Biomechanical changes in oxazolone-induced colitis in BALB/C mice

Ulcerative colitis (UC) is associated with intestinal and extra intestinal clinical manifestations. The profound organic changes in UC indicate that the colonic mechanical and mechanosensory functions are affected. The aim was to study acute morphological and biomechanical properties of the distal colon in oxazolone-induced UC in BALB/C mice. Six normal male BALB/C mice and 10 oxazolone-induced UC mice were studied. UC was induced by epicutaneous and intrarectal administration of oxazolone. The mechanical test was done as a distension experiment where the colon was distended up to 20 cmH₂O. The pressure, outer diameter and length were recorded simultaneously. Circumferential and longitudinal stresses and strains were computed. The intestinal specimens were processed for histology. The mucosa was infiltrated with acute and chronic inflammatory cells. Mucosal bleeding, irregular ulcers crypt abscess, and destruction of the epithelial border were observed. Although, the mucosa in ulcers was much thinner than in the normal controls, the mucosa and submucosa around the ulcer were thicker than in the normal controls (P<0.05). Oxazolone-induced colitis increased the circumferences and wall cross-sectional area (P<0.01), the opening angle and residual strain at the serosa increased (P<0.01). Furthermore, the circumferential and longitudinal stiffness increased in the UC wall and was most pronounced in longitudinal direction. The opening angle and residual strain was linearly correlated to the wall thickness, area and inflammation degree. In conclusion, morphological and biomechanical changes of the colon occurred during the development of UC. The increased stiffness may contribute to the abnormal function in patients with UC.     

O‐8FALSE NEGATIVE BRONCHIAL CYTOLOGY

Introduction: BK virus nephropathy is an emerging cause of renal transplant failure accounting for allograft loss in 45–50% of recipients between 2–60 months post‐transplantation. The Renal Transplant Unit in Royal Free Hospital has devised a local surveillance programme for screening renal transplant patients at weekly intervals by urinary cytology (UC) and electron microscopy (EM), and confirmation of positive results by plasma PCR and allograft biopsy. Objective: (i) To monitor the implementation of local guidelines; (ii) To compare UC with EM and (iii) To identify areas for improvement. Methods: Decoy cell and EM positive cases were retrieved from the WinPath database for new renal transplant recipients (n = 55) during 1^st November 2004 to 31^st October 2005 in Royal Free Hospital. Plasma PCR was retrieved for positive cases. Results: Up to eight samples were sent for UC at random intervals from 47 patients, and up to 7 were sent for EM from 42 patients. Eleven were UC‐positive and one was EM‐positive. PCR was not requested in UC‐positive cases and 3 out of 10 were positive retrospectively. The EM‐positive case was PCR‐positive but UC‐negative. Discussion: Urine Cytology is more appropriate and cost effective for screening than Electron Microscopy. During the period covered by this audit, samples were sent in an inconsistent fashion after allograft dysfunction rather than for screening. A screening protocol has been agreed and a re‐audit is planned.     

CD177~+中性粒细胞在溃疡性结肠炎患者外周血中的表达及临床意义

目的:通过对比CD177~+中性粒细胞在溃疡性结肠炎(UC)患者与正常对照者外周血中的表达差异,分析CD177~+中性粒细胞在溃疡性结肠炎发生发展中的临床意义。方法:收集30例UC患者及20例正常对照者外周血,采用流式细胞术检测中性粒细胞CD177~+中性粒细胞表达情况,对比两组CD177表达差异。结果:UC患者外周血中CD177~+中性粒细胞表达明显高于正常对照组(P 0. 01),中度活动UC外周血CD177~+中性粒细胞表达较轻度者明显增高(P 0. 05),UC患者外周血CD177~+中性粒细胞%与Mayo评分呈显著正相关(r=0. 384,P=0. 036)。结论:CD177~+中性粒细胞在UC患者外周血表达明显增高,且与疾病活动程度密切相关,能够反映UC患者临床疾病活动程度。     

Gegen Qinlian decoction maintains colonic mucosal homeostasis in acute/chronic ulcerative colitis via bidirectionally modulating dysregulated Notch signaling

