Protein microarray analysis identifies human cellular prion protein interactors.

AIMS: To obtain an insight into the function of cellular prion protein (PrPC), we studied PrPC-interacting proteins (PrPIPs) by analysing a protein microarray. METHODS: We identified 47 novel PrPIPs by probing an array of 5000 human proteins with recombinant human PrPC spanning amino acid residues 23-231 named PR209. RESULTS: The great majority of 47 PrPIPs were annotated as proteins involved in the recognition of nucleic acids. Coimmunoprecipitation and cell imaging in a transient expression system validated the interaction of PR209 with neuronal PrPIPs, such as FAM64A, HOXA1, PLK3 and MPG. However, the interaction did not generate proteinase K-resistant proteins. KeyMolnet, a bioinformatics tool for analysing molecular interaction on the curated knowledge database, revealed that the complex molecular network of PrPC and PrPIPs has a significant relationship with AKT, JNK and MAPK signalling pathways. CONCLUSIONS: Protein microarray is a useful tool for systematic screening and comprehensive profiling of the human PrPC interactome. Because the network of PrPC and interactors involves signalling pathways essential for regulation of cell survival, differentiation, proliferation and apoptosis, these observations suggest a logical hypothesis that dysregulation of the PrPC interactome might induce extensive neurodegeneration in prion diseases.

Endoscopic submucosal dissection allows less-invasive curative resection for gastric tube cancer after esophagectomy - a case series.

Detection of early gastric tube cancers (GTCs) has increased with more detailed surveillance endoscopy using indigo carmine dye following esophagectomy. This retrospective study clarified the clinicopathological features and application of endoscopic submucosal dissection (ESD) for GTCs. Data collected for eight GTCs treated by ESD included clinical and pathological features and outcomes following ESD. Overall, eight GTCs were identified in seven (6.3 %) of 112 patients who underwent esophagectomy and gastric tube reconstruction. Almost all lesions were macroscopically type 0-IIa with mucosal to submucosal invasion, and seven GTCs were successfully resected en bloc by ESD. Submucosal invasion to > 500 microm was observed in one case with associated delayed perforation that was treated conservatively. No local recurrences of GTCs were observed. Detailed surveillance endoscopy using indigo carmine dye appears useful for diagnosing early-stage GTC. Furthermore ESD represents a feasible alternative to conventional endoscopic mucosal resection as a minimally invasive therapy for early-stage GTC.

Evaluation of combined docetaxel and nedaplatin chemotherapy for recurrent esophageal cancer compared with conventional chemotherapy using cisplatin and 5-fluorouracil: a retrospective study.

This retrospective study evaluated the safety and efficacy of combination chemotherapy using docetaxel and nedaplatin in an outpatient setting compared with those of chemotherapy using cisplatin (CDDP) and 5-Fu under hospitalization. Subjects comprised 21 patients who had been diagnosed with recurrent esophageal squamous cell carcinoma (ESCC), with 10 patients receiving combination chemotherapy comprising CDDP and 5-fluorouracil (5-Fu) under hospitalization (FP group; n = 10), and 11 patients receiving combination chemotherapy comprising docetaxel and nedaplatin in an outpatient setting (Doc/Ned group; n = 11). In the Doc/Ned group, patients received 30 mg/m(2) of docetaxel over a 1-h infusion on day 1, followed by 40 mg/m(2) of nedaplatin over a 2-h infusion on day 1 in an outpatient setting. In the Doc/Ned group, complete response was observed in two patients (18.1%), one with liver metastasis and one with abdominal lymph node metastasis, and two (18.1%) achieved partial response. In contrast, no complete responses were obtained in the FP group, and partial response was observed in only one patient (10.0%) with local recurrence. Response rates were thus 36.3% for the Doc/Ned group and 10.0% for the FP group. With a median follow-up of 234 days in the Doc/Ned group and 279 days in the FP group, median survival time (MST) was 234 days in the Doc/Ned group and 378 days in the FP group. No significant differences in MST were identified between groups. Thus regimen based on docetaxel and nedaplatin allows administration on an outpatient basis and appears feasible for recurrent ESCC as a second-line chemotherapy.

Association of ultrasound-based measures of fetal body composition with newborn adiposity.

