Usefulness of fecal calprotectin by monoclonal antibody testing in adult Japanese with inflammatory bowel diseases: a prospective multicenter study.

BACKGROUND/AIMS: Noninvasive objective monitoring is advantageous for optimizing treatment strategies in patients inflammatory bowel disease (IBD). Fecal calprotectin (FCP) is superior to traditional biomarkers in terms of assessing the activity in patients with IBD. However, there are the differences among several FCP assays in the dynamics of FCP. In this prospective multicenter trial, we investigated the usefulness of fecal FCP measurements in adult Japanese patients with IBD by reliable enzyme immunoassay using a monoclonal antibody. METHODS: We assessed the relationship between FCP levels and disease or endoscopic activity in patients with ulcerative colitis (UC, n=64) or Crohn's disease (CD, n=46) compared with healthy controls (HCs, n=64). RESULTS: FCP levels in UC patients strongly correlated with the Disease Activity Index (rs=0.676, P<0.0001) and Mayo endoscopic subscore (MES; rs=0.677, P<0.0001). FCP levels were significantly higher even in patients with inactive UC or CD compared with HCs (P=0.0068, P<0.0001). The optimal cutoff value between MES 1 and 2 exhibited higher sensitivity (94.1%). FCP levels were significantly higher in active UC patients than in inactive patients (P<0.001), except those with proctitis. The Crohn's Disease Activity Index tended to correlate with the FCP level (rs=0.283, P=0.0565). CONCLUSIONS: Our testing method using a monoclonal antibody for FCP was well-validated and differentiated IBD patients from HCs. FCP may be a useful biomarker for objective assessment of disease activity in adult Japanese IBD patients, especially those with UC.

APOB codon 4311 polymorphism is associated with hepatitis C virus infection through altered lipid metabolism.

BACKGROUND: It has been reported that some single-nucleotide polymorphisms (SNPs) in lipid regulators such as apolipoproteins and cell surface molecules for hepatitis C virus (HCV) entry into hepatocytes are associated with HCV infection. However, it is unknown how HCV infection is affected by altered lipid metabolism resulting from the SNPs. We investigated the relationship between these SNPs and HCV infection status, and also analyzed the mechanism by which these SNPs mediate HCV infection via lipid metabolism alterations. METHODS: Serum lipid and apolipoprotein profiles were tested in 158 HCV-positive and 220 HCV-negative subjects. We selected 22 SNPs in five lipid regulator genes which were related to HCV entry into hepatocytes and to lipid metabolism (APOA1, APOB, SR-B1, LDLR, and APOE), and their polymorphisms were analyzed using the PCR-sequence-specific oligonucleotide probe-Luminex method. RESULTS: An APOB N4311S (g.41553a > g) SNP, rs1042034, was significantly associated with HCV positivity; the HCV positivity rate for the minor allele AA genotype was significantly higher than for genotype AG + GG (P = 0.016). Other SNPs except for APOB P2712L SNP rs676210, which is in linkage disequilibrium with rs1042034, showed no significant difference in genotype distribution. The serum level of low density lipoprotein-cholesterol (LDL-C) in the genotype AA group was significantly lower than in the genotype non-AA group (P = 0.032), whereas the triglyceride (TG) level was significantly higher (P = 0.007). CONCLUSION: An APOB SNP, rs1042034, is closely associated with HCV infection through lipid metabolism alteration. The minor allele AA genotype might contribute to facilitating serum LDL uptake into hepatocytes via LDLR by modifying their affinity and interaction and may have an influence on HCV infection by their entry to the liver through the LDLR.

Evaluation of annexin A5 as a biomarker for Alzheimer's disease and dementia with lewy bodies.

BACKGROUND: Alzheimer's disease (AD) differs from other forms of dementia in its relation to amyloid beta peptide (Aß42). Using a cell culture model we previously identified annexin A5, a Ca(2+), and phospholipid binding protein, as an AD biomarker. Plasma level of annexin A5 was significantly higher in AD patients compared to that in a control group. On the other hand, AD has been identified to share a number of clinical and pathological features with Dementia with Lewy bodies (DLB). The present study was done to examine whether or not plasma annexin A5 is a specific marker for AD, when being compared with the levels of DLB patients. As Apolipoprotein E (ApoE) gene subtype ε4 (ApoE-ε4) has been noticed as the probable genetic factor for AD, we also examined and compared ApoE genotype in both AD and DLB. METHODS: Blood samples were obtained from 150 patients with AD (aged 77.6 ± 6.5 years), 50 patients of DLB (79.4 ± 5.0) and 279 community-dwelling healthy elderly individuals of comparable age and sex (75.6 ± 8.1). All AD patients met NINCDS-ADRDA criteria and all DLB patients were diagnosed as probable DLB according to the latest consensus diagnostic criteria. Quantification was done using the Chemiluminescent Enzyme Immunoassay (CLEIA) Technique (SphereLight assay) using the monoclonal antibodies against annexin A5. DNA genotyping of ApoE was performed by distinguishing unique combinations of Hha1 fragments of PCR-amplified genomic DNA products. RESULTS: The plasma level of annexin A5 was significantly higher in AD patients than in the healthy individuals (control) (P < 0.0001). The plasma annexin A5 level was also significantly higher in DLB patients than in the control group (P < 0.0001). From the ROC curves with plasma annexin A5 concentrations, the mean areas under the curve were 0.863 and 0.838 for the AD/control and DLB/control, respectively. The rate of ApoE4 carrier status and the frequency of the ε4 allele were significantly higher in AD or DLB than in control and there was no significant difference between AD and DLB. CONCLUSIONS: These results suggest that both annexin A5 and ApoE4 are common markers for AD and DLB.

Investigation of annexin A5 as a biomarker for Alzheimer's disease using neuronal cell culture and mouse model.

Alzheimer's disease (AD) differs from other forms of dementia in its relation to amyloid beta peptide (Abeta). Abeta, a proteolytic product of amyloid precursor proteins (APP), has a toxic effect on neuronal cells, which involves perturbation of their Ca(2+) homeostasis. This effect implies that changes of protein expression in neuronal cells with calcium stress should provide a molecular marker for this disease. In the present study, we used the supernatant from a neuronal cell culture after incubation with or without Abeta and isolated a Ca(2+)-dependent acidic phospholipid binding fraction to perform a proteomic study. Several unique proteins were identified after incubation with Abeta. We focused on annexin A5, among these proteins, because it binds both Ca(2+) and lipids likely to be involved in calcium homeostasis. Tg2576 transgenic mice (AD model) overexpressing mutant human APP showed a significant increase of annexin A5 in the brain cortex but not in other organs, including liver, kidney, lung, and intestine. In human plasma samples, the level of annexin A5 was significantly increased in a proportion of AD patients compared with a control group (P < 0.0001 in the logistic regression analysis). From the receiver operating characteristic (ROC) curve with plasma annexin A5 concentrations, the mean area under the curve (AUC 0.898) suggests that annexin A5 is a favorable marker for AD.

A histochemical and immunohistochemical investigation of guanase and nedasin in rat and human tissues.

Human guanase is known as a specific enzyme in the liver, kidney, and brain. However, its functional significance remains poorly understood. In addition, interestingly, a different organ distribution between humans and rats was suggested. Here, we performed immunohistochemical staining with anti-human nedasin (neuronal and endocrine discs large/SAP102 associated protein), whose sequence was identical to that of guanase, antibody and histochemical staining for guanase in normal tissues of rat and human liver, kidney, and small intestine, and compared the results. Guanase activity was observed uniformly in the rat hepatocytes, biliary epithelium and vascular endothelium cells, while it was localized to the hepatocytes and biliary epithelium in the human liver. When the histochemical staining for guanase and the immunohistochemical staining for nedasin were compared, the stained regions were different in the rat liver but were almost consistent in all human tissues. Totally consistent staining results were also obtained between rats and humans in the other organization except the liver. Based upon the research reports to date, the experiments on guanase and nedasin in rat organs performed in this study are considered to have important implications in the investigation of their physiological significance.

Histochemical and immunohistochemical investigation of guanase and nedasin in human tissues.

Guanase is known as an enzyme released from the liver. Recently, cloning and sequencing of the guanase gene were reported. In addition, almost simultaneously, it was reported that an unknown protein that binds to neuronal and endocrine lethal(1)-discs large (NE-dlg), one of the membrane-associated guanylate kinase homologues (MAGUK) family proteins involved in synaptic connection between neurons, was cloned and named nedasin (NE-dlg associated protein), whose sequence was almost identical to that of guanase. We immunostained fresh frozen sections of surgically removed human liver, kidney, and small intestine with anti-nedasin antibody, and simultaneously performed histochemical staining for guanase for comparison. Histochemically, guanase activity was observed in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the mucosal epithelium on the small intestine, and in the proximal tubule on the kidney. Immunohistochemically, a brown discoloration due to DAB oxidation was seen in the cytoplasm of hepatocytes and biliary epithelium on the liver, in the proximal tubule but in the distal tubule a little on the kidney, in the mucosal epithelium on the small intestine. The stained region of the liver and the small intestine were different from that of the kidney. The different staining properties dependent on the organs were considered to be due to different isozymes. The physiological significance of guanase may vary with the isozymes, further studies are considered necessary.

13C-Urea breath test, using a new compact nondispersive isotope-selective infrared spectrophotometer: comparison with mass spectrometry.

