Anti-neutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability increasing protein (BPI): a new seromarker for inflammatory bowel disease and associated disorders

It was our purpose to determine the immunodiagnostic value of ANCA directed against BPI in diseases known to be associated with ANCA, such as ANCA-associated vasculitides, inflammatory bowel disease (IBD) and the associated condition primary sclerosing cholangitis. The immunoreactivity of recombinant BPI (rBPI) was established in order to develop an ELISA specific for rBPI. By means of this assay, BPI-ANCA were assessed in sera of 178 patients with IBD or the associated disorder primary sclerosing cholangitis, 112 patients with ANCA-associated vasculitides, and in sera of 182 disease and 140 healthy controls. BPI-ANCA were found to be closely associated with IBD and primary sclerosing cholangitis (34% and 44% of ANCA-positive sera, respectively). By contrast, BPI-ANCA positivity was low (<10%) in the double-negative sera of patients with ANCA-associated vasculitides and in disease and healthy controls. BPI-ANCA appear to constitute an important marker for IBD and primary sclerosing cholangitis, but not for the ANCA-associated vasculitides.     

Imbalanced secondary mucosal antioxidant response in inflammatory bowel disease

Intestinal mucosal damage in the inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC) involves reactive oxygen metabolites (ROMs). ROMs are neutralized by endogenous antioxidant enzymes in a carefully balanced two-step pathway. Superoxide dismutases (SODs) convert superoxide anion to hydrogen peroxide (H(2)O(2)), which is subsequently neutralized to water by catalase (CAT) or glutathione peroxidase (GPO). Remarkably changed expression levels of the three isoforms of SOD in paired non-inflamed and inflamed mucosae from CD and UC patients have been previously reported in comparison to normal control mucosa. Most notable was the strong up-regulation of Mn-SOD in inflamed epithelium. It was hypothesized that in order to provide optimal protection against ROM-mediated damage, these changes should be coordinately counterbalanced by an increased H(2)O(2)-neutralizing capacity. Therefore, the same tissue samples were used to assess the levels, activities, and/or localization of the most prominent mucosal H(2)O(2)-related antioxidants CAT, GPO, glutathione (GSH), myeloperoxidase (MPO), and metallothionein (MT). Quantitative measurements showed that in both CD and UC patients, intestinal inflammation was associated with increased activities of CAT, GPO, and MPO, whereas the mucosal GSH content was unaffected and the concentration of MT was decreased. Despite this overall increase in mucosal H(2)O(2)-metabolizing enzyme capacity, immunohistochemical analysis revealed a differentially disturbed antioxidant balance in IBD epithelium and lamina propria. In the lamina propria, the risk of direct H(2)O(2)-mediated damage seemed to be restrained by the increasing numbers of CAT- and MPO-positive monocytes/macrophages and neutrophils that infiltrated the inflamed areas. On the other hand, MPO overexpression might increase the lamina propria levels of hypochlorous acid, a stable ROM with multiple pro-inflammatory effects. In the epithelium, the number of cells that expressed CAT remained unchanged during inflammation and GPO was found in only a very low and constant number of epithelial cells. In addition, the inflamed epithelium displayed decreased expression of the hydroxyl radical (OH(*)) scavenger MT. In view of the high epithelial SOD levels in inflamed IBD epithelium, it is speculated that the efficient removal of excess H(2)O(2) is hampered in these cells, thereby increasing not only the risk of detrimental effects of H(2)O(2) directly, but also those of its extremely reactive derivatives such as OH(*). Taken together, the results suggest an imbalanced and inefficient endogenous antioxidant response in the intestinal mucosa of IBD patients, which may contribute to both the pathogenesis and the perpetuation of the inflammatory processes.     

