多标记免疫荧光染色及多光谱成像技术在组织学研究中的应用

免疫组织化学染色及成像分析是研究组织形态和组织原位抗原表达不可或缺的检测技术,广泛应用于临床病理诊断和医学及生物学研究的各个领域。组织切片样本中蕴含着丰富的信息,但是受制于传统单标记免疫组织化学染色方法的限制,通常只能对组织中的一到两种抗原进行染色分析,而且定量结果的判读往往依赖于肉眼观测,缺乏客观标准。随着蛋白组学的发展,对现代组织学分析提出了更高的要求,例如不同的蛋白共表达和共定位分析、低丰度分子的检测、异质性分析、细胞表型统计乃至复杂组织微环境的描绘等,都需要在同一张组织切片样本上同时检测多种靶标分子。这对于理解组织微环境中各种细胞间的关系,推演信号通路上下游蛋白表达的关系,制定临床诊断和治疗方案都有着非常重要的意义。本文介绍一种基于酪胺信号放大(TSA)技术衍生而来的多标记免疫荧光染色方法,能够在同一组织切片样本上复染7种以上抗原并进行区别标记,配合光谱成像技术和定量分析软件,能够将组织中蕴含的丰富信息准确的呈现出来。这套流程化的分析方案为临床诊断和基础科研提供了更高精度和更可靠的组织学数据,将免疫组织化学分析的技术水平提升到一个新的高度。

MMP14在胰腺癌中的表达及其与肿瘤免疫微环境特征的相关性研究

目的·探究基质金属蛋白酶14(matrix metalloproteinase 14,MMP14)在胰腺癌组织中的表达及其与临床特征的相关性,并分析其与胰腺癌肿瘤免疫微环境特征的相关性。方法·通过GEPIA(Gene Expression Profiling Interactive Analysis)数据平台分析MMP家族成员在30种常见肿瘤组织中的表达情况。通过R语言整合GTEx(Genotype-Tissue Expression)数据库中167例胰腺正常组织和TCGA(The Cancer Genome Atlas)数据库中178例胰腺癌组织及4例配对癌旁组织的转录组数据,比较MMP14在胰腺癌与正常组织及癌旁组织中的表达差异。使用GEPIA数据平台对不同MMP14表达水平胰腺癌患者进行生存分析。从TCGA数据库中下载178例胰腺癌患者的相关临床信息,应用Perl和R语言分析MMP14的表达与临床特征的相关性。应用R语言和GSEA v3.0进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析和基因本体论(Gene Ontology,GO)功能富集分析。采用CIBERSORT反卷积算法计算胰腺癌免疫微环境中各类免疫细胞亚群比例,分析MMP14的表达与各免疫细胞亚群比例的相关性。收集6例新鲜胰腺癌组织和配对癌旁组织,流式细胞术检测各类免疫细胞亚群;通过t-SNE降维与统计学分析,描述胰腺癌免疫微环境的特征。利用胰腺癌组织芯片进行多色荧光免疫组织化学实验,检测MMP14的表达与免疫细胞亚群的比例,验证生物信息学分析结果。结果·在生物信息学数据库中,与MMP家族其他成员相比,MMP14在多种肿瘤中高表达,且胰腺癌中MMP14的表达水平高于其他肿瘤;MMP14在胰腺癌中表达水平显著高于胰腺正常组织和癌旁组织(P=0.000),TNM分期Ⅱ期肿瘤MMP14表达水平显著高于Ⅰ期(P=0.012),病理组织学分级G2和G3期肿瘤MMP14显著高于G1期(均P=0.000)。MMP14低表达组患者生存预后显著优于高表达组(P=0.033)。KEGG和GO分析结果显示MMP14主要富集在胰腺癌和免疫相关通路。MMP14的表达水平与CD8+T细胞、单核细胞的比例呈负相关(均P<0.05),与巨噬细胞的比例呈正相关(P=0.000)。流式细胞实验结果提示胰腺癌肿瘤微环境呈现免疫抑制性。多色荧光免疫组织化学实验结果显示,MMP14在肿瘤组织中表达水平显著高于癌旁组织(P=0.000),TNM分期较晚及病理组织学分级较高的肿瘤MMP14的表达水平也相对较高(均P<0.05)。在MMP14高表达的肿瘤组织中,CD8+T细胞比例降低(P=0.001),巨噬细胞比例升高(P=0.000),与生物信息学分析结果一致。结论·与正常胰腺组织和癌旁组织相比,MMP14在胰腺癌中的表达显著升高且与患者不良预后相关;MMP14高表达的胰腺癌组织中CD8+T细胞比例降低,巨噬细胞比例升高;高表达MMP14的胰腺癌肿瘤微环境呈现高度免疫抑制性。

