ObjectiveTo characterize two strains of street rabies virus (RABV) isolated from the brain tissue of cattle from Inner Mongolia. Differences in the histopathological and ultrastructural changes in the brain tissue of infected mice were determined to reveal variation in the pathogenesis of infection between street rabies virus strains. MethodsTen-day-old mice were intracranially inoculated with one of three virus strains and brain tissue harvested when the mice were moribund. Various histopathological and ultrastructural markers of disease were then compared between the groups. ResultsInfection with the street virus strain CNM1101C resulted in severe neuronal dendrites damage, but only mild cell apoptosis, T lymphocyte infiltration and microglial activation. Infection with the other street virus strain,CNM1103C, was characterized by cell apoptosis, T lymphocyte infiltration and microglial activation as well as dendrites damage. However, in comparison, infection with the attenuated virus strain CTN caused severe T lymphocyte infiltration, microglial activation and cell apoptosis, but left the neuronal dendrites intact. ConclusionThe two street rabies virus strains isolated from cattle from Inner Mongolia had different levels of virulence and caused distinct pathological changes in infected mice. Therefore, we concluded that different pathogenic mechanisms exist between different RABV strains.

免疫组织化学技术在卵巢性索-间质肿瘤诊断中的应用及其局限性

卵巢性索-间质肿瘤(SCST)是一组由分化程度不等的"性索样"细胞、性腺间质成分及其各种黄素化细胞单一或混合构成的肿瘤."性索"原本是胚胎学名词,卵巢胚胎发育的复杂性决定了卵巢性索.间质肿瘤的多向分化,因此分类也比较复杂~([1]),这类肿瘤发病相对少见,仅占卵巢肿瘤的8%~([2]),在临床实际工作中有些病理类型并不为临床或病理医师所熟悉,影响到对患者的正确诊治.免疫组织化学已经是我们常规病理诊断工作中不可或缺的辅助手段之一.

《Antigen Retrieval Immunohistochemistry Based Research and Diagnostics》简介

抗原修复技术的问世是免疫组织化学领域的一次革命性突破,由抗原修复技术的发明者石善溶与其合作者Clive R.Taylor主编的[1]已在国外出版发行.

AIM To study the expression of cathepsin B in gastric carcinoma and its relationship with pathologic type.METHODS The cathepsin B expression in 54 specimens of human gastric adenocarcinoma was studied byimmunohistochemistry.RESULTS The cathepsin B expression was detected in 33/54 (61.1%) specimens of human gastriccarcinoma and in 3/54 (5.6%) of normal tissue (P<0.01). There was no obvious correlation between theexpression of cathepsin B and pathologic type of gastric adenocarcinoma.CONCLUSION There is a high expression of cathepsin B in human gastric adenocarcinoma.

AIM To probe into the feasibility and theoretic basis for the treatment of chronic hepatitis with hyperbaricoxygenation (HBO).METHODS Sixty cases of chronic hepatitis were randomly distributed into an experimental group (n=30)and a control group (n =30). The experimental group was treated with HBO for 6 courses. The controlgroup was treated with commonly used drugs in clinic for 60 days. The function and blood stream graph ofliver were examined and the liver biopsies were made before and after treatment. The routine paraffin slidesof liver tissue were cut, stained with HE, and observed under optical microscope. The ultrathin slides fromparaformaldehyde and glutaraldehyde fixed liver tissue were cut, stained with lead citrate, and observedunder transmission electric microscope. The HBsAg and HBcAg in the experimental group were detected bythe ABC immunohistochemical method before and after treatment.RESULTS In the experimental group the ALT, SB, γ-GT, AKP, IgG and IgM in blood (P< 0.05) and thedegeneration and necrosis of hepatocytes (P<0.05) were remarkably decreased, the mean contractive waveof blood stream in the liver (P<0.05) and the blood stream in the right ramus of janitrix (P<0.05) weresignificantly increased, and the swelling of mitochondria, the increased lysosomes, the generation of Kupffer's cells, the infiltration of lymphocytes in portal area and the capillary generation were remarkably alleviated(P<0.05), but the fibrosis and fat-storing cells did not reduce (P>0.05) in the liver, and the expression ofHBsAg and HBcAg in the liver was not lowered (P<0.05) after the treatment with HBO.CONCLUSION The treatment with HBO for chronic hepatitis was effective and recommendable.

