Low prevalence of maternal microchimerism in peripheral blood of Japanese children with type 1 diabetes.

AIM: To clarify the prevalence and degree of maternal microchimerism in Japanese children with type 1 diabetes, as well as its effect on phenotypic variation. METHODS: We studied 153 Japanese children with type 1 diabetes, including 124 children positive for ß-cell autoantibodies, and their 71 unaffected siblings. The number of circulating microchimeric cells per 105 host cells was estimated by the use of quantitative-polymerase chain reaction targeting non-transmitted maternal human leukocyte antigen alleles. The results were compared to previous data from white European people. Phenotypic comparison was performed between maternal microchimerism carriers and non-carriers with diabetes. RESULTS: Maternal microchimerism was detected in 15% of children with autoantibody-positive type 1 diabetes, 28% of children with autoantibody-negative type 1 diabetes, and 16% of unaffected siblings. There were no differences in the prevalence or levels of maternal microchimerism among the three groups or between the children with type 1 diabetes and their unaffected siblings. Furthermore, maternal microchimerism carriers and non-carriers exhibited similar phenotypes. CONCLUSIONS: Maternal microchimerism appears to be less common in Japanese children with type 1 diabetes than in white European people. Our data indicate that maternal microchimerism is unlikely to be a major trigger or a phenotypic determinant of type 1 diabetes in Japanese children and that the biological significance of maternal microchimerism in type 1 diabetes may differ among ethnic groups.

Protein-altering variants of PTPN2 in childhood-onset Type 1A diabetes.

AIM: To examine the contribution of PTPN2 coding variants to the risk of childhood-onset Type 1A diabetes. METHODS: PTPN2 mutation analysis was carried out for 169 unrelated Japanese people with childhood-onset Type 1A diabetes. We searched for coding variants that were absent or extremely rare in the general population and were scored as damaging by multiple in silico programs. We performed mRNA analysis and three-dimensional structural prediction of the detected variants, when possible. We also examined possible physical links between these variants and previously reported risk SNPs as well as clinical information from variant-positive children. RESULTS: One frameshift variant (p.Q286Yfs*24) and two probably damaging missense substitutions (p.C232W and p.R350Q) were identified in one child each. Of these, p.Q286Yfs*24 and p.C232W were hitherto unreported, while p.R350Q accounted for 2/121,122 alleles of the exome datasets. The p.Q286Yfs*24 variant did not encode stable mRNA, and p.C232W appeared to affect the structure of the tyrosine-protein phosphatase domain. The three variants were physically unrelated to known risk SNPs. The variant-positive children manifested Type 1A diabetes without additional clinical features and invariably carried risk human leukocyte antigen alleles. CONCLUSIONS: The results provide the first indication that PTPN2 variants contribute to the risk of Type 1A diabetes, independently of known risk SNPs. PTPN2 coding variants possibly induce non-specific Type 1A diabetes phenotypes in individuals with human leukocyte antigen-mediated disease susceptibility. Our findings warrant further validation.

FUT2 non-secretor status is associated with Type 1 diabetes susceptibility in Japanese children.

AIM: To examine the contribution of the FUT2 gene and ABO blood type to the development of Type 1 diabetes in Japanese children. METHODS: We analysed FUT2 variants and ABO genotypes in a total of 531 Japanese children diagnosed with Type 1 diabetes and 448 control subjects. The possible association of FUT2 variants and ABO genotypes with the onset of Type 1 diabetes was statistically examined. RESULTS: The se2 genotype (c.385A>T) of the FUT2 gene was found to confer susceptibility to Type 1A diabetes in a recessive effects model [odds ratio for se2/se2, 1.68 (95% CI 1.20-2.35); corrected P value = 0.0075]. CONCLUSIONS: The FUT2 gene contributed to the development of Type 1 diabetes in the present cohort of Japanese children.

Variants associated with autoimmune Type 1 diabetes in Japanese children: implications for age-specific effects of cis-regulatory haplotypes at 17q12-q21.