表皮细胞生长因子治疗创面出现干细胞岛现象的方法学鉴别研究

为从方法学和不同染色角度进一步确证在再生表皮的棘细胞层出现的“干细胞岛” ,采用与发现干细胞岛相似的组织学切片 ,用CD87染色法和基底膜染色对“干细胞岛”进一步鉴别。结果发现 ,CD87染色在再生表皮的基底膜呈阳性 ,基底膜染色在正常和再生表皮基底膜可以着色 ,而在发现干细胞岛的部位基底膜染色呈阴性。

Survey of molecular profiling during human colon cancer development and progression by immunohistochemical staining on tissue microarray

AIM： To explore the molecular events taking place during human colon cancer development and progression through high-throughput tissue microarray analysis. METHODS： We constructed two separate tissue microarrays containing 1.0 mm or 1.5 mm cylindrical samples acquired from 112 formalin-fixed and paraffinembedded blocks, including carcinomas （n = 85）, adenomatous polyps （n = 18）, as well as normal paracancerous colon tissues （n = 9）. Immunohistochemical staining was applied to the analysis of the consecutive tissue microarray sections with antibodies for 11 different proteins, including p53, p21, bcl-2, bax, cyclin D1, PTEN, p-Aktl, β-catenin, c-myc, nm23-h1 and Cox-2. RESULTS： The protein expressions of p53, bcl-2, bax, cyclin D1, β-catenin, c-myc, Cox-2 and nm23-h1 varied significantly among tissues from cancer, adenomatous polyps and normal colon mucosa （P = 0.003, P = 0.001, P = 0.000, P = 0.000, P = 0.034, P = 0.003, P = 0.002, and P = 0.007, respectively）. Chi-square analysis showed that the statistically significant variables were p53, p21, bax, β-catenin, c-myc, PTEN, p-Aktl, Cox-2 and nm23-h1 for histological grade （P = 0.005, P = 0.013, P = 0.044, P = 0.000, P = 0.000, P = 0.029, P = 0.000, P = 0.008, and P = 0.000, respectively）, β-catenin, comyc and p-Akt1 for lymph node metastasis （P = 0.011, P =0.005, and P = 0.032, respectively）, β-catenin, c-myc, Cox-2 and nm23-h1 for distance metastasis （P = 0.020, P = 0.000, P = 0.026, and P = 0.008, respectively）, and cyclin D1, β-catenin, c-myc, Cox-2 and nm23h1 for clinical stages （P = 0.038, P = 0.008, P = 0.000, P = 0.016, and P = 0.014, respectively）. CONCLUSION： Tissue microarray immunohistochemical staining enables high-throughput analysis of genetic alterations contributing to human colon cancer development and progression. Our results implicate the potential roles of p53, cyclin D1, bcl-2, bax, Cox-2, β-catenin and c-myc in development of human colon cancer and that of bcl-2, nm23-h1, PTEN and p-Akt1 in progression of human colon cancer.

Diagnosis of intestinal tuberculosis using a monoclonal antibody to Mycobacterium tuberculosis