Immune privilege in the gut: the establishment and maintenance of non-responsiveness to dietary antigens and commensal flora

Summary:  Immune privilege in the gut is the result of a complex interplay between the gut microbiome, gut luminal antigens, and the intestinal epithelial barrier. Composed of both physical and immunochemical components, the intestinal barrier secretes immunoregulatory mediators that promote the generation of tolerogenic antigen-presenting cells, phagocytic innate immune cells characterized by 'inflammatory anergy', and regulatory cells of the adaptive immune system. Innate immune cells mediate controlled transepithelial transport of luminal antigens as far as the mesenteric lymph nodes, where the intestinal and peripheral immune systems intersect. This promotes the generation of adaptive regulatory lymphocytes that actively suppress effector cell responses against gut luminal antigens and flora. The net result is the generation of tolerance to dietary antigens and the maintenance of gut homeostasis. Dysregulation of this complex immunoregulatory network leads to diseases such as food allergy and inflammatory bowel disease. Future therapies for these diseases will likely involve the functional restoration of the barrier and regulatory cell functions at the epithelial/luminal interface.     

Differential expression of TGF-beta1 and TGF-beta3 in serosal tissues of human intraperitoneal organs and peritoneal adhesions

Elevated local expression of transforming growth factor (TGF-beta) has been associated with increased incidence of peritoneal adhesion formation. In this study we determine whether differences in basal expression of TGF-beta in serosal tissue of peritoneal organs correlate with incidence of adhesion formation. Serosal tissue of parietal peritoneum, uterus, oviduct, ovary, omentum, large and small bowels as well as adhesions, skin, fascia, subcutaneous tissue, peritoneal fluid and serum were collected from 57 subjects with/without adhesions who were undergoing abdominal/pelvic surgery. To determine TGF-beta1 and TGF-beta3 mRNA and protein expression, total RNA and protein were isolated from these tissues and along with the fluids, subjected to quantitative RT-PCR and enzyme-linked immunosorbent assay (ELISA) respectively. Tissue sections were immunostained for TGF-beta1 and TGF-beta3 protein. We found that TGF-beta1 and TGF-beta3 mRNA and protein are expressed in these tissues and present in peritoneal fluids and serum, with considerable variations in level of their expression. Comparatively, there was more variation in TGF-beta1 than TGF-beta3 expression without age or gender relation. Adhesions express a significantly higher TGF-beta1 mRNA and have the highest TGF-beta1:TGF-beta3 ratio, with lowest concentrations and ratio detected in omentum, small and large bowels; in contrast uterus expresses higher TGF-beta3, with lowest concentrations detected in subcutaneous tissue and large bowels (P < 0.05). A similar trend was also observed for total (active + latent) TGF-beta1 protein expression, with low active TGF-beta1 that was not significantly different among the tissue extracts and fluids. However, the lowest active:total TGF-beta1 ratio was found in adhesions and ovary. In subjects with adhesions, the adhesions express significantly more TGF-beta1 compared to parietal peritoneum (P < 0.05). Immunoreactive TGF-beta1 and TGF-beta3 protein were present in various cell types in these tissues with intensity reflecting their mRNA and protein expression. In conclusion, we provided evidence that serosal tissue of various peritoneal organs and adhesions express TGF-beta1 and TGF-beta3. Since TGF-beta is expressed differently in these tissues and tissue injury often alters the expression of TGF-beta, we propose that tissues with a higher basal expression of TGF-beta may become predisposed to develop more adhesions compared to others.     