AIMS: The aim of this study was to clarify the significance of previously reported susceptibility variants in the development of autoimmune Type 1 diabetes in non-white children. Tested variants included rs2290400, which has been linked to Type 1 diabetes only in one study on white people. Haplotypes at 17q12-q21 encompassing rs2290400 are known to determine the susceptibility of early-onset asthma by affecting the expression of flanking genes. METHODS: We genotyped 63 variants in 428 Japanese people with childhood-onset autoimmune Type 1 diabetes and 457 individuals without diabetes. Possible association between variants and age at diabetes onset was examined using age-specific quantitative trait locus analysis and ordered-subset regression analysis. RESULTS: Ten variants, including rs2290400 in GSDMB, were more frequent among the people with Type 1 diabetes than those without diabetes. Of these, rs689 in INS and rs231775 in CTLA4 yielded particularly high odds ratios of 5.58 (corrected P value 0.001; 95% CI 2.15-14.47) and 1.64 (corrected P value 5.3 × 10-5 ; 95% CI 1.34-2.01), respectively. Age-specific effects on diabetes susceptibility were suggested for rs2290400; heterozygosity of the risk alleles was associated with relatively early onset of diabetes, and the allele was linked to the phenotype exclusively in the subgroup of age at onset ≤ 5.0 years. CONCLUSIONS: The results indicate that rs2290400 in GSDMB and polymorphisms in INS and CTLA4 are associated with the risk of Type 1 diabetes in Japanese children. Importantly, cis-regulatory haplotypes at 17q12-q21 encompassing rs2290400 probably determine the risk of autoimmune Type 1 diabetes predominantly in early childhood.

Decreased secretion of Paneth cell α-defensins in graft-versus-host disease.

BACKGROUND: Intestinal microbial ecology is actively regulated by Paneth cell-derived antimicrobial peptides, α-defensins. Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (SCT). We previously demonstrated that Paneth cells are targeted by GVHD, and their expression of antimicrobial peptide α-defensins is impaired, leading to a loss of physiological diversity among the microflora and development of bloodstream infection. Herein, we evaluated whether fecal levels of α-defensins could be surrogate marker of intestinal dysbiosis. METHODS: We directly measured α-defensin cryptdin-1 (Crp1) in fecal pellets of mice with GVHD by using a novel enzyme-linked immunosorbent assay. RESULTS: Fecal levels of Crp1 were significantly decreased in mice with GVHD but unchanged in mice without GVHD after SCT. These were correlated with intestinal flora diversity. CONCLUSION: We demonstrate a link between reduced secretion of Paneth cell α-defensins and dysbiosis of intestinal flora in GVHD. Fecal levels of α-defensins could be surrogate markers for intestinal microbial homeostasis.

46, XY gonadal dysgenesis: new SRY point mutation in two siblings with paternal germ line mosaicism.

Familial recurrence risks are poorly understood in cases of de novo mutations. In the event of parental germ line mosaicism, recurrence risks can be higher than generally appreciated, with implications for genetic counseling and clinical practice. In the course of treating a female with pubertal delay and hypergonadotropic hypogonadism, we identified a new missense mutation in the SRY gene, leading to somatic feminization of this karyotypically normal XY individual. We tested a younger sister despite a normal onset of puberty, who also possessed an XY karyotype and the same SRY mutation. Imaging studies in the sister revealed an ovarian tumor, which was removed. DNA from the father's blood possessed the wild type SRY sequence, and paternity testing was consistent with the given family structure. A brother was 46, XY with a wild type SRY sequence strongly suggesting paternal Y-chromosome germline mosaicism for the mutation. In disorders of sexual development (DSDs), early diagnosis is critical for optimal psychological development of the affected patients. In this case, preventive karyotypic screening allowed early diagnosis of a gonadal tumor in the sibling prior to the age of normal puberty. Our results suggest that cytological or molecular diagnosis should be applied for siblings of an affected DSD individual.

Carcinoembryonic antigen level in serum and pleural lavage fluid in non-small cell lung cancer.

BACKGROUND: This study evaluates the tumor marker index (TMI) based on carcinoembryonic antigen (CEA) levels in serum and pleural lavage fluid as a potential prognostic determinant for patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Three hundred and eighty-three consecutive NSCLC patients were reviewed retrospectively. RESULTS: The 5-year survival of patients with normal and high serum CEA levels was 71.78% and 51.38%, respectively (P < 0.0001). The 5-year survival of patients with high CEA levels in pleural lavage fluid was 25.0%, which was significantly poorer compared with that of patients with normal lavage CEA levels (78.23%, P < 0.0001). There was a 5-year survival rate of 73.75% in patients with a TMI less than or equal to 1.0 compared to a rate of only 55.12% in patients with a TMI greater than 1.0 (P < 0.001). Both univariate and multivariate analyses indicated the independent prognostic impact of the TMI. CONCLUSIONS: The TMI based on serum and lavage CEA levels might be useful for predicting the prognosis of NSCLC patients.

Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense.

Precursors of alpha-defensin peptides require activation for bactericidal activity. In mouse small intestine, matrilysin colocalized with alpha-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT-/-) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT-/- mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT-/- mice than in MAT+/+ mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which may be a common role for this metalloproteinase in its numerous epithelial sites of expression.

Neural processes of attentional inhibition of return traced with magnetoencephalography.

Inhibition of return (IOR) is a phenomenon that involves reaction times (RTs) to a spatially cued target that are longer than RTs to an uncued target when the interval between the cue and target is prolonged. Although numerous studies have examined IOR, no consensus has yet been reached regarding the neural mechanisms responsible for it. We used magnetoencephalography (MEG) and measured the human neural responses underlying the time course of IOR, applying a typical spatial cueing paradigm. The cue-target interval was 600+/-200 ms. Three experimental conditions were employed. Cued; the cue and target were presented at the same location. Uncued; the two stimuli were presented at opposite locations. Neutral; the cue stimulus was presented bilaterally. We found differences in the amplitudes of signals in the postero-temporal and bilateral temporal areas, and peak latencies in a central area between the cued and uncued conditions. These signals were localized to the extrastriate cortex, bilateral temporal-parietal junction (TPJ), and primary motor cortex, respectively. Bilateral TPJ activities are related to the identification of salient events in the sensory environment both within and independent of the current behavioral context and may play an important role in IOR in addition to extrastriate and the primary motor cortex.

[New method for localization of the small ground-glass opacity lesion in resected lung].

A small lesion showing ground-glass opacity (GGO) by preoperative computed tomography (CT) is sometimes difficult to detect after lobectomy when it locates in the central part of the lobe. In order to facilitate to identify the lesion for marking pathological specimen, we developed a new method using CT. After surgery, the resected pulmonary lobe was expanded with airflow through the bronchial stump and the target lesion was examined with CT. The laser beam of the CT on the surface of the lung is used as a guiding line for cutting. Through the application of this method for 2 clinical cases, it was found to be possible to exactly identify the GGO lesion from the surface of the resected lung enabling to visualize a fresh surface of the lesion like a CT image with minimal destruction of the structure.

Phase II study of irinotecan, leucovorin, 5-fluorouracil and tegafur/uracil for metastatic colorectal cancer.

Irinotecan combined with continuous-infusion 5-fluorouracil (5FU) has been shown to be an effective and tolerable regimen in the treatment of metastatic colorectal cancer (MCRC). Tegafur/uracil (UFT) during 5FU infusion enhances plasma 5FU concentration, mimics continuous 5FU infusion and delivers the drug to target tumor cells. We conducted a phase II trial of four-agent combined therapy for MCRC, giving patients (pts) intravenous irinotecan (30 mg/m2 on day 1), leucovorin (LV, 200 mg/m2 on day 1 and 2), 5FU (300 mg/m2 on day 1 and 2), and UFT (400 mg/day for 14 days). The main endpoint was the objective tumor response rate. Sixteen pts with a good performance status were enrolled from February 2001 to May 2002. The response rate was 19% (3 partial responses), and 13 pts had stable disease. The median time to progression was 5.2 months, and the median survival time was 20.2 months. Considering the low toxicity and reasonable cost, this regimen deserves further investigation.

