Interleukin-1beta gene in esophageal, gastric and colorectal carcinomas.

Interleukin (IL)-1 gene polymorphisms are associated with development of gastric atrophy and with increased risk of gastric carcinoma. A -31C to T base transition in the promoter region of this gene is involved in carcinogenic changes within the stomach, especially in Helicobacter pylori infected individuals. We examined association between IL-1 locus polymorphisms and risk of esophageal, gastric and colorectal carcinomas in Japanese patients with H. pylori infection. IL-1B and IL-1RN polymorphisms were analyzed in 136 controls, 75 patients with esophageal carcinoma, 186 patients with gastric carcinoma, 69 patients with colorectal carcinoma, and 18 patients with ulcerative colitis (UC). For IL-1B-511 and -31 polymorphisms were determined by fluorescence-based polymerase chain reaction single-strand conformation polymorphism analysis. For IL-1 receptor antagonist gene (IL-1RN), penta-allelic variable number of tandem repeats (VNTR) was determined by PCR-standard agarose gel electrophoresis. For gastric carcinoma, IL-1B-511 heterozygotes (OR, 0.48; 95% CI, 0.3-0.9; p=0.0115) and T carriers (OR, 0.52; 95% CI, 0.3-1.0; p=0.0185) had a significantly reduced risk of carcinoma. For colorectal carcinoma, IL-1B-511 heterozygotes (OR, 0.34; 95% CI, 0.2-0.7; p=0.0028) and T carriers (OR, 0.43; 95% CI, 0.2-0.9; p=0.0015) had a significantly low risk of carcinoma. No significant difference was observed in the frequencies of IL-1B-31C/T and IL-1RN genotypes between controls and the esophageal carcinoma patients. Our results shows that IL-1B-511C/T and T carrier state may indicate less risk for gastric and colorectal carcinoma in the Japanese population.

Pathological features and genetic alterations in colorectal carcinomas with characteristics of nonpolypoid growth.

We sought to clarify pathological features and genetic alterations in colorectal carcinomas with characteristics of nonpolypoid growth. Colorectal carcinomas resected at Showa University Hospital in Tokyo included 86 with characteristics of polypoid growth (PG) and 21 with those of nonpolypoid growth (NPG). Mutations of APC, Ki-ras, and p53 genes, as well as microsatellite instability (MSI), were analysed using fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Carcinomas with an NPG pattern were smaller than PG tumours (P<0.0001). Carcinomas with a PG pattern were more likely to harbour Ki-ras mutations (36%) than NPG tumours (0%; P<0.0001). Mutation types in the APC gene differed significantly between PG and NPG carcinomas (P=0.0189), including frameshift mutations in 66% of PG carcinomas but no NPG carcinomas. Presence of a p53 mutation at a 'hot spot' also was more likely in PG carcinomas (37%) than in NPG carcinomas (0%; P=0.0124). No significant difference in presence of MSI was evident between carcinomas with PG and NPG patterns. In conclusion, significant genetic differences were evident between carcinomas with PG and NPG patterns. Genetic changes in NPG carcinomas differed from those of the conventional adenoma-carcinoma sequence. Assuming that some nonpolypoid growth lesions transform rapidly into advanced carcinomas, 20% of all colorectal carcinomas may progress in this manner.

Detection of Grb-2-related adaptor protein gene (GRAP) and peptide molecule in salivary glands of MRL/lpr mice and patients with Sjögren's syndrome.

The pathogenesis of Sjögren's syndrome (SS) is poorly understood. In this study we used an in-house mouse spleen cDNA microarray to analyse genes in spleens from MRL/lpr (an SS mouse model) mice. We have previously demonstrated that GRAP genes were up-regulated in salivary glands of the same mice. The microarray analysis showed that seven out of 2304 genes were highly expressed in spleens from the MRL/lpr mice, one of which was the GRAP gene. In other words, the GRAP gene is highly expressed in the salivary glands and spleen of MRL/lpr mice. We also carried out immunohistochemical studies. Mouse and human Grb-2-related adaptor protein (GRAP) antigens were expressed on ductal cells and infiltrating lymphocytes in salivary glands of MRL/lpr mice and SS patients, but only weakly in controls (MRL/+ mice and individuals with salivary cysts). These results suggest that the GRAP gene might have a role in the pathogenesis of SS.