BACKGROUND: The (13)C-urea breath test ((13)C-UBT) is the most commonly used noninvasive method of detecting Helicobacter pylori infection. The isotope ratio mass spectrometer (IRMS) is the most commonly used device for this test, but the UBiT-IR300 infrared spectrophotometer, which, by comparison, is a more compact, less expensive, and easier to use analytical device, has now become widely used in the clinical setting in Japan. The objective of this study was to examine the diagnostic performance of the (13)C-UBT, using the UBiT-IR300. METHODS: A multicenter open-label study was performed, in which the (13)C-UBT was conducted using 100 mg of (13)C-urea. Analysis of (13)CO(2) in the expired breath was performed by infrared spectroscopy and mass spectrometry, and assessment of H. pylori infection was performed by culture, histological examination, and rapid urease test. RESULTS: In 255 cases of H. pylori infection diagnosed by biopsy methods, the (13)C-UBTs, performed with two different (13)C-ruea formulations, and using infrared spectroscopy for evaluation, showed a sensitivity of 97.7%, specificity of 98.0%, and accuracy of 97.8% (total number of evaluable cases, n = 505). The rate of agreement in the assessment of H. pylori infection between infrared spectroscopy and mass spectrometry was 100% ( n = 505). The regression equation for infrared spectroscopy to mass spectrometry was y = 0.9822x - 0.0809 ( n = 2542), with a correlation coefficient of r = 0.99989 ( P = 0.0001). CONCLUSIONS: Diagnosis of H. pylori infection can be performed using infrared spectroscopy as well as mass spectrometry.

Comparison between a new 13C-urea breath test, using a film-coated tablet, and the conventional 13C-urea breath test for the detection of Helicobacter pylori infection.

BACKGROUND: In Japan, urea breath-testing includes mouth rinsing with water immediately after the ingestion of (13)C-urea solution, to prevent false-positive results that are caused by oral bacteria with urease activity. Our objective was to evaluate the diagnostic performance of a urea breath test using a film-coated (13)C-urea tablet and omitting mouth rinsing. METHODS: The study was a multicenter trial comparing the solution- and tablet-based urea breath tests (UBTs). Helicobacter pylori status was determined by histology, culture, and rapid urease testing. RESULTS: Of the 255 subjects who completed the study, evaluation of the tablet-based UBT was possible in 254, and comparison of the tablet-based UBT and the solution-based UBT was possible in 250 patients. When the assessment achieved by a combination of biopsy-based methods was used as a reference standard, the sensitivity, specificity, and accuracy of the tablet-based method were determined to be 97.7%, 98.4%, and 98.0%, respectively. When the results of the solution-based UBT were used as a reference standard, the sensitivity, specificity, and accuracy of the tablet-based UBT were determined to be 96.9%, 97.6%, and 97.2%, respectively. CONCLUSIONS: The (13)C-urea tablet-based method proved to be a simple and accurate test for the diagnosis of H. Pylori infection. Mouth rinsing was not required.

Antioxidant and antiapoptotic activities of idoxifene and estradiol in hepatic fibrosis in rats.

Oxidative stress plays a causative role in the development of hepatic fibrosis and apoptosis. Estradiol (E2) is an antioxidant, and idoxifene is a tissue-specific selective estrogen receptor modulator. We have previously demonstrated that E2 inhibits hepatic fibrosis in a rat model of hepatic fibrosis induced with dimethylnitrosamine (DMN), and suppresses activation of the nuclear factor (NF)-kappaB proinflammatory transcription factor in cultured rat hepatocytes undergoing oxidative stress. This study reports on the antioxidant and antiapoptotic role of idoxifene and E2 in the DMN model of hepatic fibrosis. The DMN model rats were administered with idoxifene or E2, and were examined activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) and expression of Bcl-2 family proteins in the liver. During the course of hepatofibrogenesis after DMN treatment, serum levels of lactate dehydrogenase (LDH), a biomarker for necrosis, and hepatic levels of malondialdehyde (MDA), an end product of lipid peroxidation, increased rapidly for 3 days. On day 14, serum LDH levels normalized, and hepatic fibrosis developed with increased levels of MDA and collagen and decreased production of SOD and GPx in the liver. Fibrotic liver also showed downregulation of Bcl-2 and Bcl-X(L) expression and upregulation of Bad expression. Idoxifene and E2 suppressed DMN-mediated necrosis, lipid peroxidation, the loss of antioxidant enzyme activity, and proapoptotic status in Bcl-2 family protein expression as well as hepatic fibrosis. These findings indicate that, in addition to their antiinflammatory and antifibrotic action, idoxifene and E2 could enhance antioxidant and antiapoptotic activity in hepatic fibrosis in rats.

Idoxifene and estradiol enhance antiapoptotic activity through estrogen receptor-beta in cultured rat hepatocytes.