BackgroundGegen Qinlian decoction (GQ) is a well-known traditional Chinese medicine that has been clinically proven to be effective in treating ulcerative colitis (UC). However, its therapeutic mechanism has not been fully elucidated. Notch signaling plays an essential role in the regeneration of the intestinal epithelium.PurposeThis study was designed to ascertain the mechanism by which GQ participates in the recovery of the colonic mucosa by regulating Notch signaling in acute and chronic UC models.MethodsAcute and chronic UC mice (C57BL/6) were established with 3 and 2% dextran sulfate sodium (DSS), respectively, and treated with oral administration of GQ. The expression of the Notch target gene Hes1 and the Notch-related proteins RBP-J, MAML and Math1 was analyzed by western blotting. PTEN mRNA levels were detected by qRT-PCR. Mucin production that is characteristic of goblet cells was determined by Alcian blue/periodic acid-Schiff staining and verified by examining MUC2 mRNA levels by qRT-PCR. Cell proliferation was assayed by immunohistochemistry analysis of Ki67. HT-29 and FHC cells and Toll-like receptor 4 knockout (TLR4^−/−) acute UC mice were also used in this study.ResultsGQ restored the injured colonic mucosa in both acute and chronic UC models. We found that Notch signaling was hyperactive in acute UC mice and hypoactive in chronic UC mice. GQ downregulated Hes1, RBP-J and MAML proteins and augmented goblet cells in the acute UC models, whereas GQ upregulated Hes1, RBP-J and MAML proteins in chronic UC mice, reducing goblet cell differentiation and promoting crypt base columnar (CBC) stem cell proliferation. Hes1 mRNA was suppressed in TLR4^−/− UC mice, and GQ treatment reversed this effect. In vitro, GQ reduced Hes1 protein in Notch-activated HT29 and FHC cells but increased Hes1 protein in Notch-inhibited cells.ConclusionsGQ restored the colonic epithelium by maintaining mucosal homeostasis via bidirectional regulation of Notch signaling in acute/chronic UC models.     

Decreased colonization of fecal Clostridium coccoides/Eubacterium rectale species from ulcerative colitis patients in an in vitro dynamic gut model with mucin environment

The mucus layer in the colon, acting as a barrier to prevent invasion of pathogens, is thinner and discontinuous in patients with ulcerative colitis (UC). A recent developed in vitro dynamic gut model, the M-SHIME, was used to compare long-term colonization of the mucin layer by the microbiota from six healthy volunteers (HV) and six UC patients and thus distinguish the mucin adhered from the luminal microbiota. Although under the same nutritional conditions, short-chain fatty acid production by the luminal communities from UC patients showed a tendency toward a lower butyrate production. A more in-depth community analysis of those microbial groups known to produce butyrate revealed that the diversity of the Clostridium coccoides/Eubacterium rectale and Clostridium leptum group, and counts of Faecalibacterium prausnitzii were lower in the luminal fractions of the UC samples. Counts of Roseburia spp. were lower in the mucosal fractions of the UC samples. qPCR analysis for butyryl-CoA:acetate CoA transferase, responsible for butyrate production, displayed a lower abundance in both the luminal and mucosal fractions of the UC samples. The M-SHIME model revealed depletion in butyrate producing microbial communities not restricted to the luminal but also in the mucosal samples from UC patients compared to HV.     