BACKGROUND: Newborns exhibit substantial variation in gestational age-adjusted and sex-adjusted fat mass proportion. The antecedent characteristics of fetal body composition that are associated with newborn fat mass proportion are poorly understood. OBJECTIVE: The aim of this study was to determine whether a composite measure of fetal fat mass is prospectively associated with newborn adiposity. METHODS: In a longitudinal study of 109 low-risk pregnancies, fetal ultrasonography was performed at approximately 12, 20 and 30 weeks gestation. Estimated fetal adiposity (EFA) was derived by integrating cross-sectional arm and thigh per cent fat area and anterior abdominal wall thickness. Newborn per cent body fat was quantified by Dual Energy X-Ray Absorptiometry. The association between EFA and newborn per cent body fat was determined by multiple linear regression. RESULTS: After controlling for confounding factors, EFA at 30 weeks was significantly associated with newborn per cent body fat (standardized ß = 0.41, p < 0.001) and explained 24.0% of its variance, which was substantially higher than that explained by estimated fetal weight (8.1%). The observed effect was driven primarily by arm per cent fat area. CONCLUSIONS: A composite measure of fetal adiposity at 30 weeks gestation may constitute a better predictor of newborn per cent body fat than estimated fetal weight by conventional fetal biometry. Fetal arm fat deposition may represent an early indicator of newborn adiposity. After replication, these findings may provide a basis for an improved understanding of the ontogeny of fetal fat deposition, thereby contributing to a better understanding of its intrauterine determinants and the development of potential interventions.

Magnesium sulphate and perinatal mortality and morbidity in very-low-birthweight infants born between 24 and 32 weeks of gestation in Japan.

OBJECTIVE: Maternal exposure to magnesium sulphate has a neuroprotective effect in premature infants. This study aimed to examine this neuroprotective effect and the dose-response relationship in very-low-birthweight infants born between 24 and 32 weeks of gestation. STUDY DESIGN: A retrospective cohort study compared the rates of mortality and brain damage between three groups: no magnesium sulphate, low-dose (<50g) magnesium sulphate and high-dose (≥50g) magnesium sulphate. RESULTS: Japanese maternal and neonatal databases were linked using six key parameters from 2003 to 2007. Of 298,514 deliveries, 9101 were very-low-birthweight infants. Among these, full matching was possible for 5562 infants. Of the fully-matched infants, 3763 were born between 24 and 32 weeks of gestation, and 1813 (48%) were followed-up beyond 18 months. A multivariate analysis of the data, including gestational age, sex, fetal growth restriction, antenatal steroids and low pH (<7.1), showed that the low-dose group had no beneficial effects in terms of a reduction in mortality or incidence of brain damage (cerebral palsy or mental retardation). The high-dose group showed a significantly higher mortality rate [odds ratio (OR) 1.9, 95% confidence interval (CI) 1.2-2.9]. A stratified subgroup analysis of infants born between 28 and 32 weeks of gestation showed that survivors in the low-dose group had significantly lower rates of cerebral palsy (OR 0.4, 95% CI 0.2-0.98) and brain damage (OR 0.2, 95% CI 0.1-0.9), while the high-dose group did not show any significant changes. CONCLUSION: This study found that antepartum exposure to magnesium sulphate did not reduce the infant mortality rate or influence neurological outcomes. However, among infants born between 28 and 32 weeks of gestation, rates of cerebral palsy and brain damage were found to be significantly lower among survivors in the low-dose group.

Intragenic homozygous deletion of the WT1 gene in Wilms' tumor.

One example of intragenic homozygous deletion of the WT1 gene on chromosome 11p13 was found after screening 42 samples of Wilms' tumor DNA from Japanese patients. After construction of a restriction map for the genomic sequence covering the 3' half of the gene, the deletion was analysed at the nucleotide sequence level. The deletion occurred in the patient's germline on his paternal chromosome, and most of the short arm of his maternal chromosome 11 was subsequently lost in the tumor. The size of the deletion was about 8 kb, removing exons 6 and 7 and resulting in premature termination. The deletion seemed to be created by recombination between short homologous sequences found in an Alu repeat, with a 16-bp duplication left at the junction. This case conforms to a two-hit model for the genesis of a certain group of tumors, and supports the hypothesis that WT1 is one of the recessive oncogenes responsible for Wilms' tumor.

Elastofibromatous lesion of the stomach in a patient with elastofibroma dorsi.

A 69-year-old woman underwent partial gastrectomy because of peptic ulcer which resisted medical treatment. The resected stomach exhibited a wide thickening of the antral wall around a 1.2 X 0.8 cm ulcer. The cut surface showed a gray-white thickened submucosal layer and had a rubbery elastic consistency. Microscopically, the thickened areas consisted of abundant acellular collagen fibers containing numerous elastinophilic, thick, serrated fibers and globules, identical with the elastofibroma fibers seen in elastofibroma dorsi. Reexamination of the patient revealed bilateral subscapular masses; one of these was biopsied and proved to be an elastofibroma dorsi.