AIM:To investigate the utility of immunohistochemical(IHC) staining with an antibody to Mycobacterium tuberculosis(M.tuberculosis) for the diagnosis of intestinal tuberculosis(TB).METHODS:We retrospectively identified 10 patients(4 males and 6 females;mean age = 65.1 ± 13.6 years) with intestinal TB.Clinical characteristics,including age,gender,underlying disease,and symptoms were obtained.Chest radiograph and laboratory tests,including sputum Ziehl-Neelsen(ZN) staining,M.tuberculosis culture,and sputum polymerase chain reaction(PCR) for tubercle bacilli DNA,as well as Tuberculin skin test(TST) and QuantiFERON-TB gold test(QFT),were examined.Colonoscopic records recorded on the basis of Sato's classification were also reviewed,in addition to data from intestinal biopsies examined for histopathological findings,including hematoxylin and eosin staining,and ZN staining,as well as M.tuberculosis culture,and PCR for tubercle bacilli DNA.For the present study,archived formalin-fixed paraffin-embedded(FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M.tuberculosis complex.These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS:From the clinical data,we found that no patients were immunocompromised,and that the main symptoms were diarrhea and weight loss.Three patients displayed active pulmonary TB,six patients(60%) had a positive TST,and 4 patients(40%) had a positive QFT.Colonoscopic findings revealed that all patients had type 1 findings(linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules),all of which were located in the right hemicolon and/or terminal ileum.Seven patients(70%) had concomitant healed lesions in the ileocecal area.No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples,and both M.tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples.The histopathological data revealed that tuberculous granulomas were present in 4 cases(40%).IHC staining in archived FFPE samples with anti-M.tuberculosis monoclonal antibody revealed positive findings in 4 patients(40%);the same patients in which granulomas were detected by hematoxylin and eosin staining.M.tuberculosis antigens were found to be mostly intracellular,granular in pattern,and primarily located in the CD68 + macrophages of the granulomas.CONCLUSION:IHC staining with a monoclonal antibody to M.tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.

Overexpression of metastasis-associated in colon cancer 1 predicts a poor outcome of hepatitis B virus-related hepatocellular carcinoma

AIM: To investigate the intratumoral expression of metastasis-associated in colon cancer 1 (MACC1) and c-Met and determine their clinical values associated with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: A retrospective study admitted three hundred fifty-four patients with HBV-related HCC. The expression and distribution of MACC1 and c-Met were assessed by quantitative real-time polymerase chain reaction and immunohistochemistry staining. Prognostic factors influencing survival, metastasis and recurrence were assessed. RESULTS: Intratumoral MACC1 level was found to be associated with HCC disease progression. Both median tumor-free survival (TFS) and overall survival (OS) were significantly shorter in the postoperative HCC patients with high intratumoral MACC1 expression, as compared to those with low intratumoral MACC1 levels (TFS: 34 mo vs 48.0 mo, P < 0.001; OS: 40 mo vs 48 mo, P < 0.01). Multivariable analysis indicated that high MACC1 expression or co-expression with c-Met were independent predictors for HCC clinic outcome (P < 0.001). CONCLUSION: High intratumoral MACC1 expression can be associated with enhanced tumor progression and poor outcome of HBV-related HCC. MACC1 may serve as a prognostic biomarker for postoperative HCC.

Upregulated CD133 expression in tumorigenesis of colon cancer cells

AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging drop. Expression of CD133 and Lgr5 in spheroids of colorectal cancer cells and monolayer culture was detected by RT-qPCR. Spheroids of colorectal cancer cells were analyzed using anti-human CD133 with immunohistochemical staining. RESULTS: CD133 antigen was expressed in colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) as well as in primary and metastatic human colon cancer tissues. However, the CD133 was differently expressed in these cell lines and tissues. The expression levels of CD133 and Lgr5 were significantly higher in spheroids of parental, CD133hi and CD133-cells than in their monolayer culture at the mRNA level (P < 0.05). Immunohistochemical staining of spheroids of CD133-cells showed that CD133 was highly expressed in colorectal cancer cell lines. CONCLUSION: Upregulated CD133 expression plays a role in tumorigenesis colorectal cancer cells, which may promote the expression of other critical genes that can drive tumorigenesis.

Asymptomatic lymphangioma involving the spleen and retroperitoneum in adults

Lymphangioma, a benign neoplasm of the lymphatic system, is common in children but rare in adults. Its clinical manifestations include abdominal pain, nausea, vomiting and a palpable mass. However, abdominal sonography or abdominal computed tomography （CT） scan can also incidentally reveal lymphangioma. A larger or symptomatic lymphangioma is treated with total resection to prevent recurrence, infection, torsion and enlargement. Although lymphangioma rarely becomes malignant, its prognosis is generally good. We report a cystic lymphangioma of the spleen and retroperitoneum, which was incidentally found in a 56-year-old man who was hospitalized due to a colon mass. Physical examination showed no specific findings. Abdominal CT revealed a 5.7 cm, non-enhanced multilobulated cystic mass with multiple sepia in the spleen and a 10 cm lobulated cystic mass in the paraaortic area. Splenectomy and retroperitoneal resection of the cystic mass were conducted. The endothelium of splenic and retroperitoneal cyst was immunohistochemically stained with D2-40 antibody. The patient was finally diagnosed with splenic cystic and retroperitoneal cavernous lymphangioma.

