Tofacitinib inhibits inflammatory cytokines from ulcerative colitis and healthy mucosal explants and is associated with pSTAT1/3 reduction in T-cells.

Poor translatability of animal disease models has hampered the development of new inflammatory bowel disorder (IBD) therapeutics. We describe a preclinical, ex vivo system using freshly obtained and well-characterized human colorectal tissue from patients with ulcerative colitis (UC) and healthy control (HC) participants to test potential therapeutics for efficacy and target engagement, using the JAK/STAT inhibitor tofacitinib (TOFA) as a model therapeutic. Colorectal biopsies from HC participants and patients with UC were cultured and stimulated with multiple mitogens ± TOFA. Soluble biomarkers were detected using a 29-analyte multiplex ELISA. Target engagement in CD3+CD4+ and CD3+CD8+ T-cells was determined by flow cytometry in peripheral blood mononuclear cells (PBMCs) and isolated mucosal mononuclear cells (MMCs) following the activation of STAT1/3 phosphorylation. Data were analyzed using linear mixed-effects modeling, t test, and analysis of variance. Biomarker selection was performed using penalized and Bayesian logistic regression modeling, with results visualized using uniform manifold approximation and projection. Under baseline conditions, 27 of 29 biomarkers from patients with UC were increased versus HC participants. Explant stimulation increased biomarker release magnitude, expanding the dynamic range for efficacy and target engagement studies. Logistic regression analyses identified the most representative UC baseline and stimulated biomarkers. TOFA inhibited biomarkers dependent on JAK/STAT signaling. STAT1/3 phosphorylation in T-cells revealed compartmental differences between PBMCs and MMCs. Immunogen stimulation increases biomarker release in similar patterns for HC participants and patients with UC, while enhancing the dynamic range for pharmacological effects. This work demonstrates the power of ex vivo human colorectal tissue as preclinical tools for evaluating target engagement and downstream effects of new IBD therapeutic agents.NEW & NOTEWORTHY Using colorectal biopsy material from healthy volunteers and patients with clinically defined IBD supports translational research by informing the evaluation of therapeutic efficacy and target engagement for the development of new therapeutic entities. Combining experimental readouts from intact and dissociated tissue enhances our understanding of the tissue-resident immune system that contribute to disease pathology. Bayesian logistic regression modeling is an effective tool for predicting ex vivo explant biomarker release patterns.

The Use of Droplet Digital PCR to Quantify HIV-1 Replication in the Colorectal Explant Model.

Ex vivo explant models are used to characterize in vitro efficacy of preexposure prophylaxis (PrEP) agents. Tissue is challenged with virus in culture and HIV-1 p24 levels are quantified with enzyme-linked immunosorbent assay (ELISA) on supernatants collected throughout a 14-21-day incubation. Due to the narrow dynamic range of HIV-1 p24 kits, we evaluated whether droplet digital PCR (ddPCR) provides an alternative method to quantify HIV-1 replication in supernatant samples. We used samples from the MWRI-01 study, which evaluated the pharmacokinetic/pharmacodynamic profile of long-acting rilpivirine using the explant model (McGowan et al. Lancet HIV 2016). HIV-1 pol RNA was measured with ddPCR, either directly with a one-step method or reverse transcribed to cDNA before ddPCR (two-step method) on supernatants from the MWRI-01 study. Previously analyzed HIV-1 p24 antigen levels (Alliance; Perkin-Elmer) were available for comparison purposes. Both ddPCR methods strongly correlated with HIV-1 p24 and displayed similar patterns of HIV-1 suppression before and after rilpivirine. Compared to the p24 ELISA, two-step and one-step ddPCR reduced the amount of hands-on time by approximately one-half and two-thirds, respectively. ddPCR also required less sample and based on p24 versus ddPCR correlation, could potentially reduce the explant culture time from 14 to 10 days (r2 = 0.78, p < .001) due to the increased sensitivity of ddPCR. We demonstrate that ddPCR is a suitable alternative to HIV-1 p24 ELISA to quantify HIV-1 infection in the explant model and has the potential to decrease explant culture time.

