Tenofovir, emtricitabine, lamivudine and dolutegravir concentrations in plasma and urine following drug intake cessation in a randomized controlled directly observed pharmacokinetic trial to aid point-of-care testing.

BACKGROUND: Poor adherence to ART and pre-exposure prophylaxis (PrEP) can impact patient and public health. Point-of-care testing (POCT) may aid monitoring and adherence interventions. OBJECTIVES: We report the pharmacokinetics of tenofovir [dosed as tenofovir disoproxil (TDF) and tenofovir alafenamide (TAF)], emtricitabine (FTC), lamivudine (3TC) and dolutegravir (DTG) in plasma and urine following drug cessation to evaluate adherence targets in urine for POCT. METHODS: Subjects were randomized (1:1) to receive DTG/FTC/TAF or DTG/3TC/TDF for 15 days. Plasma and spot urine were collected on Day 15 (0-336 h post final dose). Drug concentrations were quantified using LC-MS, and non-linear mixed-effects models applied to determine drug disposition between matrices and relationship with relevant plasma [dolutegravir protein-adjusted 90% inhibitory concentration (PA-IC90 = 64 ng/mL) and minimum effective concentration (MEC = 324 ng/mL)] and urinary thresholds [tenofovir disoproxil fumarate 1500 ng/mL]. RESULTS: Of 30 individuals enrolled, 29 were included (72% female at birth, 90% Caucasian). Median (range) predicted time to plasma dolutegravir PA-IC90 and MEC were 83.5 (41.0-152) and 49.0 h (23.7-78.9), corresponding to geometric mean (90%) urine concentrations of 5.42 (4.37-6.46) and 27.4 ng/mL (22.1-32.7). Tenofovir in urine reached 1500 ng/mL by 101 h (58.6-205) with an equivalent plasma concentration of 6.20 ng/mL (4.21-8.18). CONCLUSIONS: These data support use of a urinary tenofovir threshold of <1500 ng/mL (tenofovir disoproxil fumarate-based regimens) as a marker of three or more missed doses for a POCT platform. However, due to low dolutegravir concentrations in urine, POCT would be limited to a readout of recent dolutegravir intake (one missed dose).

Immune microenvironment profiling of normal appearing colorectal mucosa biopsied over repeat patient visits reproducibly separates lynch syndrome patients based on their history of colon cancer.

Introduction: Lynch syndrome (LS) is the most common hereditary cause of colorectal cancer (CRC), increasing lifetime risk of CRC by up to 70%. Despite this higher lifetime risk, disease penetrance in LS patients is highly variable and most LS patients undergoing CRC surveillance will not develop CRC. Therefore, biomarkers that can correctly and consistently predict CRC risk in LS patients are needed to both optimize LS patient surveillance and help identify better prevention strategies that reduce risk of CRC development in the subset of high-risk LS patients. Methods: Normal-appearing colorectal tissue biopsies were obtained during repeat surveillance colonoscopies of LS patients with and without a history of CRC, healthy controls (HC), and patients with a history of sporadic CRC. Biopsies were cultured in an ex-vivo explant system and their supernatants were assayed via multiplexed ELISA to profile the local immune signaling microenvironment. High quality cytokines were identified using the rxCOV fidelity metric. These cytokines were used to perform elastic-net penalized logistic regression-based biomarker selection by computing a new measure - overall selection probability - that quantifies the ability of each marker to discriminate between patient cohorts being compared. Results: Our study demonstrated that cytokine based local immune microenvironment profiling was reproducible over repeat visits and sensitive to patient LS-status and CRC history. Furthermore, we identified sets of cytokines whose differential expression was predictive of LS-status in patients when compared to sporadic CRC patients and in identifying those LS patients with or without a history of CRC. Enrichment analysis based on these biomarkers revealed an LS and CRC status dependent constitutive inflammatory state of the normal appearing colonic mucosa. Discussion: This prospective pilot study demonstrated that immune profiling of normal appearing colonic mucosa discriminates LS patients with a prior history of CRC from those without it, as well as patients with a history of sporadic CRC from HC. Importantly, it suggests the existence of immune signatures specific to LS-status and CRC history. We anticipate that our findings have the potential to assess CRC risk in individuals with LS and help in preemptively mitigating it by optimizing surveillance and identifying candidate prevention targets. Further studies are required to validate our findings in an independent cohort of LS patients over multiple visits.

Immune Microenvironment Profiling of Normal Appearing Colorectal Mucosa Biopsied Over Repeat Patient Visits Reproduciably Separates Lynch Syndrome Patients Based on Their History of Colon Cancer.