A new tactile skin sensor for measuring skin hardness in patients with systemic sclerosis and autoimmune Raynaud's phenomenon.

We used a new tactile sensor to measure the elastic properties of skin in patients with systemic sclerosis or Raynaud's phenomenon. The sensor consists of a piezoelectric vibrator with vibration pickup to measure frequency changes when the sensor is placed on the skin. The mean frequency change at the skin surface of the proximol third phalanx in patients with systemic sclerosis was significantly lower than in age- and sex-matched controls. The results in systemic sclerosis patients were statistically correlated to the Modified Rodnan Skin Thickness Score. This technique was also used to measure the therapeutic efficacy of salpogrelate, a new specific serotonin receptor antagonist. A greater mean frequency change was seen after treatment. We conclude that this new tactile sensor is useful for quantitatively measuring skin sclerosis and may help determine the efficacy of therapeutic treatments.

Prognostic index of liver cirrhosis with ascites with and without hepatocellular carcinoma.

BACKGROUND: In Japan, the incidence of liver cirrhosis caused by hepatitis viruses is higher, and cirrhosis is more likely to be complicated by hepatocellular carcinoma, than in Western countries. The aim of this study was to predict the outcome in liver cirrhosis with ascites with and without hepatocellular carcinoma. METHODS: The subjects were 146 patients with liver cirrhosis complicated by ascites. Forty-six factors were evaluated concerning clinical laboratory parameters and extracted prognostic factors using the Cox proportional hazards model. RESULTS: The mean duration of the follow-up period was 634.9 days, during which 89 (61%) of the patients died, 27 (18.5%) survived, and 30 (20.6%) were lost to follow-up. The cumulative survival rate after the onset of ascites was 59.7% after 1 year, 44.5% after 2 years, and 29.5% after 5 years. Multivariate analysis indicated 9 factors, i.e. age, total bilirubin (T-Bil), alkaline phosphatase (ALP), blood urea nitrogen (BUN), alpha-fetoprotein (AFP), mean arterial pressure (MAP), gastrointestinal bleeding, infection, and portal vein tumor thrombosis (PVTT), as independent prognostic factors. The prognostic index (PI) was calculated by the following formula using these 9 factors. PI = 0.045 x age + 0.180 x T-Bil + 0.088 x ALP + 0.020 x BUN + 0.467 x AFP + (-0.022 x MAP) + 0.662 x gastrointestinal bleeding + 0.521 x infections + 0.882 x PVTT. CONCLUSION: Prediction of the outcome using PI based on the 9 factors provides additional information for the determination of the therapeutic approach in cirrhotic patients with ascites with and without hepatocellular carcinoma.

No major tumorigenic role for beta-catenin in serrated as opposed to conventional colorectal adenomas.

Intracellular redistribution of beta-catenin through mutation of the adenomatous polyposis coli (APC) gene has been proposed as an early tumorigenic event in most colorectal tumours. In serrated adenoma (SA), a newly recognised subtype of colorectal adenoma, APC mutations are uncommon, and the contribution of beta-catenin to tumorigenesis remains unclear. We compared intracellular localisation of beta-catenin and presence of mutations in exon 3 of beta-catenin between 45 SAs, with 71 conventional adenomas (CADs), and eight carcinomas invading the submucosa (SCAs). Widespread or focal nuclear beta-catenin expression was demonstrated in 7% of SAs (three out of 45), 61% of CADs (43 out of 71), and 88% of SCAs (seven out of eight). Cytoplasmic immunostaining for beta-catenin was demonstrated in 16% of SAs (seven out of 45), 77% of CADs (55 out of 71), and 88% of SCAs (seven out of eight). No mutation in exon 3 of beta-catenin was found in SAs or SCAs, while 7% of CADs (five out of 71) had beta-catenin mutations. No nuclear or cytoplasmic expression of beta-catenin was observed in the hyperplastic or conventionally adenomatous epithelium of mixed-type SAs. These findings suggest that beta-catenin mutation is unlikely to contribute to the tumorigenesis in SA, and that intracellular localisation of beta-catenin may not be associated with an early event of the tumour progression in most SAs.