Expression of TNFalpha by muscle fibers in biopsies from children with untreated juvenile dermatomyositis: association with the TNFalpha-308A allele

Juvenile dermatomyositis (JDM) is the most common pediatric inflammatory myopathy. In patients with JDM, the A --> G polymorphism in the tumor necrosis factor alpha (TNFalpha)-308 promoter region (TNFalpha-308A) is associated with prolonged disease course and increased production of TNFalpha by peripheral blood mononuclear cells (Arthritis Rheum. 43, 2368-2377, 2000). Magnetic resonance imaging directed biopsies from 21 white children with untreated JDM were evaluated for TNFalpha expression. Using monoclonal antibody to TNFalpha, fresh frozen sections were processed by the standard immunohistochemical technique. We investigated the association among the expression of TNFalpha by muscle fibers, disease activity, duration of untreated disease, and the TNFalpha-308 polymorphism. Untreated children with JDM who had the TNFalpha-308A allele had an increased number of TNFalpha stained muscle fibers than children with the TNFalpha-308G allele (P = 0.001). There was no association with disease activity or duration of untreated disease. We speculate that muscle fiber production of TNFalpha provides a microenvironment in which TNFalpha acts synergistically with other mediators to prolong muscle fiber damage.     

腺样体肥大患儿血清炎症介质和淋巴细胞亚群表达特征及与患儿预后的相关性

目的研究腺样体肥大患儿血清炎症介质、淋巴细胞亚群表达特征及其与患儿预后的相关性。方法选取浙江中医药大学附属第三医院收治的86例腺样体肥大患儿(观察组)和86例健康儿童(对照组)作为研究对象,采集血液标本测定血清炎症介质及淋巴细胞亚群相关指标,对比两组儿童的测定结果。分析腺样体肥大患儿的肥大程度、腺样体再增生情况,对比不同肥大程度患儿的血清指标,并分析各指标与患儿病情程度及预后(腺样体再增生)的关系。结果观察组患儿肿瘤坏死因子-α(TNF-α)、可溶性白细胞介素-2受体(sIL-2R)及白细胞介素-6(IL-6)水平分别为(492.44±50.56)mg/L、(420.48±61.79)U/ml及(33.65±4.51)pg/ml,对照组儿童分别为(301.22±41.65)mg/L、(236.55±57.90)U/ml及(16.25±3.27)pg/ml,差异均有统计学意义(t=16.004,P<0.05;t=20.311,P<0.05;t=8.995,P<0.05)。观察组CD4⁺、CD8⁺及CD4⁺/CD8⁺水平分别为(35.75±7.05)、(16.22±2.69)及(1.46±0.67),对照组分别为(34.25±4.53)、(17.62±2.35)及(1.40±0.62),差异均无统计学意义(t=1.036,P>0.05;t=0.905,P>0.05;t=0.823,P>0.05)。相比腺样体中度肥大患儿,重度肥大患儿的TNF-α、sIL-2R、IL-6、CD4⁺及CD4⁺/CD8⁺水平明显更高(P<0.05)。相比未增生患儿,腺样体再增生患儿的TNF-α、sIL-2R、IL-6、CD4⁺及CD4⁺/CD8⁺水平明显更高(P<0.05)。相关分析显示,患儿腺样体肥大程度与TNF-α、sIL-2R、IL-6、CD4⁺及CD4⁺/CD8⁺水平均呈正相关(P<0.05)。患儿腺体再增生与TNF-α、sIL-2R、IL-6、CD4⁺及CD4⁺/CD8⁺水平也呈正相关(P<0.05)。结论腺样体肥大患儿的血清炎症介质及部分T淋巴细胞亚群表达显著升高,其与腺样体肥大程度、患儿预后密切相关。     

