Composition of the microbiota in forestomach fluids and feces of Japanese Black calves with white scours.

The objective of this study was to characterize the composition of the forestomach and fecal microbiota in Japanese Black calves with white scours. Forestomach fluid, feces, and peripheral blood were collected from healthy calves ( = 5; age 10 ± 2 d) and scouring calves ( = 5; age 10 ± 1 d) on the day on which white scours occurred. The pH and concentrations of VFA, lactic acid, and ammonia nitrogen (NH-N) of the forestomach fluids were determined. Microbiota composition and gene copy numbers in the forestomach fluid and feces were analyzed by 454 pyrosequencing and quantitative real-time PCR (qPCR), respectively. The cytokine mRNA level in peripheral leukocytes was evaluated by qPCR. The pH of the forestomach fluid of the scouring calves tended to be higher than that of the healthy calves ( = 0.056). No significant difference was detected in the total VFA, lactic acid, or NH-N concentrations in the forestomach fluids of the 2 groups. Firmicutes, Bacteroidetes, and Proteobacteria were the predominant phyla in the forestomach fluid and feces. At the genus level, the relative abundance of in the forestomach fluid was significantly higher in the scouring calves ( < 0.05) and the relative abundance of in the feces was significantly higher than that in the forestomach in the healthy calves ( < 0.05). Furthermore, the bacterial diversity indices of feces were lower in the scouring calves. Quantitative PCR amplification using some of the primer pairs failed in the forestomach fluid and feces in both groups. These results suggested that fermentation in the forestomach may affect the occurrence of white scours, resulting in changes in the composition and diversity of the forestomach fluid and fecal microbiota in Japanese Black calves.

Hyperkalemia in both surgically and medically treated patients with primary aldosteronism.

Hyperkalemia is an important complication of adrenalectomy for patients with primary aldosteronism (PA). The frequency of hyperkalemia after medication using mineralocorticoid receptor antagonists (MRAs) for PA is unclear. The aim of this study is to investigate the frequency and the risk factors of hyperkalemia after surgery and medication for PA. The data of 376 patients with PA registered in a multicentre-collaborative study in Japan, including surgically treated patients (group A; n=142) and medically treated patients with MRAs (group B; n=234) were studied. The prevalence of hyperkalemic patients (serum potassium >5.0 mEq l-1) after treatment was higher in group A than group B (9.9 vs 3.8%, P<0.01). At diagnosis, the hyperkalemic patients were older and had a poorer renal function than the non-hyperkalemic patients in both groups (P<0.05). The hyperkalemic patients had severer PA in group A and milder PA in group B. The independent risk factor by a logistic regression analysis was only age in both groups. After treatment, the percentages of patients withdrawing antihypertensive drugs and the normalization of aldosterone renin ratio were not different between hyperkalemic and non-hyperkalemic patients in group A. The type and dose of MRAs and the combination of other antihypertensive drugs were not different between hyperkalemic and non-hyperkalemic patients in group B. In conclusion, the potential occurrence of hyperkalemia should be considered after medical as well as surgical treatment for PA, especially in patients with older age (>60 years) and impaired renal function (estimated glomerular filtration rate <70 ml min-1 per 1.73 m2) at diagnosis.

Significance of adrenal computed tomography in predicting laterality and indicating adrenal vein sampling in primary aldosteronism.

Although laterality assessed by computed tomography (CT) in primary aldosteronism (PA) is not always concordant with that assessed by adrenal vein sampling (AVS), it is unclear whether all patients diagnosed with PA should undergo AVS for subtype classification. The aim of the current study was to investigate the accuracy of CT in subtype classification and to develop a prediction score for bilateral subtype in patients without adrenal tumour. As part of the WAVES-J study, 393 patients with PA were analysed. Subtyping using CT was concordant with that using AVS in 68% (269/393) of patients in the total sample, and in 38% (68/156) of patients with unilateral tumours, 56% (5/9) of patients with bilateral tumours and 89% (204/228) of patients without tumour. In patients without tumour, female gender, plasma aldosterone concentration (pg ml-1) to plasma renin activity ratio ⩽550 and serum potassium ⩾3.8 mEq l-1 were shown to be independent predictors for bilateral subtype. A prediction score based on these three variables was constructed with one point attributed to each variable. A score of three points had 29% sensitivity and 96% specificity in a receiver operating characteristic curve analysis. The results suggest that although CT is not sufficiently accurate for subtype classification in patients with adrenal tumours, it is sufficient to determine bilateral subtype in patients without tumour. Moreover, using our clinical prediction score in patients without tumour could be useful in determining the necessity of AVS for subtype classification.