Growth-promoting effect of muscarinic acetylcholine receptors in colon cancer cells.

PURPOSE: G-protein-coupled receptors are known to mediate cell growth via divergent signaling pathways. It has been reported that colon cancer cells express muscarinic acetylcholine receptor (mAChR) although their functional role is largely unknown. The aim of this study is to elucidate possible mechanisms responsible for the growth-promoting effect of mAChRs in colon cancer cells by using colon cancer cell line T84. METHODS: Carbachol, a stable mAChR agonist, dose-dependently induced cell growth with a maximal effect observed at 100 microM, equipotent with 1 nM EGF. 4-DAMP, a specific antagonist of subtype 3 mAChR, inhibited the stimulatory effect by carbachol, suggesting that the growth-promoting effect was receptor-mediated. Carbachol also dose-dependently stimulated extracellular signal-regulated protein kinase (ERK) activation. This effect was inhibited by PD98059, an inhibitor of extracellular signal-regulated protein kinase kinase, which also blocked carbachol activation of cell proliferation, indicating that the p21Ras-ERK pathway is an important signaling cascade in the mitogenic effect. To investigate how mAChR activated the p21Ras-ERK pathway, transactivation of epidermal growth factor receptor (EGFR) was examined. RESULTS: Carbachol induced tyrosine phosphorylation of EGFR, which was abolished by an EGFR tyrosine kinase inhibitor AG1478. Transactivation by carbachol was also abrogated by a metalloproteinases (MMPs) inhibitor GM6001 or an EGFR-blocking antibody (LA-1), suggesting that binding of EGFR ligand(s) produced by MMPs may initiate transactivation in a manner dependent on EGFR tyrosine kinase. The tyrosine-phosphorylated EGFR was immunoprecipitated together with GRB2 and tyrosine-phosphorylated Shc, indicating that transactivated EGFR is able to generate downstream signals. AG 1478 and LA-1 inhibited carbachol stimulation of cell growth. CONCLUSIONS: Taken together, our results indicate that the growth-promoting effect of subtype 3 mAChR in colon cancer cells may depend on transactivated EGFR-ERK pathways. EGFR not only receives external stimuli but also serves as a scaffold for downstream signaling molecules.

Definitive chemoradiotherapy for patients with malignant stricture due to T3 or T4 squamous cell carcinoma of the oesophagus.

We retrospectively investigated the efficacy and feasibility of concurrent chemoradiotherapy for patients with severe dysphagia caused by oesophageal squamous cell carcinoma. Concurrent chemoradiotherapy was performed in 57 patients with T3 or T4 disease containing M1 lymph node (LYM) disease. Chemotherapy consisted of protracted infusion of 5-fluorouracil (5-FU) 400 mg m(-2) 24 h(-1) on days 1-5 and 8-12, combined with 2-h infusion of cisplatin (CDDP) 40 mg m(-2) on days 1 and 8. Radiation treatment at a dose of 30 Gy in 15 fractions of the mediastinum was administered concomitantly with chemotherapy. A course schedule with 3-week treatment and a 1 to 2-week break was applied twice, with a total radiation dose of 60 Gy, followed by two or more courses of 5-FU and CDDP. In all, 24 patients (42%) achieved a complete response, and the 3-year survival rate was 19%. Major toxicities were leukocytopenia and oesophagitis, and there were two (4%) treatment-related deaths. In contrast, 22 patients with T3 disease survived longer than 35 patients with T4 disease (P=0.001); however, the survival rate in 15 patients with M1 LYM disease did not differ significantly from that in 42 patients without M1 LYM disease (P=0.3545). Our results indicate that definitive chemoradiotherapy is potentially curative for locally advanced oesophageal carcinoma with malignant stricture. The efficacy and survival of patients treated with this regimen are related to the T factor.

Histological findings after placement of a self-expanding stent in rectal carcinoma with complete obstruction--case report.