AIM To find out if there is any difference in human primary liver carcinogenesis between Han and minorityethnic patients in Xinjiang.METHODS Expression of p53, c-erbB-2, H-rasp21 protein and proliferating cell nuclear antigen (PCNA)in tumor tissues of 50 patients (Hah 38, minorities 12) with primary hepatic carcinoma (HCC) was detectedby immunohistochemistry (LSAB).RESULTS The positive frequency of p53, c-erbB-2, H-rasp21 and PCNA expression was 46.0% (23/50,70.0% (35/50), 68.0% (34/50) and 82.0% (41/50) in tumor tissues; 4.0% (2/50), 22.0% (11/50),64.0% (32/50) and 52.0% (26/50) in peritumor respectively with a significant difference, except for H-rasp21 (P<0.05) between tumor and non-tumor tissues. Combined the three oncogenes alteration, 26%(13/50) tumor tissues had positive immunoreactivity, but peritumor and normal liver were negative. Thepositive p53, c-erbB-2, H-rasp21 protein expression was 39.5 % ( 15 / 38), 60.5 % (23 / 38) and 39.5 % ( 15 /38) in tumors of Han patients; 66.7% (8/12), 100% (12/12) and 75.0% (9/12) in minority patientsrespectively. A statistical difference between Han and minority cancer samples was observed (P< 0.05).CONCLUSION Overexpression of p53, c-erbB-2 and H-rasp21 in human primary liver carcinoma is animportant biomarker of genetic alteration. The different frequency of these oncogenetic changes may reflectsome environmental factors or/and ethnic hereditary affecting the liver carcinogenesis. The special life styleof Han, Uygur, Kazak and Mongolia nationalities in Xinjiang may also involve the etiopathogenesis of thisdisease.

AIM The hepatic content of collagens (type I, Ⅲ and Ⅶ) and laminin (LN) in rat model of experimentalliver fibrosis was observed to find out their roles in the pathogenesis of liver fibrosis.METHODS The experimental rat model was established by immunological injury induced by injectinghuman albumin. Histopathological and immunohistochemical methods were used to measure the hepaticcontent of collagens and laminin in the fibrotic rat livers.RESULTS The hepatic contents of collagens (type I, Ⅲ, Ⅶ) and LN in the fibrotic rat livers weresignificantly increased as compared with those in the control group, and they were found to be mainlylocalized in the portal space, central veins and fibrous septa. Electron microscopic study showed that pro-collagens were present around the “activated” hepatic stellate cells (HSC) and the hepatocytes atrophied.CONCLUSION Pathological deposition of collagens (type Ⅰ, Ⅲ and Ⅶ ) and laminin was the fundamentallesion of liver fibrosis. HSC may be the major cellular source of collagens (type Ⅰ, Ⅲ and Ⅶ) and laminin inthe liver tissue.

AIM To examine hepatitis C in hepatocellular carcinoma in most endemic area, Guangxi, China.METHODS Immunochemistry was performed on formalin fixed, paraffin embedded tissue sections. A total202 specimens were analysed from the high, intermediate and low HCC prevalent regions of Guangxi.RESULTS The distribution of positive hepatitis C cases from high, intermediate and low regions wasrespectively 24/63 (38%), 23/62 (37%) and 30/77 (39%), with a total of 77/202 (38.12%).CONCLUSION Hepatitis C virus is an important risk factor in the development of hepatocellularcarcinoma, but the regional difference in prevalence of this cancer is more likely influenced by hepatitis Bviral infection and aflatoxin B1 exposure. In Guangxi, infection of hepatitis B and C virus in thedevelopment of hepatocellular carcinoma may be greatly enhanced by exposure to aflatoxin.

AIM To establish a relevant animal model of human gastrointestinal cancer, which can be used forrepetitive investigations and may improve our understanding of carcinogenesis and cancer metastasis.METHODS Intact tissue of human colorectal and pancreatic cancers was transplanted in nude mice. Thebiological characteristics of the original and corresponding transplanted tumors were investigated by HEstaining, PAS staining and immunostaining. The metastases in livers and lungs of the nude mice wereinvestigated by immunostaining with biotinylated mab KL-1 and by RT-PCR using CK20 specific primers.RESULTS Nine of 16 surgical specimens grew in the nude mice subcutaneously and/or orthotopically (4 of6 colorectal and 5 of 10 pancreatic cancer). Tumor cell content of the specimens and freezing of tissuespecimens are important factors influencing the growth of transplanted tumor. In the group of fresh tumortissues with greater than 50% tumor cell content, transplantation rate was 100% (3 cases of pancreatic cancerand 3 cases of colorectal cancer). The orthotopically transplanted tumors resembled the original tumormorphologically and biologically, including TAA expression such as CEA by immunohistochemistry, andCEA level in the serum of mice. Ki-67 labeling index and the expression of TAA especially K-ras, 17-1A and RA-96, were associated with the potential of tumor growth in nude mice. Micrometastases in the lungs andlivers of tumor bearing mice could be detected by immunostaining with biotinylated mab KL-1 and CK20-sepcific RT-PCR.CONCLUSION An orthotopic transplantation model for human colon and pancreatic cancer in nude micehas been established. The sensitive detection methods with CK-immunohistochemistry and CK20-RT-PCRwere also established to study xenotransplanted human cancer and its metastatic cancer cells in the liver andlung of nude mice. This study may be helpful in understanding the mechanism of cancer metastasis and indeveloping new diagnostic methods and therapeutic strategies for metastases.Acknowledgement The authors thank Dr. J. Luettges, Department of Pathology; Kiel University, for investigating thepathological characterics of the specimens; Dr. N. Zawazawa, Institute of Immunology, Kiel University, for the quantitativemeasurement of serum of CEA.

AIM To evaluate the expression of CD44v3 and v6 protein in colorectal carcinoma and its prognosticsignificance.METHODS One hundred and twenty-one cases of formalin-fixed paraffin-embedded colorectal carcinomaspecimens were retrospectively analyzed using EnvisionTM immunohistochemical method with the monoclonalantibody CD44v3 and v6. The median follow-up time was 67.77 months and the prognostic value of theCD44v3 and CD44v6 was assessed using univariate and multivariate survival analysis.RESULTS The positive rates of CD44v3 and v6 protein were 60.3% and 57.9%, respectively. There wassignificant correlation between CD44v3 immunoreactivity and tumor location, lymph node metastasis, distantmetastasis and Duke's stage (P< 0.05, Spearman correlation test). Significant correlation between CD44v6immunoreactivity and patients' gender, lymph node metastasis, distant metastasis, Duke's stage was alsonoticed (P < 0.05, Spearman correlation test). The 5-year survival rates were 81.25% and 60.27% inCD44v3 negative and positive cases, respectively. As CD44v6, the 5-year survival rates were 80.39% and60.00% in CD44v6 negative and positive cases, respectively; these differences between the two groups ofpatients were significant (P<0.05, Log-rank test). In multivariate analysis using the Cox regression model,CD44v3 expression emerges as an independent prognostic indicator.CONCLUSION CD44v3 and v6 might play some important roles in metastasis of colorectal carcinoma, andCD44v3 expression might be a new useful independent prognostic marker of colorectal carcinoma.

AIM To investigate the role of sphincter of Boyden in bile excretion and its regulating factors.METHODS Perfusion manometry, choledocho-cineradiography, reaction of the sphincter of Boyden toendogenous cholecystokinin (CCK) and immunohistochemical quantitative analysis were performed in 16dogs to study the motility and morphology of the sphincter of Boyden in experimental (postcholecystectomy)group ( n = 8) and the control group (n = 8).RESULTS The bile duct surrounded by SB was a low-pressure lumen (10.0 ± 2.0 mmHg), in which thepressure was significantly different (P<0.01, t = 6. 195) from the basal pressure of the high-pressure area ofsphincter of Oddi (SO), its basal pressure (SOBP) was 16.9±0.5 mmHg. The SB was an enlarged ampulladuring bile excretion interval, and showed active contraction during bile excretion. Intrinsic CCK could causediastole of SO, but does not affect the systole and diastole of SB. After cholecystectomy, spastic contractionpersisted in SB, which could not be relieved by intrinsic CCK. The sensitivity to CCK of SO was decreased,and the evacuation time of media prolonged (27.0±3.4 min vs precholecystectomy 17.l±0.9min P<0.01,t =7.961). In immunohistochemistry analysis, the contents of a-actin, myosin in the SB of experimentalgroup showed no increase. Under electronic microscope, the main changes were 3D structuraldisarrangement of the cell framework, distortion of the microfilaments, swelling and aggregation ofmitochondria at the nuclear side.CONCLUSION The excretion of bile can be divided into two types, physiological bile excretion with a drivemainly caused by the contraction of SB, and the other, functional bile excretion with a drive mainly causedby the contraction of gallbladder. It seems that the function of SB was controlled by vagus, whereas SO wasmore sensitive to the intrinsic CCK, The intact gallbladder is an elemental factor of functional coordinationof SB and SO.

AIM To study the development of D (somatostatin-secreting) and P (bombesin-secreting) cells ofproventriculus from Shao ducks at different ages.METHODS Ninety Shao ducks were divided into nine groups, 10 ducks per group. The ducks were slayedgroupby group at the nine time points of week 0 (after hatching), 1, 2, 4, 6, 10, 14, 18 and 22.Proventriculus samples from each duck were collected, fixed by Bouin solution and embedded with paraffin.gections were made and stained with an avidin-biotin-peroxidase complex kit (Dako Co., Ltd. ) to visualizeD and P cells of each proventriculus. Thirty glandular lobes per duck were observed for enumeration of Dand P cells.RESULTS ① Both D and P cells were mostly oval or polygonal shape with dumpy cytoplasmic processesand located in the inner and central area of the glandular lobe. ② The D and P cells peaked at wk 18 and 6respectively. ③ There was no apparent correlation between D cells and the body weight until wk 4. Negativecorrelation was observed from wk 6 and reached a marked level at 18wk (r = -0.829, P<0.05).Individuals with maximum body weight had less D cells than those with minimum body weight from wk 10-wk 18.CONCLUSION The morphology and distribution of D and P cells in the proventriculus of Shao ducks weresimilar to Peking ducks, gooses and chickens. Both D and P cells had continuing development during thepostnatal period, though the rates of their development were different. If appeared that beyond a definitenumber of D cells, they were negatively correlated with the body weight.

AIM To study the effects of ATRA on experimental liver metastasis of gastric cancer cells.METHODS MGc80-3 and SGC-7901 cells were injectied into spleen subcapsule of nude mice, who weresubsequently administrated with ATRA every other day. Food-intake and body weight of mice were measuredweekly. After six weeks, the nude mice were executed, tumors in spleen and liver were examinedpathologically, microtumor vessel density (MVD) was accounted by immunohistochemical method and serumCEA was measured by radioimmunoassay.RESULTS Nude mice administrated with ATRA, the growth of spleen tumor and its metastatic ability toliver were inhibited, the metastatic rate was decreased by 33.3% (MGc80-3) and 50.0% (SGC-7901). SpleenMVD and liver MVD were reduced by 28.6% and 22.9% (MGc80-3), 23.7% and 37.6% (SGC-7901),respectively. The serum CEA was lowered by 43.4% (MGc80-3).CONCLUSION ATRA can effectively inhibit the experimental liver metastasis of gastric cancer cells,which is relavant with the decrease of MVD and CEA.

AIM To study the clinicopathological significance of CD15 antigen expression and its contents in humancarcinoma.METHODS CD15 expression and its contents in 45 cases of gallbladder carcinoma, 17 cases of gallbladderadenoma and 10 cases of chronic cholecystitis were evaluated by microwave-LSAB immunohistochemicaltechnique combined with image analysis.RESULTS Positive rate of CD15 and its integral absorbance (optic density) in the gallbladder carcinomawere 71% and 4.0±3.2 respectively, being significantly higher than 35% (6/17) and 0.9±0.3 in thegallbladder adenoma, 20% (2/10) and 0.4±0.1 in chronic cholecystitis (P<0.05, P<0.01). In grade Ⅱ-Ⅲ cancer, they were 90% (17/19) and 5.4±1.7, being significantly higher than 58% (15/26) and 2.2±3.1 in grade I cancer (P<0.05, P<0.01). In lymph node positive cases, they were 88% (21/24) and 6.5±3.5, which were significantly higher than 52% (11/21) and 3.3±2.1 in the negative ones (P<0.025,P<0.01).CONCLUSION CD15 expression and its contents could be involved in aggresiveness of gallbladdercarcinoma and might be a useful indicator to evaluate the malignancy and biological features, and could beconsidered as a good prognostic predictor for patients with gallbladder carcinoma.

AIM To investigate the effect of boschniakia rossica (BR) extract on expression of GST-P, p53 and p21fasproteins in early-stage chemical hepatocarcinogenesis in rats and its anti-inflammatory actions.METHODS The expression of tumor marker, placental form glutathione S-transferase (GST-P), p53 and p21ras proteins were investigated by immunohistochemical techniques and ABC method. Anti-inflammatoryactivities of BR were observed by xylene and croton oil-induced mouse ear edema, carrageenin, histamineand hot scald-induced rat pow edema, adjuvant-induced rat arthritis and cotton pellet-induced mousegranuloma formation methods.RESULTS The 500 mg/kg of BR-H2O extract fractionated from BR-Methanol extract had inhibitory effecton the formation of DEN-induced GST-P-positive foci in rat liver and the expression of mutant p53 and p21fasprotein was lower than that of hepatic preneoplastic lesions. Both CH2Cl2 and H2O extract from BR haveinhibitory effect in xylene and croton oil-induced mouse ear edema. BR-H2O extract exhibited inhibitoryeffect in carrageenin, histamine and hot scald-induced hind paw edema and adjuvant-induced arthritis in ratsand cotton pellet-induced granuloma formation in mice.CONCLUSION BR extract exhibited inhibitory effect on formation of preneoplastic hepatic foci in earlystage of rat chemical hepatocarcinogenesis. Both CH2Cl2 and H2O extract from BR exerted anti-inflammatory effect in rats and mice.

AIM To study the relationship between nitric oxide (NO), nitric oxide synthase (NOS) and humanhepatocellular carcinoma (HCC).METHODS Plsama NO2-/NO3- was measured by Griess reaction in 122 patients with chronic hepatitis(CH) and compensated liver cirrhosis (LC), among which 62 patients were complicated with HCC(CH = 28, LC = 34), and the rest 60 patients were not (CH = 29, LC = 31). Thirty healthy persons served asnormal controls (NC). There were no prominent differences among the groups in sex, age and the ratio ofCH to LC. The expression of inducible nitric oxide synthase (iNOS) in HCC (n = 40), CH (n = 30) and LC(n = 30) samples obtained from liver biopsy or operation was compared with that in normal liver tissues byusing immunohistochemistry. Ten normal liver tissue samples obtained from liver operation served as normalcontrols. The samples were fixed in formalin and embeded in paraffin. Anti-iNOS antibody (Santacruzcompany) was served as antibody-Ⅰ in immunohistochemical assay of iNOS in tissue.RESULTS Plasma NO2-/NO3- level in normal was 11.5 μmol/L±4.2μmol/L. The plasma level ofNO2 /NO3- in CH (58.6±17.4 μmol/L) and LC (38.7±10.6μmol/L) accompanied with HCC wasnotably higher than in those patients without HCC (CH: 24.8±9.4 μmol/L; LC: 22.3±8.7μmol/L,t=2.901, 2.756, P<0.01). Plasma NO2-/NO3- level in HCC accompanied with CH was significantlyhigher than in those accompanied with LC ( t = 2.216, P<0.05). Positive rate of iNOS in HCC, CH and LCwas 95%, 93% and 57% respectively. iNOS was not expressed in normal liver tissues. The expression level ofiNOS in HCC (χ2=17.4, P<0.001) and CH (χ2=11.64, P<0.025) was much higher than in LC.CONCLUSION Plasma NO2 / NO3- level significantly increased in patients with HCC and theimmunohistochemical staining of iNOS was positive. This suggests that the liver secrets NO in the higherlevel may participate in the carcinogenesis and progression of HCC.