双歧杆菌调节VIP/cAMP/PKA和mTOR通路改善溃疡性结肠炎小鼠的实验研究

摘要 目的：探讨双歧杆菌MIMBb75通过调节血管活性肠肽（VIP）/环磷酸腺苷（cAMP）/蛋白激酶A（PKA）和哺乳动物雷帕霉素靶蛋白（mTOR）通路对溃疡性结肠炎（UC）小鼠的影响。方法：BALB/c小鼠随机分为正常对照（NC）组、结肠炎模型（UC）组、Mesalazine组和MIMBb75低、高剂量组、MIMBb75高剂量+VIP antagonist组、MIMBb75高剂量+MHY1485组（每组10只）,除NC组外均采用5%葡聚糖硫酸钠（DSS）诱导UC模型。治疗结束后,观察小鼠的一般情况及UC疾病活动指数（DAI）,检测小鼠肠道组织病理损伤、结肠组织中髓过氧化物酶（MPO）活性、肠道菌群多样性（Chao指数、Shannon指数和Simpson指数）及结肠组织VIP、cAMP、PKA、水通道蛋白3（AQP3）、mTOR、核糖体蛋白S6激酶（S6K1）的mRNA和蛋白水平。结果：与UC组相比,MIMBb75低、高剂量组和Mesalazine组小鼠的体重升高、DAI评分降低,组织病理损伤得到改善,结肠长度增加,MPO活性降低,Chao指数、Shannon指数和Simpson指数升高;VIP、cAMP、PKA、AQP3的mRNA水平和VIP、cAMP、AQP3蛋白的表达及PKA的磷酸化水平升高,mTOR和S6K1 mRNA及其蛋白的磷酸化水平降低（P<0.05）。与MIMBb75高剂量组相比,MIMBb75高剂量+VIP antagonist组VIP、cAMP、PKA、AQP3的mRNA水平和VIP、cAMP、AQP3蛋白的表达及PKA的磷酸化水平降低（P<0.05）;MIMBb75高剂量+MHY1485组mTOR和S6K1 mRNA及其蛋白的磷酸化水平升高（P<0.05）。VIP antagonist和MHY1485均能逆转MIMBb75对UC小鼠的保护作用,使其结肠损伤加重,MPO活性增高（P<0.05）。结论：双歧杆菌可改善UC小鼠的结肠损伤,增加肠道菌群的多样性,这可能与激活VIP/cAMP/PKA通路、抑制mTOR通路有关。     

Intestinal macrophage/epithelial cell-derived CCL11/eotaxin-1 mediates eosinophil recruitment and function in pediatric ulcerative colitis

Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn's disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2(-/-) and eotaxin-1/2(-/-) mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80(+)CD11b(+) macrophages in DSS-induced epithelial injury and to CD68(+) intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.     

溃疡性结肠炎小鼠肠道黏膜和盲肠内容物菌群差异比较

了解溃疡性结肠炎(UC)小鼠肠道黏膜菌群的构成及其与盲肠内容物菌群的差异, 探讨肠道菌群在UC小鼠发病中的作用。

12只C57BL/6N雄性小鼠随机分为正常组、治疗组和模型组, 每组4只。采用葡聚糖硫酸钠盐(DSS)进行造模, 采用柳氮磺胺吡啶灌胃进行治疗, 造模第8天处死全部小鼠。采用16S rRNA基因高通量测序法对肠道黏膜和盲肠内容物菌群进行测序分析。

UC小鼠肠道黏膜菌群与盲肠内容物菌群相比, OTU数量和多样性均降低; Beta多样性分析显示其黏膜和盲肠内容物菌群差异较正常小鼠更大。在门和属的分类水平上, UC小鼠黏膜菌群中变形菌门、疣微菌门、魏斯菌属和肠球菌属丰度较正常小鼠升高, 厚壁菌门和拟杆菌门丰度降低; 阿克曼菌属在UC小鼠黏膜和盲肠内容物菌群中增多, 颤螺菌属在治疗组小鼠黏膜和盲肠内容物菌群中均减少。此外, 黏膜菌群中脱铁杆菌门Mucispirillum菌属丰度较高; 盲肠内容物中拟杆菌属丰度较高。物种组成分析中, 颤螺菌属、Mucispirillum、志贺菌属、魏斯菌属和肠球菌属聚类在一起。

UC小鼠黏膜菌群与盲肠内容物菌群在群落组成、多样性、优势菌群等方面存在明显差异, 黏膜菌群在UC发病机制中可能发挥着主要的作用。

     

Studies on Morphological Transformation of BALB/3T3-Derived Clones by Murine Leukemia Virus

