- 作者列表："Parakrama R","Fogel E","Chandy C","Augustine T","Coffey M","Tesfa L","Goel S","Maitra R
BACKGROUND:KRAS mutations are prevalent in 40-45% of patients with colorectal cancer (CRC) and targeting this gene has remained elusive. Viruses are well known immune sensitizing agents. The therapeutic efficacy of oncolytic reovirus in combination with chemotherapy is examined in a phase 1 study of metastatic CRC. This study evaluates the nature of immune response by determining the cytokine expression pattern in peripheral circulation along with the distribution of antigen presenting cells (APCs) and activated T lymphocytes. Further the study evaluates the alterations in exosomal and cellular microRNA levels along with the effect of reovirus on leukocyte transcriptome. METHODS:Reovirus was administered as a 60-min intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3 × 1010. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood prior to reovirus administration and post-reovirus on days 2, 8, and 15. The expression profile of 25 cytokines in plasma was assessed (post PBMC isolation) on an EMD Millipore multiplex Luminex platform. Exosome and cellular levels of miR-29a-3p was determined in pre and post reovirus treated samples. Peripheral blood mononuclear cells were stained with fluorophore labelled antibodies against CD4, CD8, CD56, CD70, and CD123, fixed and evaluated by flow cytometry. The expression of granzyme B was determined on core biopsy of one patient. Finally, Clariom D Assay was used to determine the expression of 847 immune-related genes when compared to pre reovirus treatment by RNA sequencing analysis. A change was considered if the expression level either doubled or halved and the significance was determined at a p value of 0.001. RESULTS:Cytokine assay indicated upregulation at day 8 for IL-12p40 (2.95; p = 0.05); day 15 for GM-CSF (3.56; p = 0.009), IFN-y (1.86; p = 0.0004) and IL-12p70 (2.42; p = 0.02). An overall reduction in IL-8, VEGF and RANTES/CCL5 was observed over the 15-day period. Statistically significant reductions were observed at Day 15 for IL-8 (0.457-fold, 53.3% reduction; p = 0.03) and RANTES/CC5 (0.524-fold, 47.6% reduction; p = 0.003). An overall increase in IL-6 was observed, with statistical significance at day 8 (1.98- fold; 98% increase, p = 0.00007). APCs were stimulated within 48 h and activated (CD8+ CD70+) T cells within 168 h as determine by flow cytometry. Sustained reductions in exosomal and cellular levels of miR-29a-3p (a microRNA upregulated in CRC and associated with decreased expression of the tumor suppressor WWOX gene) was documented. Reovirus administration further resulted in increases in KRAS (33x), IFNAR1 (20x), STAT3(5x), and TAP1 (4x) genes after 2 days; FGCR2A (23x) and CD244 (3x) after 8 days; KLRD1 (14x), TAP1 (2x) and CD244(2x) after 15 days. Reductions (> 0.5x) were observed in VEGFA (2x) after 2 days; CXCR2 (2x), ITGAM (3x) after 15 days. CONCLUSIONS:Reovirus has profound immunomodulatory properties that span the genomic, protein and immune cell distribution levels. This is the first study with reovirus in cancer patients that demonstrates these multi- layered effects, demonstrating how reovirus can function as an immune stimulant (augmenting the efficacy of immuno-chemo-therapeutic drugs), and an oncolytic agent. Reovirus thus functions bimodally as an oncolytic agent causing lysis of tumor cells, and facilitator of immune-mediated recognition and destruction of tumor cells.
背景: KRAS突变在 40-45% 的结直肠癌 (CRC) 患者中普遍存在，并且靶向该基因仍然难以捉摸。病毒是众所周知的免疫增敏剂。在转移性CRC的 1 期研究中检查溶瘤呼肠孤病毒联合化疗的治疗效果。本研究通过确定外周循环中的细胞因子表达模式以及抗原提呈细胞 (apc) 和活化T淋巴细胞的分布来评估免疫应答的性质。进一步，该研究评估了外泌体和细胞microRNA水平的改变以及呼肠孤病毒对白细胞转录组的影响。 方法: 呼肠孤病毒以 3 × 1010 的组织培养感染剂量 (TCID50) 每 28 天静脉输注一次 60 min，连续 5 天。在呼肠孤病毒给药前和呼肠孤病毒后第 2 、 8 和 15 天从全血中分离外周血单核细胞 (PBMC)。在EMD Millipore多重Luminex平台上评估血浆中 25 种细胞因子的表达谱 (PBMC隔离后)。在呼肠孤病毒处理前后的样本中测定miR-29a-3p的外泌体和细胞水平。外周血单核细胞用抗CD4 、CD8 、CD56 、CD70 和CD123 的荧光团标记抗体染色，固定并通过流式细胞术进行评价。在 1 例患者的核心活检中测定颗粒酶B的表达。最后，通过RNA测序分析，与呼肠孤病毒治疗前相比，使用clarlom D法测定 847 个免疫相关基因的表达。如果表达水平加倍或减半，并在p值为 0.001 时确定显著性，则考虑变化。 结果: 细胞因子检测表明，IL-12p40 在第 8 天上调 (2.95; P = 0.05); GM-CSF在第 15 天上调 (3.56; P = 0.009)，IFN-y (1.86; P = 0.0004) 和IL-12p70 (2.42; P = 0.02)。在 15 天期间观察到IL-8，VEGF和RANTES/CCL5 的总体减少。IL-8 (0.457 倍，53.3% 减少; P = 0.03) 和RANTES/CC5 (0.524 倍，47.6% 减少; P = 0.003)。观察到IL-6 总体增加，1.98 天有统计学意义 (98% 倍; 增加 0.00007，p =)。流式细胞术测定APCs在 48 h内刺激，在 168 h内激活 (CD8 + CD70 +) T细胞。记录了miR-29a-3p (一种在CRC中上调并与肿瘤抑制因子WWOX基因表达降低相关的microRNA) 的外泌体和细胞水平的持续降低。2 天后，呼肠孤病毒给药进一步导致KRAS (33x) 、IFNAR1 (20x) 、STAT3(5x) 和TAP1 (4x) 基因增加; FGCR2A (23x) 8 天后和CD244 (3x); 15 天后KLRD1 (14x) 、TAP1 (2x) 和CD244(2x)。2 天后VEGFA (2x) 减少 (> 0.5x); 15 天后CXCR2 (2x)，ITGAM (3x) 减少。 结论: 呼肠孤病毒具有深刻的免疫调节特性，跨越基因组、蛋白质和免疫细胞分布水平。这是第一项在癌症患者中使用呼肠孤病毒的研究，证明了这些多层效应，证明了呼肠孤病毒如何作为免疫刺激剂发挥作用 (增强免疫化疗药物的功效)，和溶瘤剂。呼肠孤病毒因此具有双重功能，可引起肿瘤细胞裂解，并促进免疫介导的肿瘤细胞识别和破坏。