[Surgery for the thymoma combined with pure red cell aplasia and myasthenia gravis].

UNLABELLED: Pure red cell aplasia (PRCA) and myasthenia gravis (MG) are respectively combined with thymoma, however, these 3 complications are extremely rare coexisted as a clinical triad. A 73-year-old female with mediastinal tumor found in 2000 was pointed out anemia in June 2002. As PRCA was diagnosed by the bone marrow examination, blood transfusion had been performed. By a chest computed tomography (CT), a thymoma in size of 7 x 5 cm in diameter was recognized in the anterior mediastinum. The serum level of anti-acetylcholine receptor antibody was elevated to be 35 nmol/l. MG was simultaneously diagnosed with a decreased power of neck muscle. The extended thymectomy was performed in August 2002, and pathological diagnosis disclosed a 'type AB' by World Health Organization (WHO) classification. After the operation, the decreased power of neck muscle had been improved, however, PRCA had not been remitted in the early-postoperative term. Blood transfusion had been required (2-4 units/1-2 weeks) for the postoperative 7 months' term. A cyclosporin (250 mg/day) as an adjuvant therapy was administered in April 2003. One month later, the patient's serum level of Hb had been over 10 g/dl without blood transfusion. The patient has been followed up with reducing the dose of cyclosporin. CONCLUSIONS: Surgery for a thymoma combined with PRCA and MG was effective for MG but not for PRCA in an early-postoperative term, however, a multimodality therapy with immunosuppressant as a postoperative adjuvant should bring a favorable outcome to patient's clinical data, and the postoperative long-observation must be critical in this case.

[Clinical assessment of the thoracic surgical diseases associated with von Recklinghausen's disease].

Type I neurofibromatosis (NF-I), also referred to as von Recklinghausen's disease, is an autosomal dominant disease characterized by neurofibromas and abnormal cutaneous pigmentation (café-au-lait spot). We studied retrospectively the 8 cases operated in our hospital between January 1979 to December 2002, which were complicated with von Recklinghausen's disease and a thoracic surgical disease. The patients were 6 males and 2 females and the age from 16 to 70 (the averaged age was 36 +/- 22). The thoracic diseases were consist of mediastinal tumors (n = 7) and esophageal cancer (n = 1). The operative procedures were tumorectomy (n = 6), subtotal esophagectomy (n = 1), and pericardial cystectomy (n = 1). The mediastinal tumors were neurofibroma (n = 3), malignant schawannoma (n = 1), ganglioneurinoma (n = 2), and pericardial cyst (n = 1). Malignant neoplasms were recognized in 2 cases (25%). The postoperative survival was 10 months for malignant schwannoma, and 8 months for esophageal cancer, and the others were alive. For 1 case of neurofibromas, there was observed to be the reoperated one after the postoperative recurrence. von Recklinghausen's disease are apt to be complicated with thoracic surgical neoplasms, it should be required a careful and systemic exploration especially for malignant neoplasms.

[Primary lung cancer; assessment of the disclosed 5-year survival rate by the Internet website].

