Bovine esophageal and glossal ulceration associated with Pseudomonas aeruginosa and Fusobacterium spp. in a 10-month-old Holstein heifer.

An underweight 10-month-old Holstein heifer presented with anorexia and ananastasia and was euthanized. Postmortem examination revealed extensive ulceration in the esophagus, tongue, and omasum. Histopathological examination revealed severe necrotic esophagitis, glossitis, and omasitis. Many Gram-negative bacilli were detected throughout the necrotic area in the digestive tract; these were identified as Pseudomonas aeruginosa on the basis of isolation tests, molecular examinations, and immunohistochemistry. Gram-negative long filamentous organisms in the superficial layers of the necrotic lesions reacted positively with antibodies against Fusobacterium necrophorum subsp. necrophorum. Thus, the necrotic lesions were confirmed to be associated with P. aeruginosa and Fusobacterium spp. This is the first detection of P. aeruginosa in bovine esophageal and glossal ulcers associated with Fusobacterium spp.

Molecular characterization of a novel bovine group A rotavirus.

In this study, by partial sequence analysis of the genome segments encoding VP5* and VP7, we characterized a novel bovine group A rotavirus, namely, Tak2, that was detected from adult cattle diarrhea in Tochigi Prefecture, Japan. The nucleotide (nt) and deduced amino acid (aa) sequences of the genome segments encoding VP5* and half of the amino terminal portion of VP7 of Tak2 revealed a low identity with those of group A rotaviruses carrying previously published P and G type specificities (VP5*: nt identity, 61.6%-67.6% and aa identity, 58.0%-71.4%; half of the amino terminal portion of VP7: nt identity, 57.8%-73.5% and aa identity, 61.2%-70.9%). Additionally, phylogenetic analysis of the nt sequences of the genome segments encoding VP5* and half of the amino terminal portion of VP7 revealed that Tak2 formed a branch separate from the established P and G types. These results suggested that Tak2 could possess novel P and G types yet not reported among group A rotaviruses.

Molecular characterisation of equine group A rotavirus, Nasuno, isolated in Tochigi Prefecture, Japan.

In this study, equine group A rotavirus (RV-A), Nasuno, isolated from foal diarrhoea in Tochigi Prefecture, Japan was characterised genetically by sequence analysis of the genome segments encoding VP4 and VP7. The nucleotide and deduced amino acid sequences revealed high homology with P[12] RV-As (94.0-99.3% and 94.9-99.4%) and G3 RV-As (86.9-99.5% and 91.1-99.4%). Nasuno was also classified into P[12] and G3 in the phylogenetic analysis of the nucleotide sequences of the genome segments encoding VP4 and VP7.

Serological characterization of novel P11[14],G8 bovine group A rotavirus, Sun9, isolated in Japan.

In this study, a novel bovine group A rotavirus (BoRV-A), Sun9, isolated from calf diarrhea in Tochigi Prefecture, Japan, was serologically characterized by a cross-neutralization assay, and serological surveillance by using its reassortant was performed on cattle bred in Japan. The G serotype of Sun9 was identified as G serotype 8 based on the one- or two-way serological relationships observed in Sun9 and other G8 strains. The P serotype of Sun9 was identified as P serotype 11 based on the one- or two-way serological relationships observed in Sun9, its reassortants, and the P11 lapine group A rotavirus R-2. The serological surveillance data indicated that 2.4% of the specimens appeared to possess antibodies against the P11[14] antigen. Few P11[14] bovine group A rotaviruses may exist in the Japanese cattle population.

Multidrug resistance3 is in situ detected in the liver of patients with primary biliary cirrhosis, and induced in human hepatoma cells by bezafibrate.