Prevalence and determinants of anal human papillomavirus infection in men who have sex with men and transgender women.

Human papillomavirus (HPV) prevalence varies by population. This study investigated anal HPV type detection risk by country in a population of men who have sex with men (MSM) and transgender women (TW) at risk of HIV. Sexually active HIV-1-uninfected MSM and TW were enrolled at eight sites: four in the United States (US), two in Thailand, one in Peru, and one in South Africa. Baseline anal HPV swabs were collected, and DNA typing was performed. One hundred and ninety-five participants, 76 (42%) from the US, had a mean age of 30.9 years (range 18-64). In 182 participants with results available, anal HPV infection was common with 169 (93%) with ≥1 type, 132 (73%) with ≥1 nine-valent vaccine types, and 66 (36%) with HPV 16. Participants in the US had a higher prevalence of HPV 16 (56%, p = 0.004) and HPV 6 (69%, p < 0.001) compared to the other regions. Stimulant drug use was significantly associated with HPV 6 detection. Anal HPV is highly prevalent in this population of MSM and TW sampled from four countries, with HPV 16 the most commonly detected type. The nine-valent HPV vaccine has the potential to provide significant protection if given prior to exposure.

Immunological responsiveness of intestinal tissue explants and mucosal mononuclear cells to ex vivo stimulation.

BACKGROUND: There are limited data on the immunological responsiveness of healthy intestinal tissue when it is cultured and stimulated ex vivo. Such an ex vivo model has the potential to be a valuable tool in understanding disease pathogenesis and as a preclinical tool for the assessment of candidate therapeutic agents used to treat inflammatory bowel disease (IBD). AIM: We undertook a comprehensive study to evaluate ex vivo immunological responses of intestinal tissue and isolated mucosal mononuclear cells (MMC) to a broad range of stimuli. METHODS: Colorectal biopsies (explants) were obtained from healthy participants by flexible sigmoidoscopy and were placed either directly into culture or digested to isolate MMC prior to placement in culture. Explants or MMC were treated with polyinosinic:polycytidylic acid (Poly IC), phytohemagglutinin (PHA), lipopolysacccharides from E Coli (LPS), anti-CD3/CD28 antibodies, or IL-1ß/TNF-α for 24 h. Supernatants were assayed for 40 inflammatory biomarkers using multiplexed enzyme-linked immunosorbent assay (ELISA). The isolated MMCs were further characterized using twelve color flow cytometry. RESULTS: Explants have greater weight adjusted constitutive expression of inflammatory biomarkers than MMCs. Biomarker responses varied as a function of immunogen and use of intact tissue or isolated cells. PHA applied to intact explants was the most effective agent in inducing biomarker changes. Stimulation induced activated and memory cellular phenotypes in both explants and MMCs. CONCLUSIONS: The breadth and magnitude of responses from intact and enzymatically digested intestinal tissue explants stimulated with exogenous immunogens are complex and vary by tissue form and treatment. Overall, PHA stimulation of intact explants produced the most robust responses in normal human colorectal tissue. This system could potentially serve as a preliminary model of the disease state, suitable for small scale screening of new therapeutic agents prior to using IBD patient derived tissue.

Ranpirnase Reduces HIV-1 Infection and Associated Inflammatory Changes in a Human Colorectal Explant Model.