The disclosed 5-year survival rate for lung cancer in the Internet website represents a various difference by each institution. The better inferiority of the survival has been listed in a table to compare with other institutions and has been reported in magazines and media with a lack of an enough inspection, i.e., with a sufficient considering of a risk adjustment such as patient's background, operative policy, postoperative adjuvant therapy, and statistical background. We report our outcome of the surgical treatment for primary lung cancer. Of 875 patients treated for lung cancer in our department for 23 years between January 1980 and December 2002, 115 patients containing of 42 cases in 1997 and of 48 ones in 1992 and of 25 ones in 1987 were selected and the accumulated survival analysis was treated by Kaplan-Meier method. Eighty males and 35 females were between 15 and 80-year-old (average 63.2 +/- 11.4). The pathological classification was adenocarcinoma (n=69), squamous cell carcinoma (n=32), and others (n=14). The operative procedures were pneumonectomy (n=14), bilobectomy (n=12), lobectomy (n=85), and wedge resection (n=4). The survival time was from 29 days to 182 months (median survival time was 1471+/- 1180 days, the averaged time was 49 months). The 5-year survival rate was 41.4 +/- 9.1% (n=25) in 1987, 35.6 +/- 6.2% (n=48) in 1992, and 56.0 +/- 7.0% (n=42) in 1987, respectively (log-rank test, p = 0.2555). The 10-year survival rate was 24.1 +/- 7.9% in 1987 and 8.5 +/- 3.6% in 1992, respectively. The 5-year survival rate was as follows: IA 81.0 +/- 8.6% (n=20), IB 73.7 +/- 10.1% (n=19), IIA 57.1 +/- 18.7% (n=7), IIB 55.6 +/- 16.6% (n=9), IIIA 28.6 +/- 7.6% (n=35), IIIB 15.4 +/- 10.0% (n=13), IV 16.7 +/- 10.8% (n=12), respectively. The 5-year survival rate was as follows: male 42.8 +/- 5.3% (n=80), female 63.2 +/- 7.3% (n=35), respectively (p = 0.0147). In regard to the histological classification, the 5-year survival rate was as follows: adenocarcinoma 47.2 +/- 5.9% (n=69), squamous cell carcinoma 50.8 +/- 8.9% (n=32), respectively (p = 0.9012). As a rule of the disclosure on the internet website, we report our survival data by accompanying with minimum parameters such as, patient's background, pathological types, gender, pathological stages, and mean survival rate with standard error. When we compare the 5-year survival rate with other institutes, in considering of a risk adjustment, we would carefully have to estimate the determined survival rate with a standard error.

Stiff substrates increase YAP-signaling-mediated matrix metalloproteinase-7 expression.

Abnormally stiff substrates have been shown to trigger cancer progression. However, the detailed molecular mechanisms underlying this trigger are not clear. In this study, we cultured T84 human colorectal cancer cells on plastic dishes to create a stiff substrate or on collagen-I gel to create a soft substrate. The stiff substrate enhanced the expression of matrix metalloproteinase-7 (MMP-7), an indicator of poor prognosis. In addition, we used polyacrylamide gels (2, 67 and 126 kPa) so that the MMP-7 expression on the 126-kPa gel was higher compared with that on the 2-kPa gel. Next, we investigated whether yes-associated protein (YAP) affected the MMP-7 expression. YAP knockdown decreased MMP-7 expression. Treatment with inhibitors of epidermal growth factor receptor (EGFR) and myosin regulatory light chain (MRLC) and integrin-α2 or integrin-ß1 knockdown downregulated MMP-7 expression. Finally, we demonstrated that YAP, EGFR, integrin-α2ß1 and MRLC produced a positive feedback loop that enhanced MMP-7 expression. These findings suggest that stiff substrates enhanced colorectal cancer cell viability by upregulating MMP-7 expression through a positive feedback loop.

Quantitative study of the supernormal phase of ventricular excitability in man.

The supernormal phase of excitability of the human heart was studied by means of fixed rate endocardial pacing in 11 patients with acute and chronic bradyarrhythmias. Ten of the eleven patients manifested a supernormal phase. The duration of the phase increased with increasing intensity of subthreshold stimuli and ranged from 91 to 148 percent of the Q-T interval. Subthreshold stimuli of a wide range of intensity could elicit a full response. Two types of supernormal phase, early and late in relation to the cardiac cycle, were observed. The latter was attributed to the summation of subthreshold stimuli with either spontaneous phase 4 depolarization of a ventricular ectopic pacemaker or atrial depolarization potentials. Its possible connection with Wedensky facillitation was suggested. The ventricle was less excitable after an ectopic beat than after a normally conducted beat.

Immunohistochemical analysis of nm23-H1 gene product in node-positive lung cancer and lymph nodes.