Patients with acute obstruction due to colorectal carcinomas frequently require emergency surgery. However, such emergency procedures are associated with various complications, a high mortality rate and a poor prognosis. If the obstruction could be immediately relieved, the patient could later undergo an elective operation with a much better prognosis. Recently, expanding metallic stents have been used to treat obstruction due to colorectal carcinoma. In the case reported here, we initially inserted a colonoscopic retrograde bowel drainage tube per anus to achieve decompression. We then placed a self-expanding metallic stent, since we anticipated a prolonged preoperative period due to high fever, congestive heart failure, cerebral infarction, and persistent high blood sugar concentrations. The patient had no complications for 57 days after placement of the stent, and eventually underwent an elective operation. Histologically, the side of the cancerous lesion compressed by the stent was thin and consisted solely of a serosal layer. Implantation of a metallic stent is safe for the treatment of acute malignant obstruction. Stent placement is indicated not only as a palliative treatment for inoperable or recurrent cases, but also as a preoperative procedure before elective surgical resection.

[Evaluation of immunochromatography method for rapid detection of influenza A and B viruses].

We have evaluated a new rapid detection kit for influenza A and B viruses, known as the QuickVue Influenza test (Quidel Coporation, USA); which is based on immunochromatography using virus isolates and clinical specimens. Twelve strains of influenza A and B were tested for evaluate the reactivity and detection limits of this test. The QuickVue Influenza test showed a positive result for all twelve strains of influenza virus and a negative result for fourteen different kinds of other respiratory viruses. The detection limits for six strains were 5 to 30 pfu/ml for a cell culture, 1.0 x 10(3) to 6.0 x 10(4) pfu/ml for 1st PCR, 1 to 50 pfu/ml for nested PCR, 3.0 x 10(5) to 6.0 x 10(5) pfu/ml for the QuickVue Influenza test, 1.5 x 10(5) to 1.0 x 10(6) pfu/ml for the Directigen Flu A, and 7.5 x 10(5) to 5.0 x 10(6) pfu/ml for the FLU OIA. Furthermore, the QuickVue Influenza test were clinically evaluated using 92 throat swab specimens collected from patients with influenza-like illnesses. By cell culture, influenza viruses were detected in 49 of the 92 specimens (AH1N1: 20, AH3N2: 7, B: 22); the titers of the influenza viruses were between 2.5 pfu/ml and 7.0 x 10(5) pfu/ml. Compared to cell culture, the QuickVue Influenza test showed a sensitivity of 75.5%, a specificity of 93.0%, a positive predictive value of 92.5%, a negative predictive value of 76.9%, and an efficiency value of 83.7%. On the other hand, influenza viruses were detected in 54 of the 92 specimens (AH1N1: 19, AH 3N2: 10, B: 25) by RT-PCR. Compared to RT-PCR, the QuickVue Influenza test showed a sensitivity of 72.2%, a specificity of 97.4%, a positive predictive value of 97.5%, a negative predictive value of 71.2%, and an efficiency value of 82.6%. Overall, only one throat swab specimen produced a false positive result using the QuickVue Influenza test; thus, this test appears to have a high specificity. We conclude that the QuickVue Influenza test is a simple one-step test with a sensitivity and specificity equivalent to those of other conventional diagnostic kits. The test is useful and suitable for the diagnosis of influenza and for identifying influenza patients requiring antiviral therapy.

Acquisition of susceptibility to hepatitis C virus replication in HepG2 cells by fusion with primary human hepatocytes: establishment of a quantitative assay for hepatitis C virus infectivity in a cell culture system.

Hepatitis C virus (HCV) replicates in human and chimpanzee hepatocytes. To characterize the nature of HCV and evaluate antiviral agents, the development of an HCV replication system in a cell culture is essential. We developed a cell line derived from human hepatocytes by fusing them with a hepatoblastoma cell line, HepG2, and obtained several clones. When we tested the clones for their ability to support HCV replication by nested RT-PCR, we found 1 clone (IMY-N9) that was more susceptible to HCV replication than HepG2. The negative-strand HCV RNA was detected in IMY-N9 by strand-specific RT-PCR, and viral RNA was identified in culture supernatant during the culture. Then we monitored HCV RNA titers in IMY-N9 and HepG2, respectively, by real-time detection PCR throughout the culture. A significant increase in the HCV RNA titer was observed only in IMY-N9. Serial passages of HCV culture supernatant were shown in the culture system. Furthermore, we tested several infectious materials for viral infectivity by monitoring HCV RNA titers and/or 50% tissue culture infectious dose (TCID50) of HCV on IMY-N9. In each material, HCV showed various growth patterns and a different TCID50 even though the PCR titer in each material was identical. The results showed that HCV in each material served various growth patterns and different TCID50 even though PCR titer in each material was identical. This cell line is useful for estimating viral activity and for studying cellular factors that may be necessary to HCV replication in human hepatocytes.