Comparison of renal biomarkers with glomerular filtration rate in susceptibility to the detection of gentamicin-induced acute kidney injury in dogs.

Fourteen renal biomarkers were compared with measurement of glomerular filtration rate (GFR) in detecting acute kidney injury (AKI) in beagle dogs given gentamicin (40 mg/kg/day by subcutaneous injection) for 7 consecutive days. Serum and urinary biomarkers were measured before administration of gentamicin and then on days 4 and 8 after starting administration. GFR was derived by use of a simplified equation. Increased urinary cystatin C and decreased GFR occurred from day 4 and were detected before increases in blood urea nitrogen (BUN) and serum creatinine concentrations and changes in other urinary parameters. The closest correlation was between urinary cystatin C and GFR. At termination, microscopical examination revealed extensive necrosis of proximal tubular epithelium with hyaline casts in the kidney of treated dogs. These data indicate that urinary cystatin C is the most sensitive index of kidney injury and GFR reflects the kidney functional mass.

Technical note: Use of a simplified equation for estimating glomerular filtration rate in beef cattle.

This study was performed to clarify whether a formula (Holstein equation) based on a single blood sample and the isotonic, nonionic, iodine contrast medium iodixanol in Holstein dairy cows can apply to the estimation of glomerular filtration rate (GFR) for beef cattle. To verify the application of iodixanol in beef cattle, instead of the standard tracer inulin, both agents were coadministered as a bolus intravenous injection to identical animals at doses of 10 mg of I/kg of BW and 30 mg/kg. Blood was collected 30, 60, 90, and 120 min after the injection, and the GFR was determined by the conventional multisample strategies. The GFR values from iodixanol were well consistent with those from inulin, and no effects of BW, age, or parity on GFR estimates were noted. However, the GFR in cattle weighing less than 300 kg, aged<1 yr old, largely fluctuated, presumably due to the rapid ruminal growth and dynamic changes in renal function at young adult ages. Using clinically healthy cattle and those with renal failure, the GFR values estimated from the Holstein equation were in good agreement with those by the multisample method using iodixanol (r=0.89, P=0.01). The results indicate that the simplified Holstein equation using iodixanol can be used for estimating the GFR of beef cattle in the same dose regimen as Holstein dairy cows, and provides a practical and ethical alternative.

Anti-Helicobacter pylori seropositivity: influence on severity and treatment response in patients with chronic hepatitis C.

We sought to clarify the incidence and role of Helicobacter pylori (H. pylori) seropositivity in patients with hepatitis C virus (HCV) infection and the effect of coinfection on interferon-alpha and ribavirin therapy. The presence of H. pylori was tested using a commercially available enzyme immunoassay in serum samples from 93 patients with chronic hepatitis C. Clinical features, HCV markers and response of HCV to interferon-alpha and ribavirin were compared between H. pylori-positive and H. pylori-negative patients. Anti-H. pylori antibody was detected in 45 (48%) of the 93 patients, whose median HCV-RNA level (495 vs 760 kIU/mL; P = 0.013) and platelet count (128 vs 158 x 10(3)/microL; P = 0.009) were significantly lower than in patients with HCV infection alone. Anti-H. pylori antibody levels were found to be significantly correlated with fibrosis score (P = 0.0083, r = 0.33) but inversely related to platelet count (P = 0.0037, r = -0.34). The sustained response rate for HCV clearance following interferon-alpha and ribavirin treatment did not differ between patients with and without anti-H. pylori seropositivity. The presence of H. pylori [odds ratio (OR) 8.61; 95% confidence interval (CI) 1.59-46.70] and fibrosis score (OR 30.13; 95% CI 5.44-166.78) were found by multivariate analysis to be associated with the decrease of platelet count during therapy. Coexistent H. pylori infection does not demonstrably influence the clinical course of chronic hepatitis C. A possible connection between H. pylori coinfection and thrombocytopenia was found during the treatment course, suggesting that preemptive eradication of H. pylori may facilitate completion of treatment and increased sustained virological response.

Long-term follow-up of hepatitis C virus infection: HLA class II loci influences the natural history of the disease.

