Three-dimensional microarray compared with PCR-single-strand conformation polymorphism analysis/DNA sequencing for mutation analysis of K-ras codons 12 and 13

BACKGROUND: We developed a rapid, precise, and accurate microarray-based method that uses a three-dimensional platform for detection of mutations. METHODS: We used the PamChip microarray to detect mutations in codons 12 and 13 of K-ras in 15 cell lines and 81 gastric or colorectal cancer tissues. Fluorescein isothiocyanate-labeled PCR products were analyzed with the microarray. We confirmed the microarray results with PCR-single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. RESULTS: We could correctly identify wild-type, heterozygous, and homozygous mutant genotypes with the PamChip microarray in <3.5 h. The array data were consistent with those of PCR-SSCP analysis and DNA sequencing. All 15 cell lines and 80 of 81 clinical cancer specimens (98.8%; 95% confidence interval, 96.4-100%) were genotyped accurately with the microarray, a rate better than that of direct DNA sequencing (38.9%) or SSCP (93.8%). Only one clinical specimen was misdiagnosed as homozygous for the wild-type allele. Densitometric analysis of SSCP bands indicated that the content of the mutant allele in the specimen was approximately 16%. The PamChip microarray could detect mutant alleles representing more than 25% of the SSCP band proportions. Therefore, the limit for detection of mutant alleles by the PamChip microarray was estimated to be 16-25% of the total DNA. CONCLUSIONS: The PamChip microarray is a novel three-dimensional microarray system and can be used to analyze K-ras mutations quickly and accurately. The mutation detection rate was nearly 100% and was similar to that of PCR-SSCP together with sequencing analysis, but the microarray analysis was faster and easier.     

Elsberg syndrome with eosinophilic meningoencephalitis caused by Angiostrongylus cantonensis

A 42-year-old man was admitted to our hospital with a history of fever, headache and disorientation. His cerebrospinal fluid revealed eosinophilia and his serum had an antibody against Angiostrongylus cantonensis (A. cantonensis). Then, he was diagnosed as eosinophilic meningoencephalitis caused by A. cantonensis. He was treated with repeated lumbar punctures and oral prednisolone. Although a symptom he had been suffering from at the time of his admission was urinary retention, this symptom disappeared as his general condition improved. Therefore his case was considered to be Elsberg syndrome with eosinophilic meningoencephalitis caused by A. cantonensis.     

Contribution of nitrergic nerve in canine gingival reactive hyperemia

Reactive hyperemia reflects a compensatory vasodilation response of the local vasculature in ischemic tissue. The purpose of this study is to clarify the mechanism of regulation of this response in gingival circulation by using pharmacological analysis of reactive hyperemia and histochemical analysis of gingival tissue. Application of pressure to the gingiva was used to create temporary ischemia, and gingival blood flow was measured after pressure release. Reactive hyperemia increased in proportion to the duration of pressure. Systemic hemodynamics remained unaffected by the stimulus; therefore, the gingival reactive hyperemia reflected a local adjustment in circulation. Gingival reactive hyperemia was significantly suppressed by nitric oxide (NO) synthase inhibitors, especially the neural NO synthase-selective antagonist 7-nitroindazole, but not by anticholinergic drugs, β-blockers, or antihistaminergic drugs. Moreover, immunohistochemical staining for neural NO synthase and histochemical staining for NADPH diaphorase activity were both positive in the gingival perivascular region. These histochemical and pharmacological analyses show that reactive hyperemia following pressure release is mediated by NO-induced vasodilation. Furthermore, histochemical analysis strongly suggests that NO originates from nitrergic nerves. Therefore, NO may play an important role in the neural regulation of local circulation in gingival tissue ischemia.     

