintenance therapy for CMVR can safely be discontinued in patients who have responded to HAART. Combining our results with the published literature, the risk of reactivation is estimated at 0.016 per person year of follow-up. Markers to predict relapse and the need for re-initiation of maintenance therapy are not yet identified.

Association of the frequency of respiratory illness in early childhood with a change in the distribution of blood lymphocyte subpopulations.

Little is known about the distribution of lymphocyte phenotypes in young children and the association specific phenotypes may have with respiratory illnesses. The objective of this study was to describe lymphocyte distributions in children at approximately 2 years of age and to test for associations with the frequency of respiratory illness during the first 2 years of life. We hypothesized that an increased frequency of illness would be associated with those phenotypes that reflect previous antigen exposure and/or immune activation. Seventy-three children were followed during their first 2 years of life with daily symptom diaries and twice-monthly telephone calls to ascertain the incidence of respiratory illness. After the children reached 2 years of age, the phenotypes of circulating blood lymphocytes were measured by flow cytometry. Associations between illness and phenotypes were adjusted for education level of parents; hours per week in day care; hours per week exposed to environmental tobacco smoke, mould, or water damage in bedroom; and parental history of allergy and asthma. The resulting median lymphocyte count was 4.0 x 109 per litre (standard deviation, 1.3) with a CD4/CD8 count of 2.28, consistent with published values. Illness rates were positively associated with the percentage of CD8+ CD38+ T cells (unadjusted p = .03, adjusted p = .014), CD8+ CD45RO+ T cells (unadjusted p = .06, adjusted p = .036), and CD4+ CD45RO+ T cells (unadjusted p = .01, adjusted p = .005). Our conclusions is that there is an association between the distribution of lymphocyte phenotypes and the incidence of respiratory illness early in life. Future research is recommended to determine the directionality of this association.

Normalization of natural killer cell function and phenotype with effective anti-HIV therapy and the role of IL-10.

OBJECTIVES: Natural killer (NK) cell function is likely to be important in controlling HIV infection and opportunistic pathogens. We therefore evaluated NK function and phenotype over the course of antiretroviral therapy (ART) and examined the potential mechanisms of altered NK activity in HIV infection. METHODS: We measured NK cell percentage, NK cytolytic activity (both by flow cytometry) and plasma IL-10 concentrations (by enzyme-linked immunosorbent assay) in 10 HIV-seropositive patients before and over one year of effective ART. To examine potential mechanisms of altered NK activity, we measured NK receptor expression in ART treated and untreated HIV-positive individuals by flow cytometry. As IL-10 enhances NK activity, we studied the effect of IL-10 on NK receptor expression and activity in peripheral blood mononuclear cells (PBMC) from HIV-seronegative individuals. RESULTS: NK cytolytic activity was elevated in HIV infection and decreased with ART to levels observed in HIV-negative individuals. A greater proportion of NK cells from untreated HIV-positive individuals expressed the NK receptors CD158a and CD161 than either HIV-negative volunteers or effectively treated HIV-positive patients. NK cells from PBMC incubated with IL-10 demonstrated increases in CD158a, CD161 and CD94 expression and increases in cytolytic activity. The treatment-associated decrease in NK activity paralleled a decrease in IL-10 production. CONCLUSION: The observation that IL-10 alters NK receptor expression similar to that observed in HIV infection, and the fact that NK receptor expression and activity normalize in parallel with ART-induced reduction of circulating IL-10 levels supports a role for IL-10 in NK cell activity and HIV immunopathogenesis.