The nm23-H1 gene product has been considered as an anti-metastatic protein and the level of its expression has been reported to correlate inversely with metastatic potential in some cancers. However, the expression of nm23-H1 gene product in the metastatic sites have not been studied in detail. We examined the expression of nm23-H1 gene product in surgically resected 46 pairs of primary lung cancers and metastatic lymph nodes by immunohistochemistry. The positive staining of nm23-H1 gene product in primary cancers and metastatic lymph nodes were observed in 56.5 and 67.4%, respectively. The heterogeneity of nm23-H1 gene product expression between primary cancers and metastatic lymph nodes was observed in 41.3%. No correlations were found between the nm23-H1 gene product expression in lung cancers and the patients survival. No significant association was also observed between nm23-H1 gene product expression in lymph nodes and the patients survival. There was, furthermore, no correlation between the heterogeneity of nm23-H1 gene product expression and the patients survival. In conclusion, the level of nm23-H1 gene product expression does not significantly reveal prognostic value in node-positive lung cancers. Expression of nm23-H1 gene product in metastatic lymph nodes was also unrelated to patients survival.

Expression of nm23-H1 gene product in esophageal squamous cell carcinoma and its association with vessel invasion and survival.

BACKGROUND: We assessed the nm23-H1 gene product expression and its relationship with lymphatic and blood vessel invasion in patients with esophageal squamous cell carcinoma. METHODS: Formalin-fixed and paraffin-embedded tissue sections from 45 patients who were treated surgically were used in this study. Pathologists graded lymphatic and blood vessel invasion in each of the tissue samples. Expression of nm23-Hl gene product was determined using a specific monoclonal antibody. RESULTS: Expression of nm23-H1 gene product was present in 17 (37.8%) cases. We found an inverse correlation between nm23-H1 gene product expression and lymphatic vessel invasion, whereas no correlation between nm23-H1 gene product expression and blood vessel invasion. Overall survival rate was not different between nm23-H1 gene product positive and negative patients (p = 0.21). However, reduced expression of nm23-H1 gene product was associated with shorter overall survival in patients with involved lymph nodes (p < 0.05), but not in patients without involved lymph nodes (p = 0.87). CONCLUSIONS: In patients with esophageal squamous cell carcinoma, there appears to be an inverse relationship between nm23-H1 gene product expression and lymphatic vessel invasion. Furthermore, nm23-H1 gene product expression might be a prognostic marker in patients with involved lymph nodes. Our data does not demonstrate any correlation between nm23-H1 gene product expression and blood vessel invasion.

The steady-state kinetics of the enzyme reaction tested by site-directed mutagenesis of hydrophobic residues (Val, Leu, and Cys) in the C-terminal alpha-helix of human adenylate kinase.

To elucidate whether the C-terminal region in human adenylate kinase participates in the interaction with the substrate (MgATP(2-) and/or AMP(2-)), hydrophobic residues (Val182, Val186, Cys187, Leu190, and Leu193) were substituted by site-directed mutagenesis and the steady-state kinetics of fifteen mutants were analyzed. A change in the hydrophobic residues in the C-terminal domain affects the affinity for substrates (K(m)), that is, not only for MgATP(2-) but also for AMP(2-), and the catalytic efficiency (k(cat)). The results obtained have led to the following conclusions: (i) Val182 may interact with both MgATP(2-) and AMP(2-) substrates, but to a greater extent with MgATP(2-), and play a role in catalysis. (ii) Val186 appears to play a functional role in catalysis by interacting with both MgATP(2-) and AMP(2-) to nearly the same extent. (iii) Cys187 appears to play a functional role in catalysis. (iv) Leu190 appears to interact with both MgATP(2-) and AMP(2-) substrates but to a greater extent with AMP(2-). (v) Leu193 appears to interact with both MgATP(2-) and AMP(2-) but to a greater extent with AMP(2-). The activity of all mutants decreased due to the change in substrate-affinity. The closer the residue is located to the C-terminal end, the more its mutation affects not only MgATP(2-) but also AMP(2-) substrate binding. The hydrophobic alterations disrupt hydrophobic interactions with substrates and that might destabilize the conformation of the active site. The more C-terminal part of the alpha-helix appears to interact with AMP, as if it has swung out and rotated to cover the adenine moieties. The C-terminal alpha-helix of human adenylate kinase appears to be essential for the interaction with adenine substrates by swinging out during catalysis.