Hepatitis C virus (HCV) causes various grades of chronic liver disease, ranging from an asymptomatic state to cirrhosis. To assess genetic factors of disease severity, we selected two HCV patient groups according to the following stringent criteria: (i) asymptomatic carrier state (ASC) defined by HCV infection for more than 20 years, normal alanine aminotransferase levels for the past 5 years as well as normal liver histology and/or shape and (ii) liver cirrhosis (LC) as diagnosed by clinical symptoms, liver biopsy and/or ultrasonography. A total of 103 chronically infected Japanese HCV patients (43 ASC and 60 LC) were analyzed. HLA class I and II alleles were established using low resolution DNA typing. HLA-DRB1 and DQB1 genotypes were inferred upon polymerase chain reaction-restriction fragment length polymorphism analysis. Two hundred and one anti-HCV-negative ethnically matched controls were included. The frequencies of DRB1*12 (*1201 and *1202), DQB1*0301 and DRB3*03 alleles were higher in patients with ASC than in those with LC (odds ratio (OR) 11.23, OR 4.25, and OR 3.22, respectively). The frequency of DQB1*0503 were lower in ASC patients compared to LC patients (OR 0.05). No significant differences between groups were observed for age, sex, source of infection, HCV genotype or viral loads. Our findings establish that certain HLA class II alleles strongly influence disease progression following HCV infection.

Role of (111)In-DTPA-pentetreotide scintigraphy in accurate diagnosis of neuroendocrine gastroenteropancreatic tumors.

Most gastroenteropancreatic neuroendocrine tumors contain high-affinity binding sites for somatostatin, and somatostatin-receptor scintigraphy has been introduced for the in-vivo evaluation of such tumors. We report two patients with gastroenteropancreatic neuroendocrine tumors, in whom it was quite difficult to localize the tumors by conventional techniques, and in whom we found that (111)In-DTPA-pentetreotide scintigraphy was useful for accurate information on tumor localization. In the first patient, who had gastrinoma, multiple tumors were shown in the gastrinoma triangle, but we could not clarify whether there were any tumors in the pancreatic body. The selective arterial secretin injection (SASI) test diagnosed that the gastroduodenal artery was the feeder of the gastrinomas, and (111)In-DTPA-pentetreotide scintigraphy with single-photon emission computed tomography indicated the absence of tumors in the pancreatic body. In the second patient, who had insulinoma, multiple liver tumors and a large mass in the hilum of the spleen were shown. (111)In-DTPA-pentetreotide scintigraphy was useful in determining that there was no secretion of insulin from the tumor in the hilum of the spleen. In conclusion, X-ray computed tomography is superior for detection of neuroendocrine tumors, because not all neuroendocrine tumors have somatostatin receptors; however, somatostatin receptor scanning, as well as the SASI test, may be useful for the surveillance of patients with known primary tumors, for monitoring patients with disseminated disease, and for following the treatment of these patients.

[Epidemiological trend of chronic hepatitis C].

Though patients with chronic hepatitis C after blood transfusion hepatitis decrease dramatically during the past decade, patients with it after non-transfused acute hepatitis C are still in existence. This means that new patients with chronic hepatitis C are on the decrease but not diminish. To the contrary, the number of patients with chronic hepatitis C is increasing. This depends on mainly dig up the undiscovered patients. These result in increasing the patients with hepatocellular carcinoma.

Pituitary apoplexy caused by luteinizing hormone-releasing hormone in prolactin-producing adenoma.

We report a case in which pituitary apoplexy developed shortly after an intravenous (i.v.) injection of luteinizing hormone-releasing hormone (LH-RH). A 56-year-old man with prolactin-producing pituitary tumor complained of severe headache, visual field loss and facial nerve palsy shortly after LH-RH test. Magnetic resonance image (MRI) revealed a hemorrhage in the pituitary adenoma. He showed dramatic improvement in his symptoms after decompression surgery. These findings suggest a causal relationship between the i.v. injection of LH-RH and pituitary apoplexy. Possible pituitary apoplexy should be kept in mind during pituitary testing.

Seroepidemiological study of hepatitis E virus infection in Japan using a newly developed antibody assay.

PURPOSE: A seroepidemiological study of hepatitis E virus (HEV) infection was conducted in Japan, where HEV infection is not considered endemic. METHODS: IgG and IgM class antibodies to HEV were measured with a newly developed enzyme-linked immunosorbent assay in which recombinant virus-like particles were used as an antigen. A total of 1253 individuals (401 males and 852 females; age range, 6-89 years) were enrolled from two different areas: area 1 (n = 478), in which hepatitis C was endemic; and area 2 (n = 775), in which it was not endemic. RESULTS: The HEV antibody (IgG class) positive rate was 6.7% in area 1 and 4.6% in area 2. Similarly, the HAV antibody (IgG class) positive rates were 65.3% and 72.3%. The age- and sex-specific prevalence of both HAV and HEV antibodies was quite similar in the two areas, and the HAV antibody positive rate clearly increased with age in both males and females. On the other hand, the HEV antibody positive rate showed a slight tendency to increase with age in males, but not in females. None of the 32 individuals with the HEV antibody who were interviewed had a history of visiting countries in which hepatitis E was endemic. In both areas, the mean age, percentage of males, and HAV antibody positive rate were significantly higher in the group of individuals with the HEV antibody than in the group of those without it, according to conventional statistical analyses. Of the three factors age, male sex, presence of HAV antibody, and the area factor, only male sex was statistically significant (P < 0.001) on multivariate logistic regression analysis. Two (0.2%) of the total of 1253 individuals were positive for the IgM class antibody to HEV. CONCLUSIONS: Our results suggest the possibility that HEV infection is circulating in Japan at a low level. HEV infection was associated with male sex, but not with HAV infection.

Distinct functions of the two protein tyrosine phosphatase domains of LAR (leukocyte common antigen-related) on tyrosine dephosphorylation of insulin receptor.

Most receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.

Clinical impact of genotype 1 TT virus infection in patients with chronic hepatitis C and response of TT virus to alpha-interferon.

BACKGROUND: The relationship between genotype 1 TT virus (TTV) infection and the status of chronic hepatitis C was studied. METHODS: A total of 52 patients with chronic hepatitis C who were treated with interferon (IFN)-alpha were enrolled in the present study. Of those, 12 were infected with genotype 1 TTV and 40 were uninfected. RESULTS: Clinical backgrounds, including mean age, sex, blood transfusion history, serum alanine aminotransferase (ALT) level, and the results of liver biopsy did not differ between patients with and without genotype 1 TTV infection. The distribution of hepatitis C virus (HCV) genotypes did not differ between the two groups of patients, but TTV-infected patients tended to have a lower serum HCV-RNA level than uninfected patients (median (range) 26.0 (< 1-460) vs 135 (1.2-740) kilo copies/mL, respectively; P = 0.065). Patients with a sustained response of HCV to IFN-alpha were significantly more common in TTV-infected than -uninfected patients (58 vs 23%, respectively; P = 0.018). Multivariate logistic regression analysis revealed that patients with a sustained response of HCV correlated significantly with the serum HCV-RNA level (P = 0.006), but not with the presence or absence of genotype 1 TTV infection (P = 0.161). Serum TTV-DNA decreased with IFN-alpha therapy in all 12 patients and remained negative in six patients even after treatment. There was no correlation between patients with a sustained response of HCV and the same of TTV. Serum ALT levels correlated with changes in the status of HCV viremia, but not with changes in the status of TTV viremia. CONCLUSIONS: An opposing relationship between HCV and TTV proliferation was suggested, but coinfection with genotype 1 TTV did not affect the status of chronic hepatitis C.

Persistent infection mechanism of GB virus C/hepatitis G virus differs from that of hepatitis C virus.

OBJECTIVE: Changes in the deduced amino acid sequence of the envelope 2 (E2) region of the GB virus C/hepatitis G virus (GBV-C/HGV) were analyzed to investigate whether or not the region contributes to persistent infection with the virus. METHODS: Eight patients with acute hepatitis C and 1 patient with acute hepatitis of unknown etiology were included in the study. GBV-C/HGV RNA was detected in 6 patients, including the patient with hepatitis of unknown origin. The nucleotide sequence of the E2 region of hepatitis C virus (HCV) and GBV-C/HGV was determined by direct sequencing of polymerase chain reaction products in 5 patients with HCV infection and in 6 patients with GBV-C/HGV infection twice during the period of early infection and several months or years later in each patient. RESULTS: The mean substitution rate of the deduced amino acid sequence in the E2 region was over 100 times lower (p < 0.001) in GBV-C/HGV (0.01 +/- 0.04/month/100 sites) than in HCV (2.4 +/- 1.7/month/100 sites). The amino acid sequence of the loop domain of GBV-C/HGV-E2 did not change in any of the 6 patients. On the other hand, the sequence of the hypervariable region of HCV-E2 changed remarkably (5.9 +/- 4.3/month/100 sites). No amino acid substitution in the loop domain was observed in 7 additional patients who showed persistent GBV-C/HGV viremia for more than 2 years. CONCLUSION: These results indicate that changes in the amino acid sequence of the E2 region are not involved in the mechanism of persistent GBV-C/HGV infection.

