Astrovirus-Associated Polioencephalomyelitis in an Alpaca

An 8-year-old alpaca was admitted to the emergency service of the Clinic for Ruminants in Bern due to a reduced general condition and progressive neurological signs. Despite supportive treatment, its condition deteriorated and the animal had to be euthanized. Histopathological analysis revealed a severe non-suppurative polioencephalomyelitis with neuronal necrosis, most likely of viral origin. We detected abundant neuronal labelling with antibodies directed against two different epitopes of Bovine Astrovirus CH13/NeuroS1 (BoAstV-CH13/NeuroS1), which is a common viral agent associated with non-suppurative encephalitis in Swiss cattle. These findings were further verified by detection of viral RNA by use of in-situ hybridization and real-time RT-PCR. Next generation sequencing revealed that the detected virus genome had a pairwise identity of 98.9% to the genome of BoAstV-CH13/NeuroS1. To our knowledge, this is the first report of an astrovirus-associated polioencephalomyelitis in an alpaca. These results point to the possibility of an interspecies transmission of BoAstV-CH13/NeuroS1.     

细胞学涂片及其联合细胞块免疫组化在超声内镜下细针穿刺活检术中的应用价值

目的探讨细胞学涂片及其联合细胞块免疫组化在超声内镜下细针穿刺活检术(EUS-FNA)中的应用价值。方法收集因占位性疾病行EUS-FNA的患者60例,将细胞学涂片及其联合细胞块免疫组化结果与手术病理及随访预后结果比较。结果 EUS-FNA下穿刺物细胞学涂片检查对肿瘤性占位的阳性检出率为78.6%(44/56),特异度为50.0%(2/4),准确度为76.7%(46/60);19例患者同时进行了细胞学涂片联合细胞块免疫组化检查,其对肿瘤性占位的阳性检出率为94.7%(18/19),准确度为94.7%(18/19),两者在该19例患者中对肿瘤性占位的检出率差异无统计学意义(P=0.250)。但在明确肿瘤性占位病理类型方面,细胞学涂片联合细胞块免疫组化检查优于单独细胞学涂片检查。结论在EUS-FNA下,细胞学涂片及其联合细胞块免疫组化检查对肿瘤性占位的检出率相当,联合细胞块免疫组化检查在明确病理类型上更有优势,更能指导后续治疗。     

Immunocytochemical detection of milk proteins in tracheal aspirates of ventilated infants: a pilot study

In this study, we evaluated immunocytochemical staining for milk proteins (alpha-lactalbumin and beta-lactoglobulin) in tracheal aspirates of mechanically ventilated infants, and assessed whether this staining technique supported a clinical diagnosis of aspiration in infants receiving orogastric feedings. All newborns requiring mechanical ventilation in the neonatal intensive care unit of a major tertiary care hospital were potential subjects for this study. Tracheal aspirates were obtained prior to the introduction of enteral feeding and at various time points thereafter in newborns requiring mechanical ventilation. Cells were obtained and processed for immunocytochemical staining of alpha-lactalbumin and beta-lactoglobulin. In total, 88 specimens recovered from 34 infants were adequate for staining. Alveolar macrophages recovered from most of the infants who were never fed (true negative controls) did not display immunoreactivity for milk proteins: 4/34 or 12% of infants' aspirates demonstrated presence of milk proteins before enteral feeding was commenced. Tracheal aspirates obtained from 12 infants after introduction of enteral feedings appeared to support clinical and radiological findings suggestive of aspiration events, with positive immunostaining on several occasions. These observations support our work in a murine model and demonstrate that immunocytochemical staining of tracheal aspirates for milk proteins may enhance the ability to diagnose pulmonary aspiration. Further studies are needed to define the clinical significance of our findings and the effects of single and repeated aspiration events on respiratory status.     

Immunohistochemical analysis of the distribution of measles related antigen in the intestinal mucosa in inflammatory bowel disease

BACKGROUND: Measles virus is implicated in the aetiology of Crohn's disease. This measles hypothesis is mainly supported by immunohistochemical findings that the measles related antigen is present in the intestine of patients with Crohn's disease. Recently we isolated this antigen from the intestine of a patient with Crohn's disease using a molecular cloning technique and produced the monoclonal antibody against it (designated 4F12). AIM: To discover whether the measles related antigen is uniquely present in Crohn's disease. SUBJECTS/METHODS: Colonic mucosa samples from 20 patients with Crohn's disease, 20 with ulcerative colitis, 11 with non-inflammatory bowel disease (IBD) colitis, and nine controls were immunohistochemically stained with the anti-measles monoclonal antibody 4F12. The numbers of positive cells, the ratio of positive cells to nucleated cells, and the staining intensity of the positive cells were compared. Furthermore, the distribution of the measles antigen in other human organs was examined. RESULTS: Both the number of positive cells and the ratio of positive cells to nucleated cells were significantly increased in Crohn's disease, ulcerative colitis, and non-IBD colitis compared with controls (p<0.05) but were similar among the three disease groups. The staining intensity of the positive cells was also similar among the three disease groups. Small numbers of positive cells were observed in the oesophagus, stomach, duodenum, jejunum, and lung. CONCLUSIONS: The presence of the measles related antigen in the colonic mucosa was not unique to Crohn's disease. These results, together with the observation that such a measles related antigen was derived from host protein, do not support the hypothesis that measles virus causes Crohn's disease.     