METHODS::Chronic diseases, including inflammatory bowel disease (IBD) urgently need new biomarkers as a significant proportion of patients, do not respond to current medications. Inflammation is a common factor in these diseases and microbial sensing in the intestinal tract is critical to initiate the inflammation. We have identified ELMO1 (Engulfment and Cell Motility Protein-1) as a microbial sensor in epithelial and phagocytic cells that turns on inflammatory signals. Using a stem-cell-based "gut-in-a-dish" coculture model, we studied the interactions between microbes, epithelium and monocytes in the context of IBD. To mimic the in-vivo cell physiology, enteroid-derived monolayers (EDMs) were generated from the organoids isolated from WT and ELMO1-/- mice and colonic biopsies of IBD patients. The EDMs were infected with the IBD-associated microbes to monitor the inflammatory responses. ELMO1-depleted EDMs displayed a significant reduction in bacterial internalization, a decrease in pro-inflammatory cytokine productions and monocyte recruitment. The expression of ELMO1 is elevated in the colonic epithelium and in the inflammatory infiltrates within the lamina propria of IBD patients where the higher expression is positively correlated with the elevated expression of pro-inflammatory cytokines, MCP-1 and TNF-α. MCP-1 is released from the epithelium and recruits monocytes to the site of inflammation. Once recruited, monocytes require ELMO1 to engulf the bacteria and propagate a robust TNF-α storm. These findings highlight that the dysregulated epithelial ELMO1→MCP-1 axis can serve as an early biomarker in the diagnostics of IBD and other inflammatory disorders.
METHODS:BACKGROUND:Peripheral blood eosinophilia (PBE) is a biomarker of an aggressive multiyear natural history in adults with inflammatory bowel diseases (IBDs). Additionally, PBE at diagnosis is associated with higher disease activity in pediatric-onset IBD. We sought to determine if PBE can function as a biomarker of long-term disease severity in pediatric-onset IBD patients who are followed into adulthood. METHODS:We analyzed a consented, prospective, natural history IBD registry at an adult tertiary center from 2009 to 2018. Prevalence of PBE was evaluated in both pediatric- and adult-onset IBD patients. Demographics, clinical characteristics, and health care utilization data were compared in patients with and without PBE. RESULTS:Among 2800 adult IBD patients, 23.4% had pediatric-onset disease. PBE was found in 34% of the pediatric-onset patients compared with 26.8% of the adult-onset IBD patients (P < 0.001). In the pediatric-onset IBD cohort, PBE was associated with higher rates of allergies (P < 0.0001), but not of asthma, allergic rhinitis, or primary sclerosing cholangitis. In the adult IBD patients with pediatric-onset disease, PBE was associated with higher rates of C-reactive protein elevation (P < 0.0001), erythrocyte sedimentation rate elevation (P < 0.0001), higher health care utilization, and higher average health care charges per year (P < 0.00001). CONCLUSIONS:Peripheral blood eosinophilia was more prevalent in adult IBD patients with pediatric-onset compared with adult-onset disease. Among all IBD patients with long-term follow-up, PBE defined a subgroup with more severe illness. These data suggest that PBE may be a biomarker for a high-risk subgroup with high cost trajectory and long-term severity in pediatric-onset IBD that persists into adulthood.
METHODS::Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex pathogenesis, affecting people of all ages. They are characterized by alternating phases of clinical relapse and remission, depending on the fine balance between immune cells and the gut microbiota. The cross talk between cells of the immune system and the gut microbiota can result in either tolerance or inflammation, according to multifactorial triggers, ranging from environmental factors to genetic susceptibility. Glucocorticoid (GC) administration remains the first-line treatment for IBDs, although long-term use is limited by development of serious adverse effects. Recently, new alternative pharmacological therapies have been developed, although these are not always effective in IBD patients. There is a constant demand for effective new drug targets to guarantee total remission and improve the quality of life for IBD patients. The glucocorticoid-induced leucine zipper (GILZ) has been implicated as a promising candidate for this purpose, in view of its powerful anti-inflammatory effects that mimic those of GCs while avoiding their unwanted adverse reactions. Here we present and discuss the latest findings about the involvement of GILZ in IBDs.