Protective effect of nitric oxide on hepatopulmonary syndrome from ischemia-reperfusion injury

AIM： To evaluate immunological protection of nitric ox- ide （NO） in hepatopulmonary syndrome and probable mechanisms of ischemia-reperfusion （IR） injury in rat liver transplantation, METHODS： Sixty-six healthy male Wistar rats were randomly divided into three groups （11 donor/recipi-ent pairs）. In group 11, organ preservation solution was lactated Ringer＇s solution with heparin 10 000/μL at 4℃. In groups I and 111, the pLeservation solution added, respectively, L-arginine or Ng-L-arginine methyl ester （L-NAME） （1 mmol/L） based on group 11, and recipients were injected with L-arginine or L-NAME （50 mg/kg） in the anhepatic phase. Grafted livers in each group were stored for 6 h and implanted into recipi- ents. Five rats were used for observation of postopera- tive survival in each group. The other six rats in each group were used to obtain tissue samples, and execut- ed at 3 h and 24 h after transplantation. The levels of alanine aminotransferase （ALT）, tumor necrosis factor （TNF）-a and NO metabolites （NOx） were detected, and expression of NO synthase, TNF-α and intercellular adhesion molecule 1 （ICAM-1） was examined by tri- phosphopyridine nucleotide diaphorase histochemical and immunohistochemical staining. RESULTS： By supplementing L-arginine to strengthen the NO pathway, a high survival rate was achieved and hepatic function was improved. One-week sur- viral rate of grafted liver recipients in group I was significantly increased （28.8 4±36.6 d ys 4 4±1.7 d, P 〈 0.01） as compared with groups 11 and Ill. Serum levels of ALT in group ] were 2-7 times less than those in groups 11 and 11I （P 〈 0.01）. The cyclic guanosine monophosphate （cGMP） levels in liver tissue and NOx in group I were 3-4 times higher than those of group 11 after 3 h and 24 h reperfusion, while in group ]]], they were significantly reduced as compared with those in group ]1 （P 〈 0.01）. The levels of TNF-（z in group I were significantly lower than in group Ⅱ after 3 h and 24 h reperfusion （P 〈 0.01）, while being sig- nificantly higher in group Ⅲ than group Ⅱ （P 〈 0.01）. Histopathology revealed more severe tissue damage in graft liver and lung tissues, and a more severe in- flammatory response of the recipient after using NO synthase inhibitor, while the pathological damage to grafted liver and the recipient＇s lung tissues was signifi-cantly reduced in group I after 3 h and 24 h reperfu- sion. A small amount of constitutive NO synthase （cNOS） was expressed in liver endothelial cells after 6 h cold storage, but there was no expression of inducible NO synthase （iNOS）. Expression of cNOS was particularly significant in vascular endothelial cells and liver cells at 3 h and 24 h after reperfusion in group Ⅱ but expres- sion of iNOS and ICAM-1 was low in group I. There was diffuse strong expression of ICAM-1 and TNF-α in group Ⅱ at 3 h after reperfusion. CONCLUSION： The NO/cGMP pathway may be critical in successful organ transplantation, especially in treat- ing hepatopulmonary syndrome during cold IR injury in rat orthotopic liver transplantation.

A case of hepatic angiomyolipoma difficult to distinguish from hepatocellular carcinoma

We report a case of hepatic angiomyolipoma with uncommon clinical features. A 56-year-old man presented with a hepatic tumor in the caudate lobe. The tumor was hypoechoic on ultrasonography, showed early-phase hyperattenuation on enhanced computed tomography and did not absorb iron on superparamagnetic iron oxide-enhanced magnetic resonance imaging. Hepatocellular carcinoma was highly suspected, and the patient underwent hepatic resection. Histologically, the tumor was mainly composed of smooth muscle cells and contained small amounts of adipose cells and blood vessels. On immunohistochemical staining, the smooth muscle cells were positive for a melanocytic cell-specific monoclonal antibody. In cases with uncommon features of angiomyolipoma, it is quite difficult to distinguish angiomyolipoma from hepatocellular carcinoma.