PAI-1 plays an important role in the expression of t-PA activity in the euglobulin clot lysis by controlling the concentration of free t-PA.

The antigen levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were assayed in the plasma and in the euglobulin fraction, and their contributions to the euglobulin clot lysis time (ECLT) and t-PA activity were analyzed. Total and free PAI-1 levels in both fractions showed significant positive correlation with ECLT (p less than 0.001), whereas t-PA antigen level did not have a high correlation coefficient with ECLT. t-PA activity showed significant negative correlation with ECLT (p less than 0.001) and positive correlation with free t-PA level (p less than 0.001), which was calculated by the ratio of the concentrations of t-PA-PAI-1 complex and the free PAI-1. Thus free t-PA seems to dissolve the euglobulin clot and its concentration seems to be controlled by the concentration of free PAI-1. These findings were confirmed by the analyses of the effects of C1-inactivator and antibody against t-PA to regular ECLT and kaolin activated ECLT, the latter of which was only inhibited by the addition of C1-inactivator whereas the former was inhibited by anti-t-PA antibody.

Cerebral blood flow measurement of severely head-injured patients during mild hypothermia.

In 14 patients with severe head injury, the cerebral blood flow (CBF) during mild hypothermia therapy was measured. Their Glasgow Coma Scale scores on admission were 8 or less and the intracranial pressures were greater than 20 mmHg despite conventional therapy. The CBF was measured with two-level stable xenon CT techniques. And in 11 patients, the cerebral metabolic rates for oxygen (CMRO2) was also calculated. All cases were divided into two groups according to the outcome at discharge by using the Glasgow Outcome Scale, good outcome group in 6 and poor outcome one in 8. The values of mean CBF and CMRO2 of each group were 25.6 +/- 6.6 vs 24.4 +/- 6.4 ml/100 g/min and 1.26 +/- 0.45 vs 0.79 +/- 0.31 ml/100 g/ml, respectively. There were no statistically significant differences between both groups. Single CBF measurement during this therapy may not be helpful as a factor of prognosis evaluation in patients with severe head injury.

Disseminated subcutaneous Nocardia asteroides abscesses in a patient after bone marrow transplantation.

We describe an unusual case of disseminated subcutaneous abscesses caused by Nocardia asteroides in a 17-year-old female with AML undergoing allogeneic BMT. She was receiving immunosuppressive therapy with CYA and a corticosteroid for acute GVHD, and maintenance therapy with ganciclovir for interstitial pneumonia (IP) caused by CMV, but was not neutropenic. The subcutaneous abscesses spread from the primary infection on her right anterior leg to both thighs, the left buttock, both upper arms, the left forearm and right shoulder, indicating hematogenous dissemination. Nocardia asteroides was identified from biopsy material in culture. The patient was successfully treated with a combination of trimethoprim/sulfamethoxazole (TMP/SMX) and minocycline, given for 3 months. The possibility of nocardiosis should be considered in the differential diagnosis of such patients.

Increase in levels of plasminogen activator and type-1 plasminogen activator inhibitor in human breast cancer: possible roles in tumor progression and metastasis.

We measured antigen levels of two kinds of plasminogen activators, tissue type plasminogen activator (t-PA) and urokinase type plasminogen activator (UK), as well as their primary inhibitor, type-1 plasminogen activator inhibitor (PAI-1) in the tissue extracts of benign and malignant breast tumors. Tumor tissues of 36 fibroadenomas and 39 breast cancers were examined. t-PA levels were not different in both groups. Malignant tumors contained the significantly higher levels of UK than benign tumors (p less than 0.001). Furthermore in breast cancer tissues, UK antigen levels of tumors with axillary lymph node involvements were significantly higher than those of tumors without lymph node involvements (p less than 0.05). PAI-1 antigen levels of breast cancer tissues were dramatically higher than those of fibroadenoma (p less than 0.001). PAI-1 levels of node positive carcinomas showed also values significantly higher than node negative ones (p less than 0.01). When we divided cancer tissues into three groups as node negative tumors, tumors with positive axillary nodes fewer than four and tumors with four or more positive nodes, PAI-1 levels increased corresponding to the progression of lymph node involvements (p less than 0.05). Immunohistochemical studies, using mouse monoclonal antibodies to human UK and PAI-1, showed that those immunoreactivities were diffusely distributed in the cytoplasm of human breast cancer cells. Their staining patterns were very similar to each other.

Fluctuation of TPA and PAI-1 antigen levels in plasma: difference of their fluctuation patterns between male and female.