SLIT Improves Cedar Pollinosis by Restoring IL-10 Production from Tr1 and Monocytes~IL-10 Productivity Is Critical for Becoming Allergic ~

BackgroundAllergen-specific immunotherapy (SIT) is currently used for several allergic disorders and IL-10- producing regulatory T cells (Tr1) induced by SIT suppress allergic reactions. We investigated the relation between IL-10 production and acquiring allergy.MethodsA prospective study was undertaken to evaluate the effect of SIT on IL-10 production in T cells and other cell fractions in children with pollinosis. In addition, blood samples were collected from non-allergic healthy controls and patients with pollinosis to compare the levels of IL-10 production. PBMC were cultured with pollen peptides or control allergens, and the IL-10 production from monocyte and CD4 T cell was analyzed.ResultsMonocytes and CD4 T cells from SIT group of patients produced high levels of IL-10, suggesting that the induction of IL-10 is essential for inducing T cell tolerance. IL-10 production from monocytes and T cells was significantly increased in non-allergic controls compared to patients with pollinosis. This high IL-10 production was observed even when PBMC were stimulated with antigens other than pollen peptides.ConclusionsIL-10 is critical for induction of specific T cell tolerance, and increased production of IL-10 by monocytes and T cells during inflammatory responses or after SIT may influence effector cells in allergy. Present data implicates that the low productivity of IL-10 by monocytes and T cells is closely related with sensitivity to multiple allergens, and resistance to allergic diseases. Augmentation of constitutive IL-10 production from immune system is a potential therapeutic approach for allergic disorders.     

The effect of beta-blocker on intractable ascites in cirrhotic patients undergoing hepatectomy for hepatocellular carcinoma

BACKGROUND/AIMS: Intractable ascites is one of the serious complications after hepatectomy. Only little is known about their effect on postoperative ascites in patients with liver cirrhosis although beta-blockers have been used for cirrhotic complications including ascites. METHODOLOGY: Here, we report five cases of intractable ascites after hepatectomy, which were treated by propranolol (1 mg/kg/body). RESULTS: In three patients, plasma renin activity and aldosterone concentrations were markedly increased before propranolol administration, but fell to normal levels thereafter. Ascites subsided in all subjects except one, who developed cardiac dysfunction. CONCLUSIONS: Beta-blockers might be a promising drug for intractable ascites in cirrhotic patients undergoing hepatectomy.     

Molecular epidemiologic study of Clostridium difficile infections in university hospitals: Results of a nationwide study in Japan

We conducted a nationwide molecular epidemiological study of Clostridium difficile infection (CDI) in Japan investigated the correlation between the presence of binary toxin genes and CDI severity. This is the first report on molecular epidemiological analyses for CDI in multiple university hospitals in Japan, to our knowledge. We examined 124,484 hospitalized patients in 25 national and public university hospitals in Japan between December 2013 and March 2014, investigating antimicrobial susceptibilities and toxin-related genes for C. difficile isolates from stools. Epidemiological genetic typing was performed by PCR-ribotyping and repetitive sequence-based (rep)-PCR to examine the genetic similarities. The results detected toxin A-positive, toxin B-positive, binary toxin-negative (A⁺B⁺CDT^?) detected from 135 isolates (80.8%) and toxin A-negative, toxin B-positive, binary toxin-negative (A^??B⁺CDT^?) in 23 (13.8%). Toxin A-positive, toxin B-positive, and binary toxin-positive (A⁺B⁺CDT⁺) were seen in 9 isolates (5.4%). Vancomycin (n = 81, 37.7%) or metronidazole (n = 88, 40.9%) therapies were undertaken in analyzed cases. Ribotypes detected from isolates were 017/subgroup 1, 070, 078, 126, 176, 449, 475/subgroup 1, 499, 451, 566 and newtypes. Rep-PCR classified 167 isolates into 28 cluster groups including 2–15 isolates. In addition, 2 pairs of strains isolated from different institutions belonged to the same clusters. Seven out of 9 (77.8%) of the patients with binary toxin producing strains had “mild to moderate” outcome in evaluated symptoms. In conclusion, we found that binary toxin did not show regional specificity and had no relevance to severity of CDI.