We have described a cell line, UC1-B, derived spontaneously from BALB/3T3 mouse embryo cells, which, unlike the standard BALB/3T3, are morphologically transformed and produce bizarre viral forms in response to murine leukemia virus. Although UC1-B and BALB/3T3 are morphologically similar, and both form contact-inhibited monolayers at confluence, the UC1-B cells are partially transformed because: they grow to a slightly higher saturation density than 3T3 cells, they grow in medium lacking serum growth factors, and they produce tumors in mice. Another clone, 12A-3, derived from BALB/3T3, also transforms and produces bizarre viral forms after infection with murine leukemia virus. Unlike UC1-B cells, the 12A3-8 cells are identical in growth properties to BALB/3T3; therefore, a partially altered morphology is not required for the induction of transformation by murine leukemia virus.     

Inhibition of MLV-induced Transformation in Balb/3T3 Derived Cells

UC1-B cell transformation by MuLV virus is inhibited by dimethyl-benzyl-rifampicin.     

Studies on morphological transformation of BALB/3T3-derived clones by murine leukemia virus.

We have described a cell line, UC1-B, derived spontaneously from BALB/3T3 mouse embryo cells, which, unlike the standard BALB/3T3, are morphologically transformed and produce bizarre viral forms in response to murine leukemia virus. Although UC1-B and BALB/3T3 are morphologically similar, and both form contact-inhibited monolayers at confluence, the UC1-B cells are partially transformed because: they grow to a slightly higher saturation density than 3T3 cells, they grow in medium lacking serum growth factors, and they produce tumors in mice. Another clone, 12A-3, derived from BALB/3T3, also transforms and produces bizarre viral forms after infection with murine leukemia virus. Unlike UC1-B cells, the 12A3-8 cells are identical in growth properties to BALB/3T3; therefore, a partially altered morphology is not required for the induction of transformation by murine leukemia virus.     

A uv-sensitive Chinese hamster lung fibroblast cell line (V79/UC) with a possible defect in DNA polymerase activity is deficient in DNA repair

Studies of repair enzyme activities in a uv-sensitive cell line (V79/UC) derived from Chinese hamster V79 cells have revealed levels of total DNA polymerase that are about 50% of the levels in the parental cell line. There are a number of DNA polymerase inhibitors available which allow us to distinguish between the major forms of DNA polymerase (alpha, beta, gamma, and delta) identified in mammalian cells. Enzyme assays with these inhibitors indicate that the aphidicolin-sensitive DNA polymerase is defective in the V79/UC cell line. This could be either polymerase alpha or delta, or both. The V79/UC cells do not express resistance to aphidicolin in standard toxicity studies. However, when aphidicolin is added postirradiation in survival assays designed to measure the extent of inhibitable repair, V79/UC cells do not respond with the further decrease in survival seen in the parental line. Further evidence of a polymerase-dependent repair defect is evident from alkaline elution data. In this case the V79/UC cells show the appearance of single-strand breaks following uv irradiation in the absence of any added inhibitor. Cells of the V79/M12G parental line, on the other hand, show the appearance of single-strand breaks only when aphidicolin is present.     

黏质阿克曼菌及其代谢物短链脂肪酸与溃疡性结肠炎肠黏膜屏障的相关性研究

溃疡性结肠炎(ulcerative colitis, UC)已成为当今世界范围内的发病率高、患病人数多、病程缠绵的终身难治性疾病。而黏质阿克曼菌(Akkermansia muciniphila,A.muciniphila)及其代谢物短链脂肪酸(short chain fatty acid, SCFA)是近年来发现的对UC肠黏膜屏障具有保护作用的益生菌及代谢物,但其具体作用机制有待归纳和总结。因此本文从肠黏膜机械、化学、免疫及生物屏障这四个角度综合分析近年来的相关研究,试图探讨A. muciniphila和SCFA对肠黏膜屏障的具体作用机理,为研究UC的发病机制、治疗手段提供新视角和新思路。     

Patient Perspectives on the Experience of Being Newly Diagnosed with HIV in the Emergency Department/Urgent Care Clinic of a Public Hospital