The daytime fluctuation of the fibrinolytic activity and the antigen levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) in plasma were analyzed in normal male and female volunteers. Samples were obtained at 8:30, 10:30, 12:30, 14:30 and 16:30 h. Euglobulin clot lysis time (ECLT) showed the longest time at 8:30 to 10:30 h and the shortest time at 16:30 h. The highest level of tPA was obtained at 8:30 h and the lowest at 14:30 h in men, whereas the highest value was at 8:30 h and the lowest was at 16:30 h in females. Active free PAI-1 showed highest value at 10:30 h in men and at 8:30 h in women. The lowest values obtained at 16:30 h in both men and women and were about one third of the highest values. ECLT was always shorter in females than in males and parameter such as tPA and PAI-1 were also lower in females than in males. The phase of the circadian rhythm of the fibrinolytic parameters may be advanced in females than in males.

Fluctuation of tPA and PAI-1 antigen levels in plasma: difference of their fluctuation patterns between male and female.

The circadian fluctuation of the fibrinolytic activity and the antigen levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) in plasma were analyzed in normal male and female volunteers. Samples were obtained at 8:30, 10:30, 12:30, 14:30 and 16:30 h. Euglobulin clot lysis time (ECLT) showed the longest time at 8:30 to 10:30 h and the shortest time at 16:30 h. The highest level of tPA was obtained at 8:30 h and the lowest at 14:30 h in men, whereas the highest value was at 8:30 h and the lowest was at 16:30 h in females. Free PAI-1 showed highest value at 10:30 h in men and at 8:30 h in women. The lowest values obtained at 16:30 h in both men and women and were about one third of the highest values. ECLT was always shorter in females than in males and parameter such as tPA and PAI-1 were also lower in females than in males. The phase of the circadian rhythm of the fibrinolytic parameters may be advanced in females than in males.

The concentration of tissue plasminogen activator and urokinase in plasma and tissues of patients with ovarian and uterine tumors.

The concentrations of tissue plasminogen activator (t-PA) and urokinase (UK) were measured in the plasma of patients with benign or malignant ovarian and uterine tumors. Plasma levels of t-PA were higher in patients with malignant ovarian and uterine tumors than in patients with benign tumors. Plasma UK levels were, however, not different between patients with ovarian and uterine tumors (benign or malignant) and normal persons. The concentrations of t-PA and UK of tissues of uterine myoma were lower than those in normal uterine muscular layer. UK levels of tissues of endometrial and cervical cancers were significantly higher than those in normal endometrium and uterine cervix, whereas t-PA levels were not different between these tumors compared with those in normal uterine tissues.

Biosynthesis and secretion of MHC class III gene products (complement C4 and factor B) in the exocrine pancreas.

We recently found that complement C3 is locally synthesized and secreted into the exocrine pancreas. In the present study, we attempted to demonstrate the secretion of complement C4 and factor B in the exocrine pancreas. In five samples of pancreatic fluid, both C4 and factor B proteins were detected by enzyme-linked immunosorbent assay (ELISA). Immunoblot analysis revealed the C4 and factor B molecules in pancreatic fluid to be identical with these molecules in serum. Reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis in pancreatic carcinoma cell lines suggested ductal epithelial cells to be the local production sites of these proteins in the pancreas. The secretion of C4 and factor B in ductal cell lines (PANC-1 and MIA PaCa-2) was independently regulated by interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma; C4 secretion was induced by IFN-gamma, whereas factor B secretion was induced by IL-1 beta, TNF-alpha, or IFN-gamma. These observations indicate that: (a) complement C4 and factor B are secreted into the exocrine pancreas, (b) ductal epithelial cells appear to be the site of C4 and factor B biosynthesis, and (c) local secretion of C4 and factor B in the pancreas is differentially regulated by IL-1 beta, TNF-alpha, and IFN-gamma.

Characterization of complement C3, C4, and factor B molecules in human bile.

We performed molecular analysis of complement components (C3, C4, and factor B) in human bile by sodium dodecyl sulfate-polyarylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Complement C3 was detected as a molecule composed of a 115-kDa alpha-chain linked to a 70-kDa beta-chain by disulfide bonds, and C3 levels ranged from 45 to 650 micrograms/ml (n = 15). C4 was detected as a triple chain (98-kDa alpha-chain, 73-kDa beta-chain, and 33-kDa gamma-chain) molecule linked by disulfide bonds, and C4 levels ranged from 2.5 to 60 micrograms/ml. Factor B, a component of the alternative pathway, was also detected, as an intact form. Factor B levels ranged from 0.3 to 8.0 micrograms/ml. The sizes and subunit structures of complement components in human bile were compatible with those reported in human serum. The results of a hemolytic assay indicated that complement molecules in human bile were functionally active. These molecules may participate in local immune and inflammatory responses in the biliary tract.