Bezafibrate has been empirically used for the treatment of primary biliary cirrhosis. Although its clinical efficacy has been demonstrated, the therapeutic mechanism of action of this drug remains unclear. We suggested that multi-drug resistance (MDR) 3 plays an important role as a mediator of bezafibrate. We adopted a human hepatoma cell line to elucidate the up-regulating effect of bezafibrate. To analyze the effects on mRNA, the dose effect was assessed by slot-blot study, the time course by real time PCR, and the subcellular location was determined by in situ hybridization. The results revealed that MDR3 mRNA reached a peak level 12h after the administration of bezafibrate, and dose dependency was observed. We also investigated the interaction of bile acid and bezafibrate. A low dose of chenodeoxycholic acid could become a co-effecter of bezafibrate. In the protein analysis, a precursor protein was induced by bezafibrate. Even in the cell line endogenously expressing MDR3, the expression of the protein was found to be modulated. We identified MDR3 mRNA in the livers of PBC patients using a sensitive in situ hybridization study. The present findings indicate that PBC patients can respond to bezafibrate, and therefore receive clinical benefits from this medication.

Molecular characterization of novel P[14],G8 bovine group A rotavirus, Sun9, isolated in Japan.

In this study, a novel bovine group A rotavirus (RV-A), Sun9, isolated from calf diarrhea in the Tochigi Prefecture, Japan, was characterized genetically by the sequence analysis of the genome segments encoding VP4 and VP7. The nucleotide and deduced amino acid sequences of the genome segments encoding VP4 and VP7 of Sun9 revealed high homology with P[14] human and lapine RV-As (80.2-88.7% and 90.9-94.8%) and G8 bovine and human RV-As (83.1-95.5% and 92.3-98.2%). Sun9 was also classified into P[14] and G8 in the phylogenetic analysis of the nucleotide sequences of the genome segments encoding VP4 and VP7. Although previous reports have suggested that P[14],G8 human RV-As isolated until now were obtained from the reassortment between human and bovine RV-As, or the interspecies transmission of bovine RV-A to human, no P[14],G8 bovine RV-A has yet been reported. Sun9 may be initial direct evidence of the above hypothesis.

p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts.

Hepatic stellate cells (HSCs) spontaneously transdifferentiate into myofibroblast (MFB)-phenotype on plastic dishes. This response recapitulates the features of activation in vivo. Transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by MFBs. In quiescent HSCs, TGF-beta signaling involves TGF-beta type I receptor (TbetaRI)-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. The middle linker regions of Smad2 and Smad3 also are phosphorylated by mitogen-activated protein kinase (MAPK). This study elucidates the change of Smad3-mediated signals during the transdifferentiation process. By using antibodies highly specific to the phosphorylated C-terminal region and the phosphorylated linker region of Smad3, we found that TGF-beta-dependent Smad3 phosphorylation at the C-terminal region decreased, but that the phosphorylation at the linker region increased in the process of transdifferentiation. TGF-beta activated the p38 MAPK pathway, further leading to Smad3 phosphorylation at the linker region in the cultured MFBs, irrespective of Smad2. The phosphorylation promoted hetero-complex formation and nuclear translocation of Smad3 and Smad4. Once combined with TbetaRI-phosphorylated Smad2, the Smad3 and Smad4 complex bound to plasminogen activator inhibitor-type I promoter could enhance the transcription. In addition, Smad3 phosphorylation mediated by the activated TbetaRI was impaired severely in MFBs during chronic liver injury, whereas Smad3 phosphorylation at the linker region was remarkably induced by p38 MAPK pathway. In conclusion, p38 MAPK-dependent Smad3 phosphorylation promoted extracellular matrix production in MFBs both in vitro and in vivo.

Distortion of autocrine transforming growth factor beta signal accelerates malignant potential by enhancing cell growth as well as PAI-1 and VEGF production in human hepatocellular carcinoma cells.

Resistance to growth inhibitory effects of transforming growth factor (TGF)-beta is a frequent consequence of malignant transformation. On the other hand, serum concentrations of TGF-beta, plasminogen activator inhibitor type 1 (PAI-1), and vascular endothelial growth factor (VEGF) are elevated as tumor progresses. The molecular mechanism of autocrine TGF-beta signaling and its effects on PAI-1 and VEGF production in human hepatocellular carcinoma (HCC) is unknown. TGF-beta signaling involves TGF-beta type I receptor-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. To investigate the involvement of autocrine TGF-beta signal in cell growth, PAI-1 and VEGF production of HCC, we made stable transfectants of human HCC line (HuH-7 cells) to express a mutant Smad2(3S-A), in which serine residues of SSXS motif were changed to alanine. The transfectants demonstrated an impaired Smad2 signaling. Along with the resistance to growth inhibition by TGF-beta, forced expression of Smad2(3S-A) induced endogenous TGF-beta secretion. Moreover, this increased TGF-beta enhanced ligand-dependent signaling through the activated Smad3 and Smad4 complex, and transcriptional activities of PAI-1 and VEGF genes. In conclusion, distortion of autocrine TGF-beta signals in human HCC accelerates their malignant potential by enhancing cell growth as well as PAI-1 and VEGF production.