We sought to understand patient perceptions of the emergency department/urgent care (ED/UC) HIV diagnosis experience as well as factors that may promote or discourage linkage to HIV care. We conducted in-depth interviews with patients (n=24) whose HIV infection was diagnosed in the ED/UC of a public hospital in San Francisco at least six months prior and who linked to HIV care at the hospital HIV clinic. Key diagnosis experience themes included physical discomfort and limited functionality, presence of comorbid diagnoses, a wide spectrum of HIV risk perception, and feelings of isolation and anxiety. Patients diagnosed with HIV in the ED/UC may not have their desired emotional supports with them, either because they are alone or they are with family members or friends to whom they do not want to immediately disclose. Other patients may have no one they can rely on for immediate support. Nearly all participants described compassionate disclosure of test results by ED/UC providers, although several noted logistical issues that complicated the disclosure experience. Key linkage to care themes included the importance of continuity between the testing site and HIV care, hospital admission as an opportunity for support and HIV education, and thoughtful matching by linkage staff to a primary care provider. ED/UC clinicians and testing programs should be sensitive to the unique roles of sickness, risk perception, and isolation in the ED/UC diagnosis experience, as these things may delay acceptance of HIV diagnosis. The disclosure and linkage to care experience is crucial in forming patient attitudes towards HIV and HIV care, thus staff involved in disclosure and linkage activities should be trained to deliver compassionate, informed, and thoughtful care that bridges HIV testing and treatment sites.     

龙须菜体表附生细菌的几种分离方法比较

采用超声波粉碎法、涡旋振荡法、超声波清洗法、研磨匀浆法等4种不同的方法处理龙须菜,分离其体表的附生细菌,并对分离细菌的数量、种类、形态结构、细胞壁特性等进行了观察和分析。通过对不同方法和相间方法的不同处理所得结果比较显示,超声波清洗法和研磨匀浆法对分离细菌的数量和种类效果都较差;超声波粉碎法和涡旋振荡法效果较好,尤以超声波粉碎法的30W30s处理效果最好,该方法分离到本项目4个方法13个处理获得的16个菌株中的12个菌株,龙须菜的细菌数1．75×10~6cells/g。     

NOD2/CARD15 and Toll-like 4 receptor gene polymorphism in Chilean patients with inflammatory bowel disease

Crohn's disease (CD) and ulcerative colitis (UC) are multifactorial diseases with a genetic background. Genes related to the innate immune response have been observed to be involved. Polymorphisms of Toll-like receptor 4 (TLR4) and CARD15/NOD2 are thought to be involved in the pathogenesis of inflammatory bowel disease (IBD). There is no information about the frequency of these polymorphisms in South American and Chilean populations. Aim. To investigate the distribution of CARD15/NOD2 (Arg702Trp, Gly908Arg and Leu1007fsinsC) and TLR4 (Asp299Gly) polymorphisms in Chilean patients with IBD. Methods. DNA was obtained from 22 CD, 22 UC patients and 20 healthy individuals. Genotyping was performed by allele-specific PCR and by PCR-RFLP analysis. Clinical and demographic features were characterized. Results. Among the CD patients, the clinical pattern was deemed inflammatory in 14, while five had penetrating and five stricturing, variants. One patient had esophageal involvement, five perianal, seven ileal and in 16 the colon was involved. Among the UC patients, two had proctitis, two proctosigmoiditis, four left-sided colitis and 14 pancolitis. NOD2/CARD15 analysis revealed the presence of the 702Trp allele in two CD patients (both heterozygotes), 1007fsinsC in one CD patient (heterozygote) while 908Arg was found in one UC patient. The 299Gly TLR4 allele was identified in one UC and one CD patient. Conclusion. This genetic study shows that the alleles frequently associated with IBD (1007fsinsC, 908Arg and 702Trp in NOD2/CARD15 and 299Gly TLR4) have a low incidence in Chilean, IBD patients, which is similar to European populations. It is possible that, in addition to environmental factors, other genetic polymorphisms may be involved in the pathogenesis of the disease in Chilean, IBD patients.