Oxidative stress plays a causative role in the development of hepatic fibrosis and apoptosis. Estradiol (E2) is an antioxidant, and idoxifene is a tissue-specific selective estrogen-receptor modulator. We have previously demonstrated that E2 inhibits hepatic fibrosis in rat models of hepatic fibrosis and that the actions of E2 are mediated through estrogen receptors (ERs). This study reports on the antiapoptotic role of idoxifene and E2, and the functions of ER subtypes ER-alpha and ER-beta in hepatocytes undergoing oxidative stress. Lipid peroxidation was induced in cultured rat hepatocytes with ferric nitrilotriacetate solution with idoxifene or E2. Oxidative stress-induced early apoptosis was linked to its ability to inhibit not only the expression of Bcl-2 and Bcl-XL but the production of antioxidant enzymes as well and to stimulate Bad expression. Hepatocytes possessed functional ER-beta, but not ER-alpha, to respond directly to idoxifene and E2. Idoxifene and E2 suppressed oxidative stress-induced reactive oxygen species generation and lipid peroxidation, and their antiapoptotic effects on the activation of activator protein-1 and nuclear factor-kappaB, the loss of antioxidant enzyme activity, and Bcl-2 family protein expression in early apoptotic hepatocytes were blocked by the pure ER antagonist ICI 182,780. Our results indicate that idoxifene and E2 could enhance antiapoptotic activity through ER-beta during oxidative damage in hepatocytes.

Clinical value of the determination of serum guanase activity in patients with chronic hepatitis type C.

The study examines the clinical significance of guanase (GU) measurement in patients with hepatitis C. 688 patients in whom either ALT was abnormal, or in whom HBsAg or HCVAb was detected in the serum, were enrolled into this study. The percentage of cases in which normal ALT while elevated GU was compared among the different disease groups. Then, the percentage of cases with normal ALT but elevated GU was compared between HBV and HCV groups. For the entire population, a significant correlation was observed between ALT and GU (r=0.872). The overall percentage of cases with normal ALT but elevated GU activity was 11.4%. In HCV group, 449 cases had normal ALT. Of these cases, 20.3% had elevated GU, while ALT was normal. Before 1989, no test to check donated blood for HCV antibody was available. However, screening of donated blood for high GU was associated with a reduced incidence of post-transfusion hepatitis. This is probably because following the screening, blood donated by patients with hepatitis C who had normal ALT but elevated GU was rejected. After the introduction of HCV antibody measurement, GU measurement is still useful to reveal the pathophysiological condition in-patients with chronic hepatitis type C.

Study of the causes of higher mortality rates from chronic liver diseases in Tokushima Prefecture.

Mortality rates from chronic liver diseases (CLD) such as liver cirrhosis and hepatocellular carcinoma have been reported to be higher in Tokushima prefecture, although its causes remain unclear. To clarify the causes of CLD in Tokushima prefecture, we evaluated the positive rates of HBs antigen and anti-HCV antibody and the mortality rates from CLD in patients with liver diseases and blood donors after dividing the entire Tokushima prefecture into 8 district boundaries of health centers. In addition, to evaluate the causes of the higher frequency of CLD and the relationship between the development of CLD and viruses, medical examinations were performed in 2 mountain villages in Tokushima prefecture where the drift of population was limited and the mortality rates from CLD differed from each other. As a result, it was found that HCV infection was the major cause of the higher mortality rates from CLD in Tokushima prefecture. Although there were marked regional differences in the mortality rates from CLD, they were mainly due to different rates of HCV infection.

Interferon-alpha enhances biological defense activities against oxidative stress in cultured rat hepatocytes and hepatic stellate cells.

Oxidative stress has been implicated as a cause of hepatic fibrosis, and hepatic stellate cells (HSCs), which are the most important collagen-producing cell types, have been reported to be activated by lipid peroxidation products. Antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GPx) provide a defense system that plays a critical role in protecting the cell from free radical damage, particularly lipid peroxidation. To elucidate the antioxidant activity of interferon-alpha (IFN-alpha), the effects of IFN-alpha on rat hepatocytes undergoing oxidative stress and HSCs in primary culture as well as isolated rat liver mitochondria were examined. IFN-alpha was observed to dose-dependently increase the immunoreactive protein levels of copper, zinc-and manganese-dependent SOD as well as the enzyme activities of GPx, and decrease the lipid peroxidation product levels and oxidative burst both in stressed hepatocytes and activated HSCs; GPx activities, however, were not detected in the latter cells. IFN-alpha also inhibited HSC activation and lipid peroxidation in liver mitochondria. These findings suggest that IFN-alpha may enhance biological defense activities against oxidative stress and function as a potent fibrosuppressant by protecting hepatocytes and hepatic stellate cells from lipid peroxidation in vivo.