Ranpirnase (RNP) is a low molecular weight type III endoribonuclease, which demonstrates broad antiviral and antitumor properties. We sought to characterize the antiviral activity of RNP against HIV-1 and to determine whether RNP modulates local inflammatory changes associated with HIV infection in the colorectal explant model. Colorectal explants were incubated for 2 h with HIV-1BaL, in the presence of increasing concentrations of RNP (0-60 µg/mL). After washing, explants were cultured for 14 days, with supernatant collected at days 3, 7, 10, and 14. All samples were assayed for HIV-1 p24. Additionally, 30 soluble inflammatory biomarkers were assayed in the day 3 supernatant sample. Other biopsies were stimulated with lipopolysaccharides (LPS) (10 µg/mL) in the presence of RNP and soluble biomarkers assayed at day 3. RNP inhibited productive infection of the colorectal explants with HIV-1BaL and induced a dose-dependent decrease in 15/30 biomarkers. Affected biomarkers included IP-10, MDC, MIP-1α, MIP-1ß, TARC, IL12-p40, IL-15, IL-17, IL-1α, IL-7, IFNγ, IL12-p70, IL-1ß, IL-4, IL-5, and TNF-ß. Similarly, RNP dose-dependent inhibition was demonstrated in 7/30 biomarkers after LPS stimulation, all of which overlapped with HIV-1BaL-induced biomarker changes. The ability of RNP to inhibit both colorectal explant HIV-1BaL infection and inflammatory changes associated with HIV-1 infection makes RPN a promising agent for topical rectal pre-exposure prophylaxis.

A Pilot Study of the Immunologic, Virologic, and Pathologic Consequences of Intra-anal 5% Imiquimod in HIV-1-Infected Men With High-Grade Squamous Intraepithelial Lesions.

BACKGROUND: Imiquimod can be used to treat internal anal high-grade squamous intraepithelial lesions. In HIV-1-infected individuals there is a theoretical concern for increased HIV replication in anorectal tissue secondary to imiquimod-induced mucosal inflammation. OBJECTIVE: The purpose of this study was to assess local virologic, immunologic, and pathologic effects of imiquimod treatment in HIV-infected individuals. DESIGN: This was a pilot study at a single academic center. SETTINGS: The study was conducted at the University of Pittsburgh Anal Dysplasia Clinic. PATIENTS: HIV-1-infected individuals with biopsy-confirmed internal anal high-grade squamous intraepithelial lesions were included. INTERVENTION: Imiquimod cream was prescribed for intra-anal use 3 times per week for 9 weeks. MAIN OUTCOME MEASURES: Anal human papillomavirus typing, anal and rectal tissue HIV-1 RNA and DNA quantification, cytokine gene expression, and anal histology were measured. RESULTS: Nine evaluable participants (1 participant was lost to follow-up) were all white men with a median age of 46 years (interquartile range = 12 y) and a median CD4 T-cell count of 480 cells per cubic millimeter (interquartile range = 835). All were taking antiretroviral therapy, and 7 of 9 had HIV-1 RNA <50 copies per milliliter. The median dose of imiquimod used was 27.0 (interquartile range = 3.5), and there was a median of 11 days (interquartile range = 10 d) from last dose to assessment. There was no progression to cancer, no significant change in the number of human papillomavirus types detected, and no significant change in quantifiable cytokines/HIV-1 RNA or DNA levels in anal or rectal tissue. Seven (35%) of 20 high-grade lesions resolved to low-grade squamous intraepithelial lesions. LIMITATIONS: The study was limited by the small number of participants and variable time to final assessment. CONCLUSIONS: Intra-anal imiquimod showed no evidence of immune activation or increase in HIV-1 viral replication in anal and rectal tissue and confirmed efficacy for intra-anal high-grade squamous intraepithelial lesion treatment morbidity. See Video Abstract at http://links.lww.com/DCR/A498.

Long-acting rilpivirine as potential pre-exposure prophylaxis for HIV-1 prevention (the MWRI-01 study): an open-label, phase 1, compartmental, pharmacokinetic and pharmacodynamic assessment.