中医药对糖尿病足溃疡相关细胞因子调控作用的研究进展

糖尿病足溃疡是糖尿病严重的破坏性并发症,致残率及致死率逐年增高,严重威胁人类身心健康。中医药治疗糖尿病足溃疡,注重辨证论治与整体观念相结合,不仅能改善中医证候,更能在加速创面愈合的同时减少创面的复发,一定程度上延缓了糖尿病足溃疡的进一步恶化,降低其致残率及致死率。现代研究发现,糖尿病足溃疡难以愈合与各种细胞因子如炎症因子、生长因子、趋化因子等分布异常密切相关;随着现代医学对中医药研究的不断深入,中药单体及复方调控细胞因子治疗糖尿病足溃疡已成为研究热点,该文通过对目前国内外研究现状进行归纳,得出以下总结：(1)芝麻酚、栀子苷、当归补血汤、紫朱软膏等调节肿瘤坏死因子-α(TNF-α)、白细胞介素（IL）-1、IL-6、IL-10等炎症因子,抑制创面炎症。(2)白芷、丹酚酸B、四效散、壮药拔毒生肌膏等调节血管内皮生长因子（VEGF）、血小板衍生生长因子（PDGF）、转化生长因子（TGF）、表皮生长因子（EGF）等生长因子,促进创面胶原沉积及血管新生。(3)芍药苷、隐丹参酮、蜂毒、回阳生肌汤等调节CXC趋化因子配体（CXCL）1、CXCL2、CC趋化因子配体（CCL）2、CCL3、基质细胞衍生...     

葛花、枳椇子及其配伍对小鼠急性酒精性胃黏膜损伤的保护作用及机制

目的 探讨葛花、枳椇子及其配伍对急性酒精性胃黏膜损伤的改善作用,为进一步开展葛花、枳椇子及其配伍防治酒精致多脏器损伤奠定基础。方法 采用多次灌胃给予56%红星二锅头白酒（15 mL·kg^-1）建立小鼠急性酒精性胃黏膜损伤模型,将120只ICR雄性小鼠随机分为空白组、模型组、奥美拉唑组（0.026 g·kg^-1）、葛花-枳椇子（配伍）高、中、低剂量组（29.2、14.6、7.3 g·kg^-1）、葛花组（19.5 g·kg^-1）、枳椇子组（19.5 g·kg^-1）共8个组,每组15只,动物适应性喂养1周后,按10 mL·kg^-1预给相应药物3 d,从第4天开始,给药1 h后按15 mL·kg^-1灌胃二锅头白酒,空白组给予相同体积去离子水,记录小鼠醉酒和醒酒时间,连续给药给酒3 d,末次给药1 h后摘眼球处死;气相色谱仪测定各组小鼠血清中乙醇体积分数,紫外-可见分光光度计检测各组小鼠胃黏膜中乙醇脱氢酶（ADH）活性;苏木素-伊红（HE）染色观察胃黏膜病理变化;酶联免疫吸附测定法（ELISA）检测各组小鼠血清中炎症因子含量;实时荧光定量聚合酶链式反应（Real-time PCR）检测核转录因子-κB（NF-κB）p65和NF-κB抑制蛋白α（IκBα）mRNA表达。结果 与正常组比较,模型组血清中白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）含量升高（P<0.05）,胃黏膜组织NF-κB p65 mRNA表达升高（P<0.01）,IκBα mRNA表达降低（P<0.01）;与模型组比较,奥美拉唑组、配伍高、中剂量组、葛花组醉酒时间延长（P<0.05）,配伍高、中剂量组醒酒时间缩短（P<0.05）,配伍高剂量组血清中乙醇体积分数降低（P<0.05）,奥美拉唑组、配伍高、中剂量组胃黏膜中ADH活性升高（P<0.05）,配伍各剂量组、葛花组肉眼损伤评分降低（P<0.05）,奥美拉唑组、配伍各剂量组、葛花组病理损伤评分降低（P<0.01）,各给药组血清中IL-6表达降低（P<0.05）,奥美拉唑组、配伍各剂量组、枳椇子组血清中IL-1β表达降低（P<0.05）,配伍高、中剂量组血清中TNF-α表达降低（P<0.05）,各给药组胃黏膜组织NF-κB p65 mRNA表达降低（P<0.05）,奥美拉唑组、配伍各剂量组胃黏膜组织IκBα mRNA表达增加（P<0.05）;与高剂量组比较,配伍低剂量组与枳椇子组醉酒时间缩短（P<0.01）,葛花、枳椇子组醒酒时间延长（P<0.01）,配伍中、低剂量组、葛花组、枳椇子组血清中乙醇体积分数升高（P<0.05）,配伍中、低剂量组、枳椇子组肉眼损伤积分增加（P<0.05）,配伍中、低剂量组、葛花组、枳椇子组病理损伤积分增加（P<0.01）,配伍低剂量组、葛花组、枳椇子组血清中IL-1β含量升高（P<0.01）,葛花组与枳椇子组胃黏膜组织IκBα mRNA表达量降低（P<0.05）;与配伍中剂量组比较,枳椇子组醉酒时间缩短（P<0.05）,葛花组醒酒时间延长（P<0.05）,葛花组、枳椇子组病理损伤积分增加（P<0.01）,配伍低剂量组、葛花组、枳椇子组血清中IL-1β含量升高（P<0.05）;与配伍低剂量组比较,枳椇子组病理损伤积分增加（P<0.05）。结论 葛花、枳椇子及其配伍能起到对小鼠急性酒精性胃黏膜损伤的防治作用,可能与抑制胃黏膜NF-κB信号通路的表达有关,且配伍高剂量组药效最佳。     