Protective effect of tea polyphenols against paracetamol-induced hepatotoxicity in mice is significanly correlated with cytochrome P450 suppression

AIM： To investigate the hepatoprotective activity of tea polyphenols （TP） and its relation with cytochrome P450 （CYP450） expression in mice. METHODS： Hepatic CYP450 and CYPbs levels were measured by UV-spectrophotometry in mice 2 d after intraperitoneal TP （25, 50 and 100 mg/kg per day）. Then the mice were intragastricly pre-treated with TP （100, 200 and 400 mg/kg per day） for six days before paracetamol （1000 mg/kg） was given. Their acute mortality was compared with that of control mice. The mice were pre-treated with TP （100, 200, and 400 mg/kg per day） for five days before paracetamol （500 mg/kg） was given. Hepatic CYP2E1 and CYPIA2 protein and mRNA expression levels were evaluated by Western blotting, immunohistochemical staining and transcriptase-polymerase chain reaction. RESULTS： The hepatic CYP450 and CYPb5 levels in mice of TP-treated groups （100, 200 and 400 mg/kg per day） were decreased in a dose-dependent manner compared with those in the negative control mice.TP significantly attenuated the paracetamol-induced hepatic injury and dramatically reduced the mortality of paracetamol-treated mice. Furthermore, TP reduced CYP2E1 and CYPIA2 expression at both protein and mRNA levels in a dose-dependent manner. CONCLUSION： TP possess potential hepatoprotective properties and can suppress CYP450 expression.

Embryonic hepatocyte transplantation for hepatic cirrhosis:Efficacy and mechanism of action

AIM： To investigate the efficacy and mechanism of action of allogeneic embryonic hepatocyte transplantation for the treatment of hepatic cirrhosis. METHODS： Rat embryonic hepatocytes were characterized by examining cell markers. Wistar rats with CCl4-induced cirrhosis were randomly divided into two groups： a model group receiving continuous CCl4, and a cell transplantation group receiving continuous CCh and transplanted with embryonic fluorescent-labeled hepatocytes. In addition, a normal control group was composed of healthy rats. All rats were sacrificed after 2 wk following the initiation of the cell transplant. Ul- trasound, pathological analyses and serum biochemical tests were used to evaluate the efficacy of embryonic hepatocyte transplantation. To analyze the recovery status of cirrhotic hepatocytes and the signaling pathways influenced by embryonic hepatocyte transplantation, real-time polymerase chain reaction was performed to examine the mRNA expression of stellate activation-associated protein （STAP）, c-myb, ~ smooth muscle actin （^-SMA） and endothelin-1 （ET-1）. West- ern blotting and immunohistochemistry were employed to detect ^-SMA and ET-1 protein expression in hepatic tissues. RESULTS： Gross morphological, ultrasound and his- topathological examinations, serum biochemical tests and radioimmunoassays demonstrated that hepatic cir- rhosis was successfully established in the Wistar rats. Stem cell factor receptor （c-kit）, hepatocyte growth factor receptor （c-Met）, Nestin, ~ fetal protein, albu- min and cytokeratin19 markers were observed in the rat embryonic hepatocytes. Following embryonic hepa- tocyte transplantation, there was a significant reversal in the gross appearance, ultrasound findings, histo- pathological properties, and serum biochemical param- eters of the rat liver. In addition, after the activation of hepatic stellate cells and STAP signaling, ^-SMA, c-myb and ET-1 mRNA levels became significantly lower than in the untreated cirrhotic group （P 〈 0.05）. These levels, however, were not statistically different from those of the normal healthy group. Immunohisto- chemical staining and Western blot analyses revealed that ^-SMA and ET-1 protein expression levels in the transplantation group were significantly lower than in the untreated cirrhotic group, but being not statistically different from the normal group. CONCLUSION： Transplantation of embryonic hepatocytes in rats has therapeutic effects on cirrhosis. The described treatment may significantly reduce the expression of STAP and ET-1.