BACKGROUND: Long-acting injectable antiretroviral agents are being developed for HIV-1 prevention. The MWRI-01 study was done to characterise the safety, acceptability, and pharmacokinetic and pharmacodynamic profile of long-acting rilpivirine. METHODS: We did a phase 1 open-label study at the University of Pittsburgh. We enrolled healthy individuals (aged 18-45 years) who were seronegative for HIV-1. Participants were assigned alternately one intramuscular dose of either 1200 mg or 600 mg long-acting rilpivirine, beginning with the 1200 mg dose. We obtained plasma specimens, genital and rectal fluids, and tissue samples (rectal, cervical, and vaginal) before and after exposure to long-acting rilpivirine for assessment of pharmacokinetics and ex-vivo biopsy challenge with HIV-1. Our primary objective was to characterise product safety, and the analysis included all enrolled participants. This trial is registered with ClinicalTrials.gov, number NCT01656018. FINDINGS: 36 participants were enrolled into the study, of whom 24 were women and 12 men. 12 women and six men received each dose. 204 adverse events were reported among the 36 participants, of which 200 (98%) were grade 1-2. The most common adverse event was injection site reaction. All grade 3 and 4 adverse events were deemed not related to rilpivirine. Geometric mean (90% CI) concentrations in plasma of rilpivirine at day 28 post dose were 53 ng/mL (38-67) in women and 43 ng/mL (23-63) in men for the 1200 mg dose and 28 ng/mL (19-37) in women and 17 ng/mL (9-24) in men for the 600 mg dose. The tissue-to-plasma ratio for rilpivirine in rectal tissue was about two-fold higher than in vaginal and cervical tissue (1·10-1·53 vs 0·61-0·72 and 0·50-0·71, respectively). Exposure to long-acting rilpivirine suppressed viral replication significantly in rectal tissue (p<0·0001), and this suppression persisted for up to 4 months. By contrast, no viral suppression was seen in cervical or vaginal tissue. INTERPRETATION: Ongoing research will characterise longer term safety and acceptability of multiple injections and help ascertain whether long-acting rilpivirine should advance to assessment of efficacy in preventing HIV-1 infection. FUNDING: Bill & Melinda Gates Foundation.

Prevalence of Anal Human Papillomavirus Vaccine Types in the Bangkok Men Who Have Sex With Men Cohort Study.

BACKGROUND: The quadrivalent human papillomavirus (qHPV) and 9 valent (nHPV) vaccine are licensed for males to prevent anal HPV-associated dysplasia and cancer caused by HPV types 6, 11, 16, and 18 (qHPV) and additional types 33, 35, 45, 52, and 58 (nHPV), respectively. Both conditions are common in HIV-infected and HIV-uninfected men who have sex with men (MSM). It is not well documented which anal HPV vaccine types are most prevalent in Southeast Asia. METHODS: A convenience sample of 400 anal swabs were obtained from 200 HIV-infected and 200 HIV-uninfected sexually active Bangkok MSM Cohort Study participants. After swab collection in PreservCyt (Cytyc Corp, Marlborough, MA), the media was stored at -80°C until processing. DNA was extracted, amplified by polymerase chain reaction, denatured, and then hybridized to probes for 37 HPV types and ß-globin. RESULTS: The mean participant age was 25.6 years (range, 18-55 years); the mean CD4 T-cell count was 410 cells/mm in the HIV-infected participants. Among all swab samples, 386 (192 HIV-positive and 194 HIV-negative) had adequate ß-globin for HPV genotype testing. Anal HPV type was detected in 44.3% of participants whose samples underwent genotype testing. Both qHPV and nHPV types were more frequently detected in HIV-infected compared with HIV-uninfected (42.2% vs. 23.2% [P < 0.01], 50.0% vs. 24.2% [P < 0.01]), respectively). There were no significant relationships between social behaviors (alcohol use, drug use) or sexual behaviors (number of partners, condom usage, sexual positioning) and anal HPV prevalence. CONCLUSIONS: The prevalence of anal vaccine HPV types in Thai MSM was similar to that reported in MSM from Western populations and has a similar distribution by HIV status. Targeting young MSM with vaccination could offer protection against HPV vaccine types.