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.

中文翻译：

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托法替尼抑制溃疡性结肠炎和健康粘膜外植体的炎症细胞因子，并与 T 细胞中 P-STAT1/3 减少相关


简介：动物疾病模型的可转化性较差阻碍了新的炎症性肠病（IBD）疗法的开发。我们描述了一种临床前离体系统，使用来自溃疡性结肠炎（UC）和健康对照（HC）参与者的新鲜获得且特征良好的人类结直肠组织，使用 JAK/STAT 抑制剂托法替尼（TOFA）测试潜在疗法的功效和靶标参与度。 ）作为模型治疗。方法：培养来自 HC 和 UC 的结直肠活检组织，并用多种有丝分裂原+/-TOFA 刺激。使用 29 种分析物多重 ELISA 检测可溶性生物标志物。 STAT1/3 磷酸化激活后，通过流式细胞术测定 PBMC 和分离的粘膜单核细胞 (MMC) 中 CD3 ⁺ CD4 ⁺和 CD3 ⁺ CD8 ⁺ T 细胞的靶标参与情况。使用线性混合效应模型、t 检验和方差分析来分析数据。使用惩罚和贝叶斯逻辑回归模型进行生物标志物选择，并使用均匀流形近似和投影（UMAP）将结果可视化。结果：在基线条件下，UC 中 27/29 的生物标志物相对于 HC 有所增加。外植体刺激增加了生物标志物的释放幅度，扩大了功效和靶点参与研究的动态范围。逻辑回归分析确定了最具代表性的 UC 基线和刺激生物标志物。 TOFA 抑制依赖于 JAK/STAT 信号传导的生物标志物。 T 细胞中的 STAT1/3 磷酸化揭示了 PBMC 和 MMC 之间的区室差异。结论：免疫原刺激以相似的模式增加 HC 和 UC 生物标志物的释放，同时增强治疗效果研究的动态范围。 这项工作证明了离体人类结直肠组织作为评估新 IBD 治疗药物的靶点参与和下游效应的临床前工具的力量。