Clinical and Pathological Features of Riedel's Thyroiditis

Objective To evaluate the clinical and pathological features of Riedel's thyroiditis(RT),and current diagnostic and treatment methods for that disease.Methods Five RT cases identified by surgery and pathological examinations at Peking Union Medical College Hospital from 1985 to 2009 were analyzed and compared with the cases reported in the literature in terms of clinical and pathological features.Immunohistochemical staining of kappa and lambda light chains was carried out for RT tissues from all the five patients.Results All the five cases were females,aged 45-55 years.Elevation of serum thyroid autoantibodies was found in only one patient,who had longer disease duration than the others.Pathological examination revealed invasive fibrosclerosis of the thyroid follicles,thyroid capsule,and the surrounding tissues.In RT tissues,the number of cells containing lambda chains was a little higher than those containing kappa chains.Conclusions RT is a rare disease which might be more common in middle-aged females than in other populations.Pathological features include the destruction of thyroid follicle,extension into surrounding tissues by inflammatory cells and fibrous tissues.Immunohistochemical staining of kappa and lambda chains could help diagnose RT.

Nuclear β-catenin expression as a prognostic factor in advanced colorectal carcinoma

AIM： To investigate the changing pattern of β-catenin expression and its prognostic value in advanced colorectal cancer （CRC）. METHODS： Archival tumor samples were analyzed for β-catenin using immunohistochemistry （IHC） in 95 patients with advanced CRC. RESULTS： Membranous β-catenin expression was found in the normal colorectal epithelium. Almost 100% of CRC cases showed membranous and cytoplasmic expression, and 55 （58%） cases showed nuclear expression. In univariate （Kaplan-Meier） survival analysis, only the nuclear index （NI） was a significant predictor of disease free survival （DFS） （P = 0.023; n = 35）, with a NI above the median associated with longer DFS （34.2 too） than those with a NI below the median （15.5 too） （P = 0.045, ANOVA）. The other indices were not significant predictors of DFS, and none of the three tested indices （for membranous, cytoplasmic, or nuclear expression） predicted diseasespecific survival （DSS）. However, when dichotomized as positive or negative nuclear expression, the former was a significant predictor of more favorable DFS （P = 0.041） and DSS （P = 0.046）. CONCLUSION： Nuclear β-catenin expression provides additional information in predicting patient outcome in advanced CRC.

An unusual enteropathy-associated T-cell lymphoma with MYC translocation arising in a Japanese patient:A case report

Enteropathy-associated T-cell lymphoma （EATL） is a rare peripheral T-cell lymphoma classified into 2 types, with or without celiac disease, based on histology. Type 2 EATL is less commonly associated with celiac disease, in which cells are characterized by being monomorphic and small- to medium-sized. Cells are characterized by CD8 and CD56 expression and c-MYC oncogene locus gain. We present an atypical case of type 2 EATL in the jejunum, with human T-lymphotropic virus-1 that was CD4- CDS＋ CD56- CD30- CD25- TIA-I＋ and granzyme B＋ on immunohistological staining. It also displayed translocation of chromosome 8p24 （c-MYC）, as de- termined by fluorescent/n situ hybridization. Mucosalspreading and intraepithelial invasion by lymphoma with villous atrophy were detected adjacent to the mucosal layer. The lymphoma may be derived from in- traepithelial CD8＋ T cells, similar to celiac disease.

Influence of Shenqing Recipe on Morphology and Quantity of Colonic Interstitial Cells of Cajal in Trinitrobenzene Sulfonic Acid Induced Rat Colitis