Regulation of complement C3 synthesis by interleukin-1 and transforming growth factor-beta in rat non-transformed intestinal epithelial cell line, IEC-6.

Intestinal epithelial cells are an important source of many biologically active molecules that modulate immune responses in the mucosa. The purpose of this study was to demonstrate the synthesis of complement C3 component in the rat non-transformed crypt-like intestinal epithelial cell line, IEC-6. Unstimulated IEC-6 cells secreted a low level of C3 protein and showed weak expression of C3 mRNA. The addition of interleukin (IL)-1 beta induced a dose- and time-dependent increase in C3 production. These effects of IL-1 beta were observed at a concentration as low as 0.01 ng/ml and reached a plateau at a concentration of 5 ng/ml. The effects were observed at the mRNA level as early as 6 h after the beginning of incubation. Transforming growth factor (TGF)-beta alone had no effect. However, TGF-beta at low concentrations (0.001-1 ng/ml) enhanced the effect of IL-1 beta in increasing C3 production; this enhancement was not observed at high concentrations (5-10 ng/ml). These effects of TGF-beta were also observed at the mRNA level. The present findings indicate that intestinal epithelial cells are indeed capable of synthesizing complement C3 in response to IL-1 beta and TGF-beta.

HLA-DR antigen expression in squamous epithelial dysplasia and squamous cell carcinoma of the esophagus: an immunohistochemical study.

To clarify the biologic significance of esophageal squamous epithelial dysplasia, especially the similarity to carcinoma in situ, immunohistochemical investigation of HLA-DR antigen expression and lymphocyte infiltration was performed. HLA-DR antigen was expressed in 12 of the 35 invasive carcinomas (34.4%), 23 of the 38 intraepithelial carcinomas (60.5%), 21 of the 50 areas of dysplasia (42.0%) and only 2 of the 625 specimens of non-cancerous squamous epithelium (0.3%). The HLA-DR-positive rate of dysplasia localized continuous to HLA-DR-positive carcinoma was 68.4%, which was significantly higher than that for HLA-DR positive dysplasia localized continuous to HLA-DR negative cancer (11.1%) (p<0.05). In areas of dysplasia and intraepithelial carcinoma, T cell infiltration was significantly increased at the sites of HLA-DR antigen expression (P<0.01). B cell infiltration was also more common in areas of positive expression. These results suggest that HLA-DR antigen is associated with the local immune response to squamous epithelial dysplasia, and that HLA-DR antigen expression may prevent tumor invasion similarly to its role in intraepithelial carcinoma.

The prognostic significance of the cytophotometric DNA content and its relationship with the argyrophilic nucleolar organizer regions (AgNOR) and proliferating cell nuclear antigen (PCNA) in oesophageal cancer.

We examined the DNA pattern, AgNOR number and PCNA-positive ratio (PCNA ratio) from biopsy specimens of oesophageal carcinoma, and attempted to identify any prognostic factors for oesophageal carcinoma. DNA analysis: the cell nuclear DNA content was measured and the distribution pattern of the DNA content was grouped into four types according to the ploidy pattern (n = 182). The survival rate of patients with high-ploidy tumours (type III and IV) was shorter than that for low-ploidy tumours (type II), (P < 0.05). AgNOR number (n = 99) and PCNA ratio (n = 41): the AgNOR number and PCNA ratio were measured from 100 cancer cells in each specimen. Both the high-AgNOR-number patients (> or = 5) and the high-PCNA-ratio patients (> or = 35) demonstrated poorer prognoses than the low-rate patients (P < 0.05). These results suggest that the DNA pattern, AgNOR number and PCNA ratio may thus reflect the proliferative activity of tumours and therefore also offer the possibility of interdependence among these three factors.

Histological suggestions of 'paratransformation' in oesophageal squamous cell carcinoma.

We have postulated that oesophageal squamous cell carcinoma arises from multifocal areas and not from one cell, and we present the circumstantial evidence for field carcinogenesis in oesophageal cancer. Among 290 cases examined with oesophageal squamous cell carcinoma, lymphatic permeation or intramural metastasis was evident in 110 cases. Of these carcinomatous transformation of the epithelium adjacent to either lymphatic vessel permeation or intramural metastasis of the squamous cell carcinoma was recognized in six cases. It is considered that the cancer tissue either in the lymphatic vessel or intramural metastasis promotes a malignant transformation of the squamous epithelium over these foci. 'Paratransformation' is therefore thought to be one of the potential models of origin for oesophageal squamous cell carcinoma.