Neutrophil chemotaxis in bile duct-obstructed rats, and effect of internal biliary drainage.

BACKGROUND/AIMS: Our previous studies demonstrated enhanced neutrophil chemotaxis in bile duct-ligated, obstructive jaundice rats. In the present study, we produced a reversible obstructive jaundice model in rats. The efficacy of the present model in producing sufficient bile flow blockade and subsequent internal biliary drainage was assessed. Furthermore, the effect of internal biliary drainage on neutrophil chemotaxis was evaluated. METHODOLOGY: Bile duct was obstructed with a polyester tape attached with a stainless steel coil. Internal biliary drainage was performed by removing the tape. Rats were subjected to either 10 days' bile duct obstruction or 4 days' bile duct obstruction followed by 6 days' internal biliary drainage. Some animals underwent conventional bile duct ligation and dissection for 4 or 10 days. Neutrophil chemotaxis was evaluated with a modified Boyden method using interleukin-8 (recombinant rat Gro-beta) as chemoattractant. RESULTS: The present technique produced sufficient obstructive jaundice as evidenced by increases in serum alanine aminotransferase and total bilirubin throughout the observation period, the values of which were insignificant with those induced by the conventional method. Internal biliary drainage effectively normalized these values. Similarly, neutrophil chemotaxis was enhanced with both procedures, and increased neutrophil chemotaxis was significantly decreased after drainage. CONCLUSIONS: The present reversible obstructive jaundice method is as efficacious as the conventional method for producing obstructive jaundice, and internal biliary drainage could be readily available. With the present model, neutrophil overactivity in obstructive jaundice was effectively alleviated by internal biliary drainage. The result may support the role of preoperative biliary drainage in the prevention of postoperative septic complications.

Altered response to inflammatory cytokines in hepatic energy metabolism in inducible nitric oxide synthase knockout mice.

BACKGROUND/AIMS: Production of nitric oxide (NO) in the liver is believed to be a critical factor for carbohydrate and energy metabolism in endotoxin shock. The present study focuses on the involvement of NO produced by inducible nitric oxide synthase (iNOS) in glycogen synthesis and energy metabolism stimulated by insulin. METHODS: Primary hepatocytes prepared from wild-type and iNOS knockout (iNOS(-/-)) mice were employed. RESULTS: Incubation of wild-type hepatocytes with a combination of cytokines (interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) and lipopolysaccharide (cytokines/LPS) inhibited insulin-stimulated glycogen synthesis and adenosine triphosphate (ATP) increase, and decreased the ketone body ratio (KBR) at 8-12 h, concomitant with expression of iNOS protein and NO production. While the glycogen synthesis was suppressed by cytokines/LPS, reduction of the ATP increase and a decrease in KBR by cytokines/LPS were not observed in iNOS(-/-) hepatocytes. Further, N(G)-monomethyl-L-arginine, a NOS inhibitor, reversed the inhibition of ATP increase and decrease in KBR by cytokines/LPS, but not the inhibition of glycogen synthesis. Conversely, addition of S-nitroso-N-acetylpenicillamine, a NO donor, inhibited the insulin-stimulated ATP increase synthesis in iNOS(-/-) hepatocytes, but not the insulin-stimulated glycogen synthesis. CONCLUSIONS: These results demonstrate that NO mediates the suppression of insulin-stimulated energy metabolism, but not glycogen synthesis, in cytokines/LPS-treated hepatocytes.

The effect of biliary decompression on antibiotic biliary excretion.