Objective To observe the influence of Shenqing Recipe (SQR), a kind of Traditional Chinese Medicine, on the morphology and quantity of colonic interstitial ceils of Cajal (ICC) in trinitrobenzene sulfonic acid (TNBS)-induced rat colitis, and to investigate the possible mechanism of SQR in regulating intestinal dynamics. Methods Sixty rats were randomly divided into normal control, model Ⅰ , model Ⅱ, mesalazine, and high-dose, and low-dose SQR groups with 10 rats in each group. TNBS (10 mg) dissolved in 50% ethanol was instilled into the lumen of the rat colon of the latter five groups to induce colitis. On the 4th day after administration of TNBS, each treatment group was administered one of the following formulations by enteroclysis gavage once a day for 7 days: 600 mg·kg^-1·d^-1 mesalazine, 2.4g^-1·d^-1 SQR, and 1.2g^-1·d^-1 SQR. Model Ⅱ rats received normal saline solution. After 7 days colonic samples were collected. While the colonic samples of model I group were collected on the 3rd day after TNBS administered. Ultrastructure of ICC in the damaged colonic tissues was observed with transmission electron microscope. Expression of c-kit protein in colonic tissue was determined by immunohistochemical staining and Western blot. Results The ultrastructure of colonic ICC in the rat model of TNBS-induced colitis showed a severe injury, and administration of SQR or mesalazine reduced the severity of injury. Similarly, the expression of c-kit protein of TNBS-induced colitis rat model was significantly decreased compared with the normal control group (P〈0.05). Treatment with SQR or mesalazine significantly increased the expression of c-kit protein compared with the administration of control formulations (P〈0.05), especially the high-dose SQR group. Conclusion SQR could alleviate and repair the injured ICC, and improve its quantity, which might be involved in regulating intestinal motility.

Matrix metalloproteinase-9 contributes to parenchymal hemorrhage and necrosis in the remnant liver after extended hepatectomy in mice

AIM： To investigate the effect of matrix metallopro- teinase-9 （MMP-9） on the remnant liver after massivehepatectomy in the mouse.METHODS： Age-matched, C57BL/6 wild-type （WT）, MMP-9（-/-）, and tissue inhibitors of metalloprotein- ases （TIMP）-1（-/-） mice were used. The mice received 80%-partial hepatectomy （PH）. Samples were obtained at 6 h after 80%-PH, and we used histology, immuno- histochemical staining, western blotting analysis and zymography to investigate the effect of PH on MMP-9. The role of MMP-9 after PH was investigated using a monoclonal antibody and MMP inhibitor.RESULTS： We examined the remnant liver 6 h after 80%-PH and found that MMP-9 deficiency attenuated the formation of hemorrhage and necrosis. There were significantly fewer and smaller hemorrhagic and ne- crotic lesions in MMP-9（-/-） remnant livers compared with WT and TIMP-1（-/-） livers （P 〈 0.01）, with no dif- ference between WT and TIMP-1（-/-） mice. Serum ala- nine aminotransaminase levels were significantly lower in MMP-9（-/-） mice compared with those in TIMP-I（-/-） mice （WT： 476± 83 IU/L, MMP-9（-/-）： 392 ± 30 IU/L, TIMP-I（-/-）： 673 ± 73 IU/L, P 〈 0.01）. Western blot- ting and gelatin zymography demonstrated a lack of MMP-9 expression and activity in MMP-9（-/-） mice, which was in contrast to WT and TIMP-1（-/-） mice. No change in MMP-2 expression was observed in any of the study groups. Similar to MMP-9（-/-） mice, when WT mice were treated with MMP-9 monoclonal antibody or the synthetic inhibitor GM6001, hemorrhagic and necrotic lesions were significantly smaller and fewer than in control mice （P 〈 0.05）. These results suggest that MMP-9 plays an important role in the development of parenchymal hemorrhage and necrosis in the small remnant liver.CONCLUSION： Successful MMP-9 inhibition attenuates the formation of hemorrhage and necrosis and mightbe a potential therapy to ameliorate liver injury after massive hepatectomy.

Potential therapeutic significance of increased expression of aryl hydrocarbon receptor in human gastric cancer

AIM： To determine the functional significance of aryl hydrocarbon receptor （AhR） in gastric carcinogenesis, and to explore the possible role of AhR in gastric cancer （GC） treatment. METHODS： RT-PCR, real-time PCR, and Western blotting were performed to detect AhR expression in 39 GC tissues and five GC cell lines. AhR protein was detected by immunohistochemistry （IHC） in 290 samples： 30 chronic superficial gastritis （CSG）, 30 chronic atrophic gastritis （CAG）, 30 intestinal metapiasia （IN）, 30 atypical hyperplasia （AH）, and 70 GC. The AhR agonist tetrachlorodibenzo-para-dioxin （TCDD） was used to treat AGS cells. MTr assay and flow cytometric analysis were performed to measure the viability, cell cycle and apoptosis of AGS cells.RESULTS： AhR expression was significantly increased in GC tissues and GC cell lines. IHC results indicated that the levels of AhR expression gradually increased, with the lowest levels in CSG, followed by CAG, IM, AH and GC. AhR expression and nuclear translocation were significantly higher in GC than in precancerous tissues. TCDD inhibited proliferation of AGS cells via induction of growth arrest at the G1-S phase. CONCLUSION： AhR plays an important role in gastric carcinogenesis. AhR may be a potential therapeutic target for GC treatment.