Mechanism of growth-inhibitory effect of cisplatin on human pancreatic cancer cells and status of p53 gene.

Pancreatic cancer is a devastating malignant tumor in humans and the development of new modalities of treatment is needed. We studied the mechanism of the growth-inhibitory effect of cisplatin (CDDP) on human pancreatic cancer cells in connection with the status of the p53 gene and expression of the bcl-2 family. COLO-357 cells with wild-type p53 gene and T3M4, Panc-1 and AsPC-1 cells with mutant-p53 gene were used. Growth of these cells was inhibited by CDDP in a dose-dependent manner in both serum-deprived and serum-supplemented conditions. CDDP induced apoptosis of COLO-357 and T3M4 cells in the serum-supplemented condition, whereas necrosis of these cells was induced by CDDP at high concentrations in the serum-deprived condition. Although expression of bax mRNA and its protein product were enhanced, while bcl-2 protein was decreased by CDDP in COLO-357 cells, expression of mRNA of the bcl-2 family and protein product were not influenced by CDDP in T3M4 cells. Increased expression of bax and reduced expression of bcl-2 are involved in the growth-inhibitory effect of CDDP on pancreatic cancer cells with wild-type p53 gene.

A case of autoimmune hepatitis with a high titer of antimitochondrial antibody and normal gamma-globulinemia.

We report here a patient with chronic active hepatitis who had no markers for hepatitis viruses and no hyper-gamma-globulinemia, but had high titers of antimitochondrial antibody. Serum levels of alkaline phosphatase were normal, and antinuclear antibody, antismooth muscle antibody, and antiliver kidney microsome antibody tested negative. The titers of antimitochondrial antibody exceeded 1:640, and the positivity for anti-M2 was ascertained by using both ELISA and immunoblot with beef-heart mitochondria and a recombinant pyruvate dehydrogenase E2 subunit as antigens. This patient responded to ursodeoxycholic acid (UDCA) therapy in the beginning, but her hepatitis flared up during UDCA therapy. In contrast, she responded completely to corticosteroid therapy. The clinical course and histological findings of this patient strongly suggest that this patient has autoimmune hepatitis.

The presence of a newly identified infectious agent (SEN virus) in patients with liver diseases and in blood donors in Japan.

The existence of the newly discovered SEN virus (SENV) was investigated in 379 Japanese patients with liver diseases and in 277 blood donors, to determine whether SENV is associated with non-A-E hepatitis. SENV DNA was detected by seminested polymerase chain reaction, with primers directed to 2 SENV strains: SENV-H and SENV-D. SENV was detected in 7 (32%) of 22 patients with fulminant hepatitis, in 15 (17%) of 86 patients with acute hepatitis, in 38 (27%) of 139 patients with chronic hepatitis, in 29 (31%) of 93 patients with liver cirrhosis, in 5 (33%) of 15 patients with autoimmune hepatitis, in 11 (46%) of 24 patients with primary biliary cirrhosis, and in 27 blood donors (10%). Infection occurred more frequently in patients with liver diseases than in blood donors; however, there were no significant differences in SENV-positive rates between patients with non-A-C hepatitis and those with acute or chronic hepatitis due to known hepatitis virus or nonviral liver disease. This study did not suggest SENV as a possible causative agent of non-A-C hepatitis.

[A case of esophageal carcinoma with multiple liver metastases successfully treated with nedaplatin (NDP) and 5-fluorouracil (5-FU) combined with radiotherapy].

