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Background Adherence is critical for successful topical, vaginally delivered anti-retroviral (ARV)-based HIV pre-exposure prophylaxis (PrEP). Quantitating systemic or tissue ARV levels through LC–MS/MS is currently viewed as the most reliable measure of adherence. However, for placebo-controlled trials, this is a high cost analysis that measures adherence only in the drug treatment group. A desirable marker of adherence is one that is measured in both placebo and drug treatment groups using a simple on-site clinical laboratory test, which allows necessary interventions for supporting participant adherence. Our objective was to develop adherence markers for four vaginal placebo products currently used as microbicide delivery systems: gel, film, insert, and intravaginal ring. Excipient and spectroscopy-based approaches were used for preclinical development of the placebo markers and subsequently validated by the CONRAD 135 study. The study collected vaginal swabs collected each day for 1 week post vaginal application of gel, film, or insert in the clinic with or without sex. Intravaginal rings were collected after 1 day, 7, and 30 days of use. Results Placebo gel, film, and insert in vaginal swabs were successfully detected by specific excipient colorimetric or probe-based assays for hydroxyethylcellulose, glycerin, and sorbitol respectively, as well as spectroscopy-based prediction models. The range of detection for gel, film, and insert in swabs collected up to 16 h post vaginal application was 70-100% of the total swabs per time point, with some markers showing potential for longer duration. Decreasing residual glycerin levels and increasing bioanalyte penetration of vaginally used intravaginal rings showed significant changes between 1 and 30 days of use. Conclusions We demonstrated clinical proof-of-concept that adherence markers for placebo product can be measured using simple, lower cost approaches. Measuring adherence in both placebo and drug arms of a HIV PrEP study would better inform future trial designs. Electronic supplementary material The online version of this article (10.1186/s40169-018-0213-6) contains supplementary material, which is available to authorized users.

Objective The primary objective of this pilot study is to determine and compare the residence time in the vagina of biomarkers of semen exposure for up to 15 days post exposure. The biomarkers are prostate-specific antigen (PSA), Y chromosome DNA, the sex determining region of the Y chromosome (SRY) and testis-specific protein Y-encoded 4 (TSPY4). The secondary objectives are to determine if biomarker concentrations differed between intercourse and inoculation groups, to establish whether the sampling frequency post exposure affected biomarker concentrations and decay profile and to determine if biomarker concentrations in vaginal swabs obtained by the participant at home were similar to swabs obtained by the nurse in the clinic. Study design We randomized healthy women to unprotected intercourse (n=17) versus vaginal inoculation with the male partner’s semen in the clinic (n=16). Women were then further randomized to have vaginal swabs obtained at either 7 or 4 time points after semen exposure, up to 15 days post exposure, either obtained at home by the participant or in the clinic by the research nurse. Results PSA and SRY were markers of recent semen exposure. TSPY4 was detectable in approximately 50% of participants at 15 days post exposure. Unprotected intercourse resulted in significantly higher concentrations of select biomarkers. Sampling frequency and home versus clinic sampling had no significant effect on biomarker concentrations. Conclusions Objective biomarkers of recent or distant semen exposure may have great utility for verifying protocol compliance in a variety of clinical trials.

Abstract. Renal pathology in mice that are transgenic for the murine albumin enhancer/promoter linked to a full-length porcine transforming growth factor-β1 (TGF-β1) gene has been described previously. In these mice, transgene expression is limited to the liver and the plasma level of TGF-β is increased. The earliest renal pathologic change is glomerulosclerosis, at 3 wk of age, and this is followed by tubulointerstitial fibrosis. In this study, it was hypothesized that circulating TGF-β1 increases renal extracellular matrix accumulation and activates local TGF-β gene expression. Immunostaining at 5 wk revealed increased amounts of collagen I and III within the mesangium, glomerular capillary loops, and interstitium, while the amount of collagen IV was normal. Similarly, Northern analysis showed increased expression of mRNA encoding collagen I and III, as well as biglycan and decorin, while the expression of collagen IV was unchanged. These changes began as early as 1 wk of age, a time before the appearance of glomerulosclerosis. To evaluate matrix degradation, collagenase IV activity was evaluated by gelatin zymography and an increase in matrix metalloproteinase-2 was found. Finally, the production of tissue inhibitors of metalloproteinase was evaluated. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA was increased 18-fold, while TIMP-2 and TIMP-3 were unchanged. In 2-wk-old transgenic kidney, local expression of TGF-β1, β2, and β3 protein was similar to wild-type mice. In 5-wk-old transgenic mice, TGF-β1 and β2 protein was present in increased amounts within glomeruli, and renal TGF-β1 mRNA was increased threefold. It is concluded that elevated levels of circulating TGF-β1 may act on the kidney to increase matrix protein production and decrease matrix remodeling. Only after glomerulosclerosis is established does local glomerular overproduction of TGF-β become manifest.

An intrafollicular increase in proteolytic activity drives ovulatory events. Surprisingly, the periovulatory expression profile of the membrane-type matrix metalloproteinases (MT-MMPs), unique proteases anchored to the cell surface, has not been extensively examined. Expression profiles of the MT-MMPs were investigated in ovarian tissue from well-characterized rat and macaque periovulatory models and naturally cycling women across the periovulatory period. Among the six known MT-MMPs, mRNA expression of Mmp14, Mmp16, and Mmp25 was increased after human chorionic gonadotropin (hCG) administration in rats. In human granulosa cells, mRNA expression of MMP14 and MMP16 increased following hCG treatment. In contrast, mRNA levels of MMP16 and MMP25 in human theca cells were unchanged before ovulation but declined by the postovulatory stage. In macaque granulosa cells, hCG increased mRNA for MMP16 but not MMP14. Immunoblotting showed that protein levels of MMP14 and MMP16 in rats increased, similar to their mRNA expression. In macaque granulosa cells, only the active form of the MMP14 protein increased after hCG, unlike its mRNA or the proprotein. By immunohistochemistry, both MMP14 and MMP16 localized to the different ovarian cell types in rats and humans. Treatment with hCG resulted in intense immunoreactivity of MMP14 and MMP16 proteins in the granulosa and theca cells. The present study shows that MMP14 and MMP16 are increased by hCG administration in the ovulating follicle, demonstrating that these MMPs are conserved among rats, macaques, and humans. These findings suggest that MT-MMPs could have an important role in promoting ovulation and remodeling of the ovulated follicle into the corpus luteum.

Multipurpose prevention technologies (MPTs), which prevent sexually transmitted infection(s) and unintended pregnancy, are highly desirable to women. In this randomized, placebo-controlled, phase I study, women used a placebo or tenofovir (TFV) and levonorgestrel (LNG) intravaginal ring (IVR), either continuously or cyclically (three, 28-day cycles with a 3 day interruption in between each cycle), for 90 days. Sixty-eight women were screened; 47 were randomized to 4 arms: TFV/LNG or placebo IVRs used continuously or cyclically (4:4:1:1). Safety was assessed by adverse events and changes from baseline in mucosal histology and immune mediators. TFV concentrations were evaluated in multiple compartments. LNG concentration was determined in serum. Modeled TFV pharmacodynamic antiviral activity was evaluated in vaginal and rectal fluids and cervicovaginal tissue ex vivo. LNG pharmacodynamics was assessed with cervical mucus quality and anovulation. All IVRs were safe with no serious adverse events nor significant changes in genital tract histology, immune cell density or secreted soluble proteins from baseline. Median vaginal fluid TFV concentrations were >500 ng/mg throughout 90d. TFV-diphosphate tissue concentrations exceeded 1,000 fmol/mg within 72hrs of IVR insertion. Mean serum LNG concentrations exceeded 200 pg/mL within 2h of TFV/LNG use, decreasing quickly after IVR removal. Vaginal fluid of women using TFV-containing IVRs had significantly greater inhibitory activity (87–98% versus 10% at baseline; p<0.01) against HIV replication in vitro. There was a >10-fold reduction in HIV p24 antigen production from ectocervical tissues after TFV/LNG exposure. TFV/LNG IVR users had significantly higher rates of anovulation, lower Insler scores and poorer/abnormal cervical mucus sperm penetration. Most TFV/LNG IVR users reported no change in menstrual cycles or fewer days of and/or lighter bleeding. All IVRs were safe. Active rings delivered high TFV concentrations locally. LNG caused changes in cervical mucus, sperm penetration, and ovulation compatible with contraceptive efficacy. Trial registration: ClinicalTrials.gov #NCT03279120.

BackgroundThe topical HIV prevention (microbicides) field is in acute need of a method to rapidly and objectively measure adherence to product use in clinical trials. Infrared (IR) spectroscopy has been used in many pharmaceutical and forensic applications but has yet to be applied to adherence monitoring. In this study, we report on efforts to test the feasibility of using IR spectroscopy as a means to measure residual active or placebo vaginal product, semen exposure and vaginal insertion from a single swab.MethodsA portable IR spectrometer equipped with diamond attenuated total reflectance (ATR) was used to capture spectra of unused vs. vaginally-used swabs, vaginal swabs containing semen, and vaginal swabs to which either tenofovir-containing or matching placebo products (vaginal gel or insert) were added. Spectral data obtained from swabs placed directly on the spectrometer were divided into calibration and testing sets for developing and validating discriminant models set up to provide yes/no predictions of: vaginal vs. non-vaginal use, presence vs. no presence of each test product, and presence vs. no presence of semen. Further validation of models was performed using vaginal swabs collected from a clinical study evaluating vaginally administered placebo insert formulations.ResultsFor each discriminant model developed to predict vaginal vs. non-vaginal use, presence vs. no presence of each test product, and presence vs. no presence of semen, classified validation samples not included in the model development were correctly identified into their respective classes with minimal prediction error. Clinically obtained vaginal swabs collected 15–60 minutes after placebo insert use were also correctly identified, further validating the models.ConclusionOur findings demonstrate the proof of concept that IR spectroscopy can be a method for rapid detection and characterization of microbicide products and biological fluids present in vaginal swabs. This novel method has potential to support real-time, on-site adherence monitoring in clinical or field settings.

The role of hyperglycemia in diabetic changes of the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) has not been clearly established. We therefore determined whether glucose modulates IGFBP synthesis and stability in vitro. Porcine vascular smooth muscle cells (pSMC) cultured in low glucose (pSMC-L) had 2.1-fold more IGFBP-4 in the conditioned medium compared with pSMC cultured in high glucose (pSMC-H) (P < 0.01). In contrast, IGFBP-2 levels remained constant. Although pSMC-H and pSMC-L cultures expressed similar levels of IGFBP-4 messenger RNA, in vitro protease assays demonstrated an increase in IGFBP-4 proteolysis in pSMC-H conditioned medium compared with pSMC-L conditioned medium (P < 0.01). The protease had properties similar to a previously characterized IGFBP-4 protease. The addition of 20 mM mannitol to pSMC-L cultures did not decrease IGFBP-4 levels, suggesting that the difference in IGFBP-4 proteolysis was not osmotically induced. The change was not due to selection bias, because cultures that were initially isolated from aortic explants in high and low glucose still exhibited the glucose-dependent difference in IGFBP-4 proteolytic activity. The results suggest that high glucose acts on pSMC to induce a change in IGFBP-4 proteolytic activity, which results in increased IGF-I availability to its receptors thereby enhancing the SMC proliferative response.