Diagnostic value of antigenemia assay for cytomegalovirus gastrointestinal disease in immunocompromised patients

AIM:To investigate the utility of the cytomegalovirus(CMV)antigenemia assay for the diagnosis of CMV gastrointestinal disease(GID). METHODS:One hundred and thirty immunocompromised patients were enrolled in this study.Patients with a history of anti-CMV treatment and who had not undergone examination using the antigenemia assay were excluded.CMV-GID was defined as the detection of large cells with intranuclear inclusions alone or associated with granular cytoplasmic inclusions by biopsy.Biopsy sections were stained with hematoxylin and eosin and immunohistochemically stained with anti-CMV.We evaluated the association between CMV-GID and patient characteristics(symptoms,underlying disease,medication,leukocyte counts,and antigenemia assay).All patients were checked with an human immunodeficiency virus(HIV)antibody test before endoscopic examination.White blood cell(WBC)counts were obtained from medical records within 1 wk of endoscopy.Leukopenia was defined as a total WBC count<5000 cells/mm 3 . For HIV patients,we also checked CD4+counts from medical records. RESULTS:A total of 99 patients were retrospectively selected for analysis.Of the immunocompromised patients,19 had malignant disease,18 had autoimmune disease,19 had disorders of biochemical homeostasis, three had undergone transplantation,and 45 had HIV infection.A total of 50 patients had received immunosuppressive therapy.No patients had inflammatory bowel disease.Fifty-five patients were diagnosed as having CMV-GID.Univariate analysis indicated an association between HIV infection,leukopenia,and positive antigenemia and CMV-GID(P<0.05).Multivariate analysis using logistic regression revealed that HIV infection and positive antigenemia were the only independent factors related to CMV-GID(P<0.01).The sensitivity,specificity,positive predictive value,and negative predictive value of antigenemia for CMV-GID were 65.4%,93.6%, 91.9%,and 71.0%,respectively.In a subgroup analy-sis,patients with leukopenia displayed low sensitivity and high specificity.Minimal differences in accuracy were seen among patients with or without leukopenia. HIV-infected patients displayed low sensitivity and high specificity.Accuracy barely differed between HIV-positive and-negative patients.In HIV-infected patients, CD4 count<50 cells/μL resulted in low sensitivity and high specificity.Differences in accuracy among patients were minor,regardless of CD4 count.In patients who had undergone both quantitative real-time polymerase chain reaction(PCR)and antigenemia assay,real-time PCR was slightly more accurate in terms of sensitivity than the antigenemia assay;however,this difference was not statistically significant(P=0.312). CONCLUSION:If the antigenemia test is positive,endoscopic lesions are acceptable for the diagnosis of CMVGID without biopsy.The accuracy is not affected by HIV infection and leukopenia.Either PCR or the antigenemia assay are valid.

Novel ABCB11 mutations in a Thai infant with progressive familial intrahepatic cholestasis

Progressive familial intrahepatic cholestasis （PFIC） type 2 is caused by mutations in ABCB11, which encodes bile salt export pump （BSEP）. We report a Thai female infant who presented with progressive cholestatic jaundice since 1 mo of age, with normal serum y-glutamyltransferase. Immunohistochemical staining of the liver did not demonstrate BSEP along the canaliculi, while multidrug resistance protein 3 was expressed adequately. Novel mutations in ABCB11, a four-nucleotide deletion in exon 3, c.90_93delGAAA, and a single-nucleotide insertion in exon 5, c.249_250insT, were identified, with confirmation in her parents. These mutations were predicted to lead to synthesis of truncated forms of BSEP. Immunostaining and mutation analysis thus established the diagnosis of PFIC type