Occult lymph node metastases detected by cytokeratin immunohistochemistry predict recurrence in “node-negative” colorectal cancer

There is controversy about the prognostic significance of occult lymph node metastases detected by immunohistochemistry with the anti-cytokeratin antibody CAM 5.2. The aim of this study was to characterize occult lymph node metastases in colorectal carcinomas that might be associated with a higher risk of recurrence. Three hundred fifty-eight lymph nodes from 10 recurrent and 9 nonrecurrent cases of colorectal carcinoma were examined. All these patients had been reported originally as having no lympho node metastases by routine hematoxylin and eosin staining. Three 10-μm sections or ten 3-μm sections (30-μm total thickness) from each lymph node were stained with CAM 5.2 and examined for the presence of occult lymph node metastases. Occult metastases were detected in 67 of 175 lymph nodes from the recurrent cases, and in 23 of 183 lymph nodes from the nonrecurrent cases. The frequency of positive nodes was significantly higher in the recurrent cases. The recurrent cases had metastases in nodes more distant from the main tumor than did the nonrecurrent cases. Detection of occult lymph node metastases with cytokeratin immunohistochemistry may make it possible to identify patients with a higher risk of recurrence after the removal of a primary colorectal tumor.     

Matrix metalloproteinase 9 (MMP-9) in patients with rheumatoid arthritis

Matrix metalloproteinase 9 (MMP-9) degrades type IV collagen, gelatin, type V collagen and type XI collagen. We measured proMMP-9 and proMMP-9-TIMP-1 complex in sera and joint fluids by sandwich ELISA, and immunohistochemically examined the expression of this enzyme in joint tissues from patients with rheumatoid arthritis (RA). ProMMP-9 was purified from the culture medium of HT 1080 cells by the three steps of chromatography. Purified proMMP-9 and activated MMP-9 by aminophenylmercuric acetate showed two bands of 92 and 67 kDa on gelatin zymography. We raised two monoclonal antibody clones, named 2G9 and 8G7, against proMMP-9. 2G9 and 8G7 reacted with proMMP-9 in western blotting and these clones reacted not only with proMMP-9, but also with proMMP-9-TIMP-1 complex in sandwich ELISA, respectively. The proMMP-9 concentration in 86 sera (749.4±940.2 ng/ml) and 54 joint fluids (4539.9±7681.5 ng/ml) from patients with RA was significantly higher than those of patients with osteoarthritis (15 sera: 139.0±149.6 ng/ml; 16 joint fluids: 655.0±1982.8 ng/ml) and control (37 sera: 266.7±120.4 ng/ml; three joint fluids: 0 ng/ml). The immunohistochemistry with 2G9 monoclonal antibody showed that proMMP-9 were expressed in the neutrophils and the monocytes-macrophages which diffusely infiltrated in the sublining layer of rheumatoid synovium. In addition, the osteoclasts along subchondral bone were also intensively stained. The proMMP-9 concentration in joint fluids from 39 RA patients was positively correlated to the count of proMMP-9 positive cells in RA synovium (r=0.607) and to the score of diffuse infiltrates of lymphocytes (r=0.720). However, it did not show correlation to the stage and the class defined by Steinbrocker and to the other clinical laboratory data. Our results suggest that proMMP-9 actively participates in joint destruction of RA through the expression of neutrophils and monocytes-macrophages and is regulated by lymphocytes.     