BACKGROUND/AIMS: Raised biliary pressure may affect antibiotic biliary excretion. We evaluated whether biliary decompression for patients with biliary obstruction could improve antibiotic biliary excretion. METHODOLOGY: Eight patients with common bile duct obstruction undergoing endoscopic nasobiliary drainage were evaluated. During endoscopic cannulation, biliary pressure above the obstruction and antibiotic concentrations in the bile and peripheral blood were determined 60 min after the intravenous antibiotic (panipenem) administration. RESULTS: Biliary pressure was initially elevated above normal in all the patients, but normalized after biliary drainage for 5 to 7 days. At the initial endoscopic retrograde cholangiopancreatography, the aspirated bile contained low or undetectable levels of the antibiotic, but the mean bile panipenem concentration and the mean bile/plasma ratio of panipenem concentrations significantly improved after biliary decompression. CONCLUSIONS: The results suggest an important role of biliary pressure in determining antibiotic transfer into the bile.

Long-term prognosis of primary biliary cirrhosis (PBC) in Japan and analysis of the factors of stage progression in asymptomatic PBC (a-PBC).

Objective: Based on data from a national survey of primary biliary cirrhosis (PBC), the pathology and prognosis of PBC in Japan were clarified. In particular, we tried to perform multivariate analysis of factors useful in determining prognosis of asymptomatic PBC (a-PBC). Methods: The survey was performed 10 times. Responses from 3778 of 4361 registered patients (416 institutions) were investigated (survey period: January 1968-December 1998). At the time of diagnosis, patients were classified as a-PBC or symptomatic PBC (s1-PBC; pruritus only, s2-PBC; jaundice and serum bilirubin level above 2 mg/dl). The survival rate was obtained by the Kaplan-Meier method. Logistic regression analysis was used in multivariate analysis of prognostic factors of a-PBC. Results: There were no significant differences in clinical findings from those in previous reports. The 5-year survival rates of patients with a-PBC, s1-PBC, and s2-PBC at the time of diagnosis were 97, 88, and 53%, respectively. Patients with a-PBC at the time of diagnosis were divided into groups: those in whom the disease progressed to s2-PBC (8%) and did not progress to s2-PBC (92%) at the final examination, and the prognosis was compared between groups. The prognosis was significantly poorer in the s2-PBC progression group. As a result of multivariate analysis for prediction of prognosis, levels at diagnosis of total serum bilirubin (T-Bil), albumin (Alb), total cholesterol (T-Cho), histological stage, and presence or absence of ursodeoxycholic acid (UDCA) administration were selected as significant factors (P<0.00001). Conclusion: Serum T-Bil, Alb, T-Cho, and histological stage at the time of diagnosis and presence or absence of UDCA administration were considered useful early prognostic indicators in patients diagnosed as having a-PBC whose prognosis may deteriorate with progression to s2-PBC.

Differential regulation of TGF-beta signal in hepatic stellate cells between acute and chronic rat liver injury.

During chronic liver injury, transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells (HSCs). On the other hand, Smad 7 was recently shown to antagonize the TGF-beta-induced activation of signal-transducing Smads (2 and 3). In this study, we investigated the regulatory mechanisms of the TGF-beta signals in rat HSCs during acute liver injury and myofibroblasts (MFBs) during chronic liver injury, focusing on the roles of Smad 2 and antagonistic Smad 7. In acute liver injury, HSC-derived TGF-beta increased plasminogen activator inhibitor type 1 (PAI-1) and alpha2(I) procollagen (COL1A2) transcripts. Smad 2 in HSCs during liver injury and primary cultured HSCs were activated by an autocrine mechanism, because high levels of Smad 2 phosphorylation and induction of PAI-1 transcript by TGF-beta were observed in HSCs. Thereafter, Smad 7 induced by TGF-beta negatively regulated the Smad 2 action. These results indicated that endogenous TGFbeta-mediated Smad 7 in HSCs terminated the fibrotic signals mediated by signal-transducing Smads, and might be involved in the transient response to autocrine TGF-beta signal after acute liver injury. By contrast, Smad 7 was not induced by the autocrine TGF-beta signal, and constitutive Smad 2 activation was observed in MFBs throughout chronic liver injury, although Smad 7 could inhibit the TGF-beta signal requiring Smad 2 phosphorylation by activated TGF-beta receptor in cultured MFBs. This constitutive phosphorylation of Smad 2 by endogenