LRRN4 and UPK3B are markers of primary mesothelial cells.

BACKGROUND: Mesothelioma is a highly malignant tumor that is primarily caused by occupational or environmental exposure to asbestos fibers. Despite worldwide restrictions on asbestos usage, further cases are expected as diagnosis is typically 20-40 years after exposure. Once diagnosed there is a very poor prognosis with a median survival rate of 9 months. Considering this the development of early pre clinical diagnostic markers may help improve clinical outcomes. METHODOLOGY: Microarray expression arrays on mesothelium and other tissues dissected from mice were used to identify candidate mesothelial lineage markers. Candidates were further tested by qRTPCR and in-situ hybridization across a mouse tissue panel. Two candidate biomarkers with the potential for secretion, uroplakin 3B (UPK3B), and leucine rich repeat neuronal 4 (LRRN4) and one commercialized mesothelioma marker, mesothelin (MSLN) were then chosen for validation across a panel of normal human primary cells, 16 established mesothelioma cell lines, 10 lung cancer lines, and a further set of 8 unrelated cancer cell lines. CONCLUSIONS: Within the primary cell panel, LRRN4 was only detected in primary mesothelial cells, but MSLN and UPK3B were also detected in other cell types. MSLN was detected in bronchial epithelial cells and alveolar epithelial cells and UPK3B was detected in retinal pigment epithelial cells and urothelial cells. Testing the cell line panel, MSLN was detected in 15 of the 16 mesothelioma cells lines, whereas LRRN4 was only detected in 8 and UPK3B in 6. Interestingly MSLN levels appear to be upregulated in the mesothelioma lines compared to the primary mesothelial cells, while LRRN4 and UPK3B, are either lost or down-regulated. Despite the higher fraction of mesothelioma lines positive for MSLN, it was also detected at high levels in 2 lung cancer lines and 3 other unrelated cancer lines derived from papillotubular adenocarcinoma, signet ring carcinoma and transitional cell carcinoma.

Changes in serum sex hormone profiles after short-term low-dose administration of dehydroepiandrosterone (DHEA) to young and elderly persons.

In man, serum concentrations of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) decrease with age after the twenties. For this reason, the decline in DHEA and DHEAS concentrations may be related to the development of some chronic diseases that are prevalent in the older age population. In this study, we evaluate the benefit and safety level of DHEA administration to men as a hormone replacement therapy. Twenty-two healthy Japanese males (age 26-63; mean +/- SD, 41.0 +/- 10.0 yrs.) received 25 mg DHEA once a day orally in the morning for two weeks. Serum concentrations of steroid hormones and cytokines were measured before and after the DHEA administration. Glucose tolerance and insulin resistance were also assessed before and after the DHEA administration using a 75 g oral glucose tolerance test and homeostasis model assessment (HOMA-R), respectively. Serum DHEA and DHEAS levels were significantly elevated after the DHEA administration for all ages of test subjects. In subjects who were older than 41 yrs. (older group) serum androstenedione and estradiol levels were elevated after the DHEA administration. Significant negative correlations were observed between the serum DHEA concentration and the serum concentration of fasting insulin, HOMA-R, leptin, and high-sensitivity C-reactive protein for all subjects. Daily administration of 25 mg DHEA increased the serum DHEA, DHEAS, androstenedione, and estradiol levels of the subjects of the older group to the same level as that of younger subjects.

Development and progression of retinopathy after inpatient management of diabetes.

OBJECTIVE: To examine factors that affect the development of retinopathy after short-term inpatient management of diabetes. PATIENTS AND METHODS: The subjects were 143 patients with type 2 diabetes who were admitted for inpatient management of diabetes, and did not have retinopathy of the right eye at admission, and had an HbA1c level of > or =8.0%. We studied the characteristics of patients who developed retinopathy within one year after discharge. RESULTS: Between the admission date and one year after discharge, twenty-six patients developed retinopathy and the retinopathy subsequently regressed in 5 patients. The 26 patients who developed retinopathy had a significantly longer duration of diabetes (p<0.005), had a higher fasting blood glucose level at admission (p=0.06), and received insulin therapy during the admission at a higher rate (p=0.06) than the 117 patients without retinopathy. The magnitude of the reduction in HbA1c level at 3 months after discharge was smaller in the 13 patients who developed retinopathy within 3 months after discharge than in the 130 patients who did not. Among the 26 patients who developed retinopathy, the HbA1c level at one year after discharge of the 5 patients whose retinopathy regressed was lower than that of the 21 patients whose retinopathy did not regress (p=0.06). CONCLUSIONS: A long duration of diabetes, high fasting blood glucose level at admission, and treatment with insulin were associated with the development of retinopathy. Patients with these characteristics should undergo frequent fundus examinations after correction of hyperglycemia. The retinopathy was likely to improve if patients maintained strict glycemic control after discharge.

mRNA expression of inducible nitric oxide synthase, endothelial nitric oxide synthase and vascular endothelial growth factor in esophageal mucosa biopsy specimens from patients with reflux esophagitis.

BACKGROUND/AIMS: Nitric oxide (NO) production is elevated in the intestine and may contribute to intestinal injury during inflammation. However, how the expression of inducible NO synthase (iNOS) mRNA and endothelial NO synthase (eNOS) mRNA in the esophageal mucosa contribute to mucosal damage caused by reflux esophagitis remains unknown. Since vascular endothelial growth factor (VEGF) exerts its action on microcirculation, contributing to angiogenesis and inflammation, we examined the role of VEGF together with iNOS and eNOS on development of reflux esophagitis. METHODOLOGY: The mRNA expression levels of iNOS, eNOS and VEGF were measured in biopsy specimens from 25 patients with reflux esophagitis, using TaqMan PCR and reverse transcription PCR. RESULTS: The expression of iNOS mRNA in the esophageal mucosa increased parallel to the severity of the esophagitis. There were no significant differences between both eNOS and VEGF mRNA expression levels and the severity of the esophagitis. The existence of gastric mucosal atrophy, hiatus hernia, therapy and Helicobacter pylori infection did not affect the levels of mRNA expression. CONCLUSIONS: The accumulation of NO, produced by iNOS, was considered to be related to the exacerbation of reflux esophagitis. Therapeutic intervention that reduces NO production may thus be of use in preventing development of esophageal mucosal injury in patients with reflux esophagitis.

Pleural effusions following endoscopic injection sclerotherapy for cirrhotic patients with esophageal varices.

BACKGROUND/AIMS: Endoscopic injection sclerotherapy is in widespread use for patients with esophageal varices. It is well known that pleural effusions are among complications following endoscopic sclerotherapy. However, there are few studies regarding the proportion of patients developing pleural effusions after sclerotherapy. METHODOLOGY: Between August 1991 and September 1998, 575 endoscopic injection sclerotherapies were carried out in 128 patients. Chest radiographs were obtained prior to and 24 hours after all procedures. We also obtained other clinical data from all patients. RESULTS: In total, 17.7% of post-sclerotherapy patients were diagnosed as having small amounts of pleural effusions. Logistic regression revealed pleural effusions after sclerotherapy to be associated with ascites, chest pain for 24 hours, total volume of sclerosant and submucosal injection of more than 4mL of sclerosant. In parallel with injection of an increasing amount of submucosal sclerosant, the proportion of patients with pleural effusion increased. CONCLUSIONS: Pleural effusions were related to ascites, chest pain for 24 hours, total sclerosant volume and submucosal injection of sclerosant.

Efficacy of a triple therapy with rabeprazole, amoxicillin, and faropenem as second-line treatment after failure of initial Helicobacter pylori eradication therapy.

BACKGROUND/AIMS: Triple therapy consisting of lansoprazole, amoxicillin, and clarithromycin (LAC regimen) is widely used to eradicate Helicobacter pylori in Japan. However, the need for appropriate treatment after failure of initial therapy to eradicate H. pylori has been increasing. We therefore assessed the efficacy of a combination of rabeprazole, amoxicillin, and faropenem for second-line eradication therapy. METHODOLOGY: The subjects were 116 patients positive for H. pylori infection. Patients initially received lansoprazole 60 mg/day, amoxicillin 1500 mg/day and clarithromycin 400 mg/day in two divided doses for 7 days. Patients in whom eradication treatment failed were given rabeprazole 20 mg/day and amoxicillin 1500 mg/day in two divided doses, and faropenem 600 mg/day in three divided doses (RAF regimen) for 7 consecutive days. H. pylori status was assessed by the 13C-urea breath test combined with rapid urease test or H. pylori culture method 8 weeks after completion of therapy. Susceptibility to clarithromycin was determined by the agar dilution method, and genetic polymorphism of CYP2C19 was analyzed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The initial H. pylori eradication rate with the LAC regimen was 76.4% (84/110). Assessment of the CYP2C19 genotypes of the patients in whom eradication therapy failed revealed that homozygous extensive metabolizers accounted for 70.0% (16/23) and heterozygous extensive metabolizers for 30.0% (7/23), with no poor metabolizers. The acquired resistance rate for clarithromycin was 52.0% (12/23). The success rate of re-eradication with the RAF regimen was 91.3% (21/23) with no serious adverse effects. CONCLUSIONS: Triple therapy comprising rabeprazole, amoxicillin, and faropenem is effective for second-line eradication treatment of H. pylori infection, regardless of the genetic polymorphism of CYP2C19 or the presence of resistance to clarithromycin.

Marked attenuation of production of collagen type I from cardiac fibroblasts by dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) is a type of adrenal steroid. The concentrations of DHEA and its sulfate (DHEA-S) in serum reach a peak between the ages of 25 and 30 yr and thereafter decline steadily. It was reported that DHEA-S concentration in humans is inversely related to death from cardiovascular diseases. In this study, we examined the effects of DHEA on regulation of collagen mRNA and collagen synthesis in cultured cardiac fibroblasts. Treatment with DHEA (10(-6) M) resulted in a significant decrease in procollagen type I mRNA expression compared with controls. This was accompanied by a significant decrease in procollagen type I protein accumulation in the medium and also a significant decrease in procollagen type I protein synthesis in the cellular matrix. Furthermore, to confirm in vitro results, we administered DHEA to Sprague-Dawley rats, which were treated with angiotensin II for 8 wk to induce cardiac damage. Procollagen type I mRNA expression was significantly decreased and cardiac fibrosis significantly inhibited in DHEA-treated rat hearts without lowering the systolic blood pressure. These results strongly indicate that DHEA can directly attenuate collagen type I synthesis at the transcriptional level in vivo and in vitro in cardiac fibroblasts.

Effects of dehydroepiandrosterone on gluconeogenic enzymes and glucose uptake in human hepatoma cell line, HepG2.

Dehydroepiandrosterone (DHEA), the most abundant human adrenal steroid, improves insulin sensitivity and obesity in human and model animals. In a previous study, we reported that orally administered DHEA suppresses the elevated activities of hepatic gluconeogenic enzymes like glucose-6-phosphatase (G6Pase) in C57BL/KsJ-db/db mice. However, the molecular mechanisms by which DHEA ameliorates insulin resistance are not clearly understood. In the present study, we cultured the human hepatoma cell line HepG2 with DHEA and measured the enzyme activity and protein expression of G6Pase to investigate the direct effect of DHEA on glucose metabolism in hepatocytes. DHEA significantly suppressed both the activity and protein expression of G6Pase. Moreover, DHEA decreased the gene expression of G6Pase and phosphoenolpyruvate carboxykinase, both of which were maximal at 1 microM DHEA, whereas the mRNA level of glucose-6-phosphate translocase was unchanged. Furthermore, DHEA enhanced 2-deoxyglucose uptake, although its effect was much smaller than that of insulin. These results suggest that DHEA may act at multiple steps in the regulation of glucose metabolism in the liver.

sPAR-3, a splicing variant of PAR-3, shows cellular localization and an expression pattern different from that of PAR-3 during enterocyte polarization.

PAR-3 (partitioning-defective) is a scaffold-like PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain-containing protein that forms a complex with PAR-6 and atypical PKC, localizes to tight junctions, and contributes to the formation of functional tight junctions. There are several alternatively spliced isoforms of PAR-3, although their physiological significance remains unknown. In this study, we show that one of the major isoforms of PAR-3, sPAR-3, is predominantly expressed in the Caco-2 cells derived from colon carcinoma and is used as a model to investigate the events involved in the epithelial cell differentiation and cell polarity development. During the polarization of Caco-2 cells, the expression of PAR-3 increases as do those of other cell-cell junction proteins, whereas the expression of sPAR-3 decreases. Biochemical characterization revealed that sPAR-3 associates with atypical PKC, as does PAR-3. On the other hand, immunofluorescence microscopy revealed that sPAR-3 does not concentrate at the cell-cell contact region in fully polarized cells, whereas it concentrates at premature cell-cell junctions. This makes a contrast to PAR-3, which concentrates at tight junctions in fully polarized cells. These results provide evidence suggesting the difference in the role between sPAR-3 and PAR-3 in epithelial cells.

A novel therapy for acute hepatitis utilizing dehydroepiandrosterone in the murine model of hepatitis.

Dehydroepiandrosterone (DHEA), one of the major androgens secreted by the adrenal cortex, has been shown to have potential immunoreguratory properties. In this study, we examined the effect of DHEA in a mouse model of hepatitis. Mice were treated with DHEA and injected with concanavalin A (Con A) or lipopolysaccharide (LPS)/D-galactosamine (GalN). Cytokine expression was measured by quantitative RT-PCR and ELISA. Apoptosis was detected by the TUNEL method and by DNA fragmentation analysis. In the DHEA-treated mice, the serum levels of ALT and expression of inflammatory mediators were significantly decreased. The number of apoptotic cells was also much lower than that observed in control, untreated mouse liver tissue. There were fewer tumor necrosis factor-alpha (TNF-alpha)-induced apoptotic cells in H4IIE hepatoma cells treated with DHEA than in non-treated cells. DHEA decreased the expression levels of mRNA transcripts encoding TNF-alpha and iNOS. These results suggest that DHEA can reduce T-cell-mediated injury in the liver as manifest by inhibition of the expression of several inflammatory mediators and hepatocyte apoptosis. DHEA should, thus, be considered as a novel candidate for the therapy of liver injury.

Insulin-induced cell cycle progression is impaired in chinese hamster ovary cells overexpressing insulin receptor substrate-3.

To analyze the roles of insulin receptor substrate (IRS) proteins in insulin-stimulated cell cycle progression, we examined the functions of rat IRS-1 and IRS-3 in Chinese hamster ovary cells overexpressing the human insulin receptor. In this type of cell overexpressing IRS-1 or IRS-3, we showed that: 1) overexpression of IRS-3, but not IRS-1, suppressed the G1/S transition induced by insulin; 2) IRS-3 was more preferentially localized to the nucleus than IRS-1; 3) phosphorylation of glycogen synthase kinase 3 and MAPK/ERK was unaffected by IRS-3 overexpression, whereas that of protein kinase B was enhanced by either IRS; 4) overexpressed IRS-3 suppressed cyclin D1 expression in response to insulin; 5) among the signaling molecules regulating cyclin D1 expression, activation of the small G protein Ral was unchanged, whereas insulin-induced gene expression of c-myc, a critical component for growth control and cell cycle progression, was suppressed by overexpressed IRS-3; and 6) insulin-induced expression of p21, a cyclin-dependent kinase inhibitor, was decreased by overexpressed IRS-3. These findings imply that: 1) IRS-3 may play a unique role in mitogenesis by inhibiting insulin-stimulated cell cycle progression via a decrease in cyclin D1 and p21 expressions as well as suppression of c-myc mRNA induction in a manner independent of the activation of MAPK, protein kinase B, glycogen synthase kinase 3 and Ral; and 2) the interaction of IRS-3 with nuclear proteins may be involved in this process.

Aldosterone stimulates gene expression of hepatic gluconeogenic enzymes through the glucocorticoid receptor in a manner independent of the protein kinase B cascade.

Primary aldosteronism is associated with glucose intolerance and diabetes, which is due in part to impaired insulin release caused by reduction of potassium, although other possibilities remain to be elucidated. To evaluate the in vivo effects of aldosterone on glucose metabolism, a single dose of aldosterone was administered to mice, which resulted in elevation of the blood glucose level. In primary cultured mouse hepatocytes, the gene expression of gluconeogenic enzymes such as glucose-6-phosphatase (G6Pase), fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase increased in response to aldosterone in a dose-dependent manner even at a concentration similar to a physiological condition (10(-9) M). The inhibitory effect of insulin on G6Pase gene expression was partially suppressed by aldosterone. Furthermore, aldosterone enhanced G6Pase promoter activity in human hepatoma cell line HepG2, which was prevented by co-treatment with a glucocorticoid antagonist RU-486, but not a mineralocorticoid antagonist spironolactone. In contrast, aldosterone had no effects on major insulin signaling pathways including insulin receptor substrate-1, protein kinase B, and forkhead transcription factor. These results suggest that aldosterone may affect the inhibitory effect of insulin on hepatic gluconeogenesis through the glucocorticoid receptor, which may be one of the causes of impaired glucose metabolism in primary aldosteronism.