T cell repertoire in the liver of patients with primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune chronic liver disease characterized by the destruction of the bile ducts with an accumulation of lymphocytes. To investigate the roles of T cells accumulating around the bile ducts, we analyzed the clonality of alphabeta T cell populations in the livers of patients with PBC by size spectratyping and sequencing of the T cell receptor (TCR) Vbeta transcripts.TCR Vbeta spectratyping of PBC patients showed several skewed complementarity determining region 3 (CDR3) size patterns suggestive of clonal predominance as well as Gaussian-like patterns suggestive of polyclonal expansion. We observed Vbeta4 clones sharing the Gly (G)-G motif in the CDR3 nDn regions and a Vbeta4-Jbeta2.7 combination in three patients bearing HLA-DR2 and -DQ1. G-Leu (L)-Ala (A) or G-L motifs were also seen in the nDn regions of Vbeta17 with Jbeta2.1 of the two patients having HLA-A26. However, there were no whole CDR3-shared clones in any of the patients. In conclusion, we have observed that T cell clones are heterogeneous in each patient, but that they have some common motifs in the TCR Vbeta CDR3. We strongly suggest that these clonally expanded T cells might be involved in the immunopathogenesis of PBC.

Detection of CD45iota mRNA in murine Th1 but not Th2 clones.

CD45, a prototype of the receptor-like protein tyrosine phosphatase (PTPase) family, is one of the essential molecules in signal transduction through T cell receptors. Because at least 8 types of CD45 isoforms can potentially be produced by alternative mRNA splicing of exons 4, 5, and 6, the analyses at the transcription and protein levels of CD45 during the development and differentiation of T cells have been performed using RT-PCR and isoform-specific monoclonal antibodies, respectively. We report here that the ninth and smallest isoform of CD45, designated as CD45iota (CD45t), which is alternatively spiced from exons 4, 5, and 6 as well as exon 7, is present in the fetal thymus and splenic T cells of mice, and in murine Th1 clones, but not in Th2 clones. The expression of full-length CD45t mRNA as the functional CD45 PTPase was confirmed by RT-PCR analysis. Furthermore, the expression vector of CD45t was constructed, and its expression was detected in combination with anti-pan CD45 mAb and our newly established anti-LAR/CD45 PTPase domain mAb. These results suggested that CD45t might be an important isoform of CD45 for differentiation signaling of Th cells, and might be used as a marker to distinguish between Th1 and Th2 cells.

T cell repertoire in the liver of patients with autoimmune hepatitis.

Despite a large number of T cells infiltrating into the liver of patients with autoimmune hepatitis (AIH), little is known about their roles or target antigens. To investigate the roles of these T cells in the pathogenesis of AIH, we have studied the clonality of alphabeta T cell populations in liver tissue by size spectratyping the complementarity-determining region (CDR)3 size lengths of T cell receptor (TCR) Vbeta-chain transcripts. Analysis of nine AIH patients who had the HLA DR4 haplotype showed clonal expansion in all samples. More than two T cell clones expanded in most patients. Although the expression of the TCR Vbeta genes was different among the nine patients, clonal expansion of T cells expressing either TCR Vbeta2, 3, 4, 16, or 22 was observed in two patients or more. TCR Vbeta4 clones expanded in 5 cases. Cloning and sequencing of TCR Vbeta CDR3 from PCR products revealed no whole CDR3-shared clones among different patients. In conclusion, several T cell clonotypes first recognize target antigens, then expand and accumulate in the liver of AIH patients. These suggest heterogeneity of autoantigens and the complexity of AIH immunopathogenesis in individual patients.

Relationship of 31P MR spectroscopy to the histopathological grading of chronic hepatitis and response to therapy.

PURPOSE: In vivo phosphorus-31 MR spectroscopy (31P MRS) was performed in the human liver in order to investigate the relation between: the ratios of phosphorus metabolites in the liver; the histopathological grading of chronic hepatitis; and the response to therapy. MATERIAL AND METHODS: Hepatic 31P MRS using the DRESS method (depth-resolved surface-coil spectroscopy) was carried out in 45 patients with chronic viral hepatitis or autoimmune hepatitis, and in 16 control subjects. We measured the ratios of the peak areas of phosphomonoesters (PME), inorganic phosphate (Pi), or phosphodiesters (PDE) to the peak area of beta-adenosine triphosphate (ATP). RESULTS: The PDE/ATP ratio of patients with chronic hepatitis or liver cirrhosis was lower than that of control subjects (liver cirrhosis = 0.74; chronic active hepatitis = 1.13-1.21; normal = 1.43); only a small difference was found in the PME/ATP and Pi/ATP ratios. There was no correlation between the spectra and histopathological grading or response to therapy, but the response to therapy was poor when a reduced PDE/ATP ratio was present. CONCLUSION: The PDE/ATP ratio measured by 31P MRS makes it possible to identify the transition of chronic active hepatitis into liver cirrhosis with a poor response to therapy.