慢溃宁方对DSS诱导溃疡性结肠炎小鼠NLRP3/Caspase-1/GSDMD细胞焦亡通路和肠道菌群的影响

目的 探讨慢溃宁方对葡聚糖硫酸钠（DSS）诱导的溃疡性结肠炎（UC）小鼠肠道菌群的调控作用,和对NOD样受体（NLR）P3/胱天蛋白酶（Caspase）-1/Gasdermin D（GSDMD）细胞焦亡通路所介导炎症的影响。方法 SPF级C57BL/6小鼠60只,随机分为空白组、模型组、慢溃宁方组（20 g·kg^-1）、美沙拉秦组（0.266 g·kg^-1）,各组15只。小鼠通过自由饮用3%DSS溶液,7 d构建UC模型。造模开始12 h后,治疗组每天灌胃给药,其余组灌胃等体积生理盐水。记录小鼠每日体质量等情况,并评估计算疾病活动指数（DAI）。第8天麻醉后,脱臼颈椎处死小鼠,收集结肠和粪便,记量结肠长度;观察结肠组织苏木素-伊红（HE）染色后的病理学改变;结肠中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）水平用酶联免疫吸附测定法（ELISA）检测;基于16S rRNA测序技术检测各组小鼠粪便中肠道菌群的差异;NLRP3/Caspase-1/GSDMD蛋白结肠组织中含量用蛋白免疫印迹法（Western blot）检测。结果 相较于空白组,模型组小鼠的DAI升高（P<0.01）,结肠长度显著缩短（P<0.01）,结肠黏膜损伤严重,TNFα、IL-1β、IL-18水平均明显升高（P<0.01）,且结肠组织中NLRP3/Caspase-1/GSDMD蛋白含量显著升高（P<0.01）,肠道菌群结构改变,门水平上的放线菌门、拟杆菌门与变形菌门丰度减少,厚壁菌门丰度增加;属水平上的乳酸杆菌、普雷沃氏菌和鼠杆菌属丰度减少,拟杆菌属、芽孢杆菌、毛螺旋菌属NK4A136丰度增加。慢溃宁方组、美沙拉秦组相较于模型组,第3天后DAI显著降低（P<0.01）,结肠长度均显著增加（P<0.01）,结肠的炎症浸润及黏膜结构损伤减轻,且结肠TNF-α、IL-1β、IL-18水平均显著下降（P<0.01）,NLRP3/Caspase-1/GSDMD蛋白在结肠组织中的含量明显降低（P<0.05,P<0.01）,门水平上的变形菌门和拟杆菌门丰度增加,厚壁菌门丰度减少。结论 慢溃宁方可通过抑制细胞经典焦亡通路,缓解UC小鼠结肠炎症反应,减轻结肠损伤,并对肠道菌群紊乱有改善作用。