AIM To determine the role of Helicobacter pylori in altering gastric mucin synthesis and define how thprocess relates to H. pylori-related diseases.METHODS Analyses of human gastric tissues using immunohistochemistry and in situ hybridizatiodocument the role of H. pylori in altering the composition and distribution of gastric mucins.RESULTS These data indicate a decrease in the product of the MUC5 (MUC5AC) gene and aberraexpression of MUC6 in the surface epithelium of H. pylori-infected patients. A normal pattern was restorby H. pylori eradication. Inhibition of mucin synthesis including MUC5AC and MUCl mucins by H. pvlohas been established in vitro using biochemical and Western blot analyses. This effect is not due to inhibitiof glycosylation, but results from inhibition of synthesis of mucin core structures. In vitro experiments usiinhibitors of mucin synthesis indicate that cell surface mucins decrease adhesion of H. pylori to gastepithelial cells.CONCLUSION Inhibition of mucin synthesis by H. pylori in vivo can disrupt the protective mucous layand facilitate bacterial adhesion, which may lead to increased inflammation in thc gastric epithelium.

AIM To study the biopathologic characteristics of the transitional mucosa (TM) adjacent to rectalcarcinoma and the resecting length of bowel.METHODS Immunohistochemical and mucin histochemical methods were used in 81 cases to observe theexpression of PCNA and the changing regulations of TM adjacent to rectal carcinoma.RESULTS The PCNA expression rate was the highest in cancer and gradually decreased in atypicaldysplasia, TM and normal mucosa (P<0.01). The range of TM adjacent to the mucinous adenocarcinomawas more extensive than that to the papilla adenocarcinoma and the tubular adenocarcinoma (P< 0.01 ). Therange of TM adjacent to the rectal carcinoma in Dukes C stage was more extensive than that in stage A, B(P<0.01, P<0.05).CONCLUSION The TM of rectal carcinoma possesses certain potential of malignancy. The range of TMadjacent to rectal carcinoma is closely related to the pathological type and advancement of rectal carcinoma.

AIM To determine NO, NO synthase (NOS) and NOSmRNA of the esophageal carcinoma cells (SHEEC1)in apoptotic process induced by As2O3 and to explore the relationship between NO and apoptosis.METHODS The apoptosis of the cell line (SHEEC1) was induced by arsenite (As2O3, 5 μmol/L and10 μmol/L). In the process, at 2 h, 4 h, 8 h, 16 h and 24 h after administration of As2O3, NO production incultural medium was detected quantitatively by spectrophotometry; NOS Ⅱ was detected byimmunohistochemistry and NOS mRNA by in situ hybridization (ISH). The cells at endpoint of theexperiment were examined under transmitted electron microscope (TEM) for apoptosis.RESULTS The amount of NO released from SHEEC1 were increased from the basal condition (0.68×10-2μmol/L) up to the high level (2.38×10-2μmol/L) at h 16. The increment of NOS Ⅱ was found afteradministration of As2O3; the intracytoplasmic ISH signals of NOSmRNA in small size was found firstly at4 h, and then became highly predominant. Apoptotic changes of SHEEC1 occurred at 24 h under TEM.CONCLUSION After administration of As2O3, NO released from cultured SHEEC1 cells was detected withincreasing amount up to 16 h. The expression of NOS H and transcription of NOSmRNA are upregulated.The present findings suggest a concept that the NO may be a mediated and effective factor in apoptosisinduced by As2O3,

AIM To study the combined expression of gastrointestinal hormone substance P and anti-apoptosis gene Bcl-2 in gastric carcinoma and its significance.METHODS Substance P and Bcl-2 protein expression was examined by the S-P immunohistochemicalmethod in 33 cases of gastric carcinoma, 17 adjacent the carcinoma and 13 normal gastric mucoma.RESULTS Positive expression of SP in gastric carcinoma was higher than that of both adjacent and normalmucosa (P < 0.001). There was no statistical difference in the positive expression between adjacent andnormal mucosa (P > 0.05). The expression of bcl-2 both in gastric carcinoma and adjacent tissues werehigher than that of normal gastric mucosa (P< 0.05-0.01). But the positive expression of Bcl-2 had nostatistical significance between gastric carcinoma and adjacent tissues.CONCLUSION Both gastrointestinal hormone SP and Bcl-2 gene have synergistic expression in gastriccarcinoma, indicating that they all take part in the occurrence of gastric carcinoma. Abnormal expression ofBcl-2 gene occurred in benign gastric pathological changes, once they become carcinoma, the positiveexpression of cell is no more increased, possibly because that there is no more increase of the intensity of Bcl-2 inhibition of cell apoptosis.