Human CD69 ⁺ CCR5 ⁺ T cells in mucosal tissues are poised to maintain barrier immunity in healthy and inflamed tissues.

Imaging chromatin organization at the molecular-scale resolution remains an important endeavor in basic and translational research. Stochastic optical reconstruction microscopy (STORM) is a powerful superresolution imaging technique to visualize nanoscale molecular organization down to the resolution of ~20 to 30 nm. Despite the substantial progress in imaging chromatin organization in cells and model systems, its routine application on assessing pathological tissue remains limited. It is, in part, hampered by the lack of simple labels that consistently generates high-quality STORM images on the highly processed clinical tissue. We developed a fast, simple, and robust small-molecule fluorescent probe—cyanine 5–conjugated Hoechst—for routine superresolution imaging of nanoscale nuclear architecture on clinical tissue. We demonstrated the biological and clinical significance of imaging superresolved chromatin structure in cancer development and its potential clinical utility for cancer risk stratification.

Introduction: Poor translatability of animal disease models has hampered development of new inflammatory bowel disorders (IBD) therapeutics. We describe a preclinical, ex vivo system using freshly obtained and well-characterized human colorectal tissue from 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. Methods: Colorectal biopsies from HC and 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 PBMC and isolated mucosal mononuclear cells (MMC) following activation of STAT1/3 phosphorylation. Data were analyzed using linear mixed effect 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 (UMAP). Results: Under baseline conditions, 27/29 biomarkers from UC were increased versus HC. 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 PBMC and MMC. Conclusions: Immunogen stimulation increases biomarker release in similar patterns for HC and UC, while enhancing the dynamic range for therapeutic efficacy studies. 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.

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-1_BaL, 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-1_BaL and induced a dose-dependent decrease in 15/30 biomarkers. Affected biomarkers included IP-10, MDC, MIP-1α, MIP-1β, TARC, IL12-p40, IL-15, IL-17, IL-1α, IL-7, IFNγ, IL12-p70, IL-1β, IL-4, IL-5, and TNF-β. Similarly, RNP dose-dependent inhibition was demonstrated in 7/30 biomarkers after LPS stimulation, all of which overlapped with HIV-1_BaL-induced biomarker changes. The ability of RNP to inhibit both colorectal explant HIV-1_BaL infection and inflammatory changes associated with HIV-1 infection makes RPN a promising agent for topical rectal pre-exposure prophylaxis.