Introduction: Lynch syndrome (LS) is the most common hereditary cause of colorectal cancer (CRC), increasing lifetime risk of CRC by up to 70%. Despite this higher lifetime risk, disease penetrance in LS patients is highly variable and most LS patients undergoing CRC surveillance will not develop CRC. Therefore, biomarkers that can correctly and consistently predict CRC risk in LS patients are needed to both optimize LS patient surveillance and help identify better prevention strategies that reduce risk of CRC development in the subset of high-risk LS patients. Methods: Normal-appearing colorectal tissue biopsies were obtained during repeat surveillance colonoscopies of LS patients with and without a history of CRC, healthy controls (HC), and patients with a history of sporadic CRC. Biopsies were cultured in an ex-vivo explant system and their supernatants were assayed via multiplexed ELISA to profile the local immune signaling microenvironment. High quality cytokine signatures were identified using rx COV fidelity metric. These signatures were used to perform biomarker selection by computing their selection probability based on penalized logistic regression. Results: Our study demonstrated that cytokine based local immune microenvironment profiling was reproducible over repeat visits and sensitive to patient LS-status and CRC history. Furthermore, we identified sets of biomarkers whose differential expression was predictive of LS-status in patients when compared to sporadic CRC patients and in identifying those LS patients with or without a history of CRC. Enrichment analysis based on these biomarkers revealed an LS and CRC status dependent constitutive inflammatory state of the normal appearing colonic mucosa. Discussion: This prospective pilot study demonstrated that immune profiling of normal appearing colonic mucosa discriminates LS patients with a prior history of CRC from those without it, as well as patients with a history of sporadic CRC from HC. Importantly, it suggests existence of immune signatures specific to LS-status and CRC history. We anticipate that our findings have the potential to assess CRC risk in individuals with LS and help in preemptively mitigating it by optimizing surveillance and identifying candidate prevention targets. Further studies are required to validate our findings in an independent cohort of LS patients over multiple visits.

rxCOV is a quantitative metric for assessing immunoassay analyte fidelity.

Immunoassay based bioanalytical measurements are widely used in a variety of biomedical research and clinical settings. In these settings they are assumed to faithfully represent the experimental conditions being tested and the sample groups being compared. Although significant technical advances have been made in improving sensitivity and quality of the measurements, currently no metrics exist that objectively quantify the fidelity of the measured analytes with respect to noise associated with the specific assay. Here we introduce ratio of cross-coefficient-of-variation (rxCOV), a fidelity metric for objectively assessing immunoassay analyte measurement quality when comparing its differential expression between different sample groups or experimental conditions. We derive the metric from first principles and establish its feasibility and applicability using simulated and experimental data. We show that rxCOV assesses fidelity independent of statistical significance, and importantly, identifies when latter is meaningful. We also discuss its importance in the context of averaging experimental replicates for increasing signal to noise ratio. Finally, we demonstrate its application in a Lynch Syndrome case study. We conclude by discussing its applicability to multiplexed immunoassays, other biosensing assays, and to paired and unpaired data. We anticipate rxCOV to be adopted as a simple and easy-to-use fidelity metric for performing robust and reproducible biomedical research.

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.

Brief Report: Dipyridamole Decreases Gut Mucosal Regulatory T-Cell Frequencies Among People With HIV on Antiretroviral Therapy.

BACKGROUND: We had previously conducted a double-blind, randomized placebo-controlled, partial cross-over trial showing that 12 weeks of dipyridamole decreased CD8 T-cell activation among treated HIV(+) individuals by increasing extracellular adenosine levels. METHODS: In this substudy, rectosigmoid biopsies were obtained from 18 participants (9 per arm), to determine whether 12 weeks of dipyridamole affects mucosal immune cells. Participants randomized to placebo were then switched to dipyridamole for 12 weeks while the treatment arm continued dipyridamole for another 12 weeks. We evaluated T-cell frequencies and plasma markers of microbial translocation and intestinal epithelial integrity. Linear regression models on log-transformed outcomes were used for the primary 12-week analysis. RESULTS: Participants receiving dipyridamole had a median 70.2% decrease from baseline in regulatory T cells (P = 0.007) and an 11.3% increase in CD8 T cells (P = 0.05). There was a nonsignificant 10.80% decrease in plasma intestinal fatty acid binding protein levels in the dipyridamole arm compared with a 9.51% increase in the placebo arm. There were no significant differences in plasma levels of ß-D-glucan. In pooled analyses, there continued to be a significant decrease in regulatory T cells (-44%; P = 0.004). There was also a trend for decreased CD4 and CD8 T-cell activation. CONCLUSION: Increasing extracellular adenosine levels using dipyridamole in virally suppressed HIV (+) individuals on antiretroviral therapy can affect regulation of gut mucosal immunity.