Fusobacterium varium localized in the colonic mucosa of patients with ulcerative colitis stimulates species-specific antibody

BACKGROUND: Microbial agents are a possible cause of ulcerative colitis. We have previously reported evidence of bacteria invading the colonic mucosa of patients with ulcerative colitis. We have isolated bacteria from inflamed colonic mucosa, examined the localization of the species in the mucosa, and assayed for serum antibodies to the bacteria. METHODS: Cohorts of 31 per group were enrolled from patients with active ulcerative colitis, Crohn's disease, ischemic colitis, and colon adenomas. A group of 31 healthy controls were also studied. The presence of bacteria in biopsies of patients with ulcerative colitis was analyzed by both isolation and immunohistochemistry. Sera from patients were tested for bacterial antibodies using both Western blots and enzyme-linked immunosorbent assay (ELISA). RESULTS: Only sera from patients with ulcerative colitis gave specific reactions with Fusobacterium varium in Western blot assays. The detection rate of specific bands was higher for patients with ulcerative colitis (61%) than for subjects with either Crohn's disease (13%) or healthy controls (29%) (P < 0.001 and P = 0.021, respectively). The ELISA showed that the mean optical densities with extracts of F. varium as antigen were significantly higher for ulcerative colitis patients than for subjects with either Crohn's disease or healthy controls (P < 0.001). Immunohistochemical detection of F. varium in colonic mucosa was significantly higher in patients with ulcerative colitis (84%) than for subjects with either Crohn's disease (16%) or other controls (3-13%) (P < 0.001). CONCLUSIONS: Fusobacterium varium bacteria were present in a significant number of patients with active ulcerative colitis, and should be tested in therapeutic trials in order to confirm the causal relationship between F. varium and ulcerative colitis.     

Estrogen receptor alpha and imprinting of the neonatal mouse ventral prostate by estrogen

Exposure to estrogen in the neonatal period affects prostatic growth and leads to an increased incidence of prostatic intraepithelial neoplasia in later life. The effects of neonatal estrogen are clearly dependent on estrogen receptor (ER) alpha because they do not occur in ERalpha-knockout mice. Because ERalpha is expressed in the stroma, but not in the epithelium, of the adult ventral prostate, the concept of indirect estrogen effects through stromal signaling has been proposed. Here, we show that during the first 4 weeks of life, there are profound and rapid changes in the ER profile in the mouse ventral prostate. ERalpha is abundant in the stroma during week 1, but by week 2 it is exclusively epithelial, and then by week 4, ERalpha is lost and ERbeta is dominant in the prostatic epithelium. The presence of ERalpha is associated with a high proliferation index, and ERbeta is associated with quiescence. Branching morphogenesis was altered in ERalpha-/-, but not in ERbeta-/-, mice. We conclude that imprinting and branching morphogenesis of the ventral prostate are mediated by estrogen acting directly on epithelial and stromal ERalpha during the first 2 weeks of life.     

血管内皮生长因子对培养内皮细胞增殖和移行的影响

目的　研究低氧培养心肌细胞分泌出的血管内皮生长因子 (VEGF)蛋白对培养内皮细胞增殖和移行的影响。方法　将培养的人脐静脉内皮细胞 (HUVECs)分为 4组 :正常对照组 ,低氧培养组 ,含VEGF的低氧培养心肌细胞分泌液组 (分泌液组 ) ,分泌液 +低氧培养组。分别采用流式细胞仪、免疫组织化学等方法检测VEGF对HUVECs细胞周期、增殖细胞核抗原表达、氚一亮氨酸掺入量及HUVECs移行的影响。结果　VEGF增加正常培养和低氧培养HUVECs由静止期 DNA合成前期向DNA合成期的转换 ,使DNA合成期及DNA合成后期 分裂期细胞数增加 ,从而促进HUVECs增殖 ,并使常氧和低氧培养HUVECs增殖细胞核抗原的表达、氚一亮氨酸掺入量及移行细胞数目增加。低氧抑制HUVECs的增殖和移行 ,VEGF可拮抗低氧对HUVECs增殖、移行的抑制作用。结论　低氧培养心肌细胞分泌出的VEGF蛋白不仅增加常氧培养HUVECs增殖、移行 ,而且能够拮抗低氧的抑制作用     

胃癌胎盘型GST－π表达的意义

目的研究胃癌组织新标志酶ＧＳＴ＿π的表达和它的临床意义。方法胃癌组织６２例用抗ＧＳＴ＿π抗体，按ＡＢＣ法进行免疫组织化学检测癌组织中ＧＳＴ＿π活性，并与正常胃粘膜，异型增生及肠上皮化生进行比较。结果胃癌组织６２例中ＧＳＴ＿π阳性率８７１％；２７例正常胃粘膜ＧＳＴ＿π阳性率只１１１％（Ｐ＜０００１）；异型增生和肠上皮化生各２３例，其ＧＳＴ＿π阳性率分别为８２６％和８６９％，与正常胃粘膜比较Ｐ＜０００１。结论ＧＳＴ＿π在胃癌组织中的胎儿型表达为研究消化道肿瘤早期诊断和发病机理提供了新的酶学标志。具有中等度异型增生和不全结肠型上皮化生的慢性萎缩性胃炎阳性率也较高，可列为胃切除手术的相对适应证。