A 78-year-old male patient had esophageal carcinoma with multiple liver metastases. Chemoradiotherapy was performed. The chemotherapy consisted of protracted infusion of 5-fluorouracil (5-FU), combined with infusion of nedaplatin (NDP). Radiation of the mediastinum was administered concomitantly with chemotherapy. The patient showed a complete response (CR) of the primary lesion and a partial response (PR) of the liver metastasis for 11 months. Since liver metastasis recurred after initial treatment, chemotherapy consisting of NDP infusion combined with vindesine sulfate (VDS) infusion was performed. The patient again showed PR. Grade 3 leukocytopenia occurred during treatment, but there were no major toxicities such as thrombocytopenia, nausea, renal dysfunction or esophagitis. Survival time was one year and 7 months. In conclusion, concurrent chemoradiotherapy including NDP is effective and safe for patients with esophageal carcinoma accompanied by multiple liver metastasis. This nonsurgical approach may be an option for standard care in such cases.

Validation of color Doppler EUS for azygos blood flow measurement in patients with cirrhosis: application to the acute hemodynamic effects of somatostatin, octreotide, or placebo.

BACKGROUND: Color Doppler EUS (CD-EUS) allows minimally invasive measurement of azygos blood flow (AzBF) in portal hypertension, but further validation of the method is needed. Because a limited number of patients has been studied, the acute hemodynamic effects of somatostatin and octreotide on AzBF and gastric mucosal perfusion are poorly defined in portal hypertension. METHODS: A double-blind hemodynamic study was designed to assess rapid changes in AzBF over a 60-minute period after intravenous administration of somatostatin, octreotide, and placebo in 30 stable patients with biopsy-proven cirrhosis. AzBF was measured by using both CD-EUS and the invasive thermal dilution technique in the first 10 patients (phase 1). Then, with CD-EUS alone, the hemodynamic study was extended to a further 20 patients (phase 2). In addition, gastric mucosal perfusion changes were assessed by using laser Doppler flowmetry at endoscopy. RESULTS: In phase 1, the 2 methods for AzBF measurement showed significant correlations both for baseline values (r = 0.685) and for AzBF changes over 60 minutes after drug administration (r = 0.733). In phase 2, a reduction was observed in AzBF 10 minutes after octreotide or somatostatin administration (-47% and -23%, p < 0.0001 vs. placebo, p = 0.058 vs. placebo, respectively). After 60 minutes of somatostatin infusion, AzBF increased 27% over placebo values (p < 0.04). Gastric mucosal perfusion was transiently reduced 5 minutes after octreotide or somatostatin (-21% and -32%, respectively, p < 0.02 vs. placebo). CONCLUSIONS: This is the first study to validate CD-EUS AzBF measurement with reference to the invasive thermodilution technique in cirrhosis. It confirmed the transient effects of somatostatin and octreotide on both AzBF and gastric mucosal perfusion. In addition, a significant rebound phenomenon after 60 minutes of continuous intravenous somatostatin infusion was observed.

Prognostic value of p53 mutations in patients with locally advanced esophageal carcinoma treated with definitive chemoradiotherapy.

PURPOSE: A significant correlation has been found between p53 mutation and response to chemotherapy or radiotherapy. To determine the prognostic value of p53 mutation in patients with locally advanced esophageal carcinoma treated with definitive chemoradiotherapy, p53 mutation was analyzed using the biopsied specimens taken for diagnosis. METHODS: Concurrent chemoradiotherapy was performed for 40 patients with severe dysphagia caused by esophageal squamous cell carcinoma associated with T3 or T4 disease. Chemotherapy consisted of protracted infusion of 5-fluorouracil, combined with an infusion of cisplatinum. Radiation treatment of the mediastinum was administered concomitantly with chemotherapy. The p53 gene mutation was detected by fluorescence-based polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) methods. DNA sequences were determined for DNA fragments with shifted peaks by SSCP methods. RESULTS: Of the 40 patients, 15 had T3 disease and 25 had T4 disease; 11 patients had M1 lymph node (LYM) disease. Of the 40 patients, 13 (33%) achieved a complete response. The median survival time was 14 months, and the 2-year survival rate was 20%. Among the 40 tumor samples, p53 mutation was detected in 24 tumors (60%). The survival rate in the 24 patients with p53 mutation did not differ significantly from that in the 16 patients without p53 mutation. In contrast, the 15 patients with T3 disease survived longer than the 25 patients with T4 disease (P = 0.016); however, the survival rate in the 11 patients with M1 LYM disease did not differ significantly from that in the 29 patients without M1 LYM disease. CONCLUSION: Concurrent chemoradiotherapy is potentially curative for locally advanced esophageal carcinoma, but p53 genetic abnormality has no impact on prognosis.

Decreased expression of signaling lymphocytic-activation molecule-associated protein (SAP) transcripts in T cells from patients with rheumatoid arthritis.

The function of Epstein-Barr virus (EBV)-specific cytotoxic T cells is disturbed in rheumatoid arthritis (RA) patients but the mechanism for this disturbance has remained unknown. In a recent study searching for the causative gene of X-linked lymphoproliferative syndrome, the gene possibly linked to EBV-specific cytotoxic T cells or NK cell-mediated cytotoxic activity to EBV-infected cells was discovered, and its product is now referred to as signaling lymphocytic-activation molecule-associated protein (SAP) or Src homology 2 domain-containing protein (SH2D1A). In the present study, we attempted to investigate the involvement of the SAP gene in RA using a quantitative real-time PCR; the expression level of SAP transcripts in peripheral leukocytes or T cells was examined for patients with RA. The expression level of SAP transcripts in peripheral leukocytes of 21 RA patients was significantly lower than that of 13 normal individuals (P = 0.0007), four patients with palindromic RA, 11 with inactive systemic lupus erythematosus (SLE) or 17 with chronic renal diseases. The decreased expression of SAP transcripts in RA patients was also observed in peripheral CD2(+) T cells compared with normal individuals. There was no mutation in the coding region of SAP cDNAs derived from peripheral leukocytes of five RA patients. The decreased expression of SAP transcripts in peripheral leukocytes or T cells of RA patients might lead to the failure of the immune system to eliminate the EBV-infected synovial lining cells in joints of RA patients. Our findings have suggested that decreased expression of the SAP gene might be involved in the onset or progress of RA.

Frequent down-regulation of E-cadherin by genetic and epigenetic changes in the malignant progression of hepatocellular carcinomas.

E-cadherin mediates cell-cell adhesion by associating with catenins. Loss of E-cadherin function by genetic or epigenetic alteration of the E-cadherin gene (CDH1) leads to tumorigenesis. To study the involvement of E-cadherin dysfunction in liver tumorigenesis, we examined the allelic loss and methylation of 5'-CpG sites of CDH1 in hepatocellular carcinomas (HCCs). Loss of heterozygosity (LOH) of CDH1 and adjacent 16q22-23 loci was observed in 13 of 30 (43%) HCCs. Methylation of the 5'-CpG of CDH1 was analyzed by Southern blot hybridization, and hypermethylation was observed in 8 of the 24 (33%) HCCs examined. The amount of E-cadherin mRNA was analyzed by RNase protection assay, and a decrease in E-cadherin mRNA was observed in 10 of the 23 cases examined. A reduction in E-cadherin was found in 10 of 21 HCCs using immunoblot analysis. The amount of E-cadherin was comparable to that of E-cadherin mRNA. Down-regulation of E-cadherin was common in cases with LOH but rare in cases with methylated promoter. These results suggest that hypermethylation of the CDH1 promoter is present in a small cell population in the tumor, thus the methylation status is liable to vary according to individual cell condition. Hypermethylation was observed in early stage HCCs, whereas LOH was found frequently in more malignant tumors. Down-regulation of E-cadherin is closely related to the progression of HCCs and is stably induced by LOH of CDH1.

Derivatization in liquid chromatography/mass spectrometric analysis of neurosteroids.

Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances. However, some compounds do not show enough sensitivity in LC/MS and soft-ionization methods commonly used in LC/MS, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), sometimes do not give satisfactory structural information. This report presents an overview of the derivatization methods in the LC/MS analysis of neurosteroids or neuroactive neurosteroids, which are synthesized and accumulated in the nervous system. The derivatization of pregnenolone 3-sulfate, one of these steroids, with 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole gave a satisfactory sensitivity during the quantitative analysis using LC/ESI-MS. The obtained results are much lower than those previously obtained using gas chromatography/mass spectrometry or radioimmunoassay. On the other hand, the derivatization to acetate was useful for the treatment of labile catechol estrogens in rat brains and gave enough structural information in LC/APCI-MS, which confirmed the existence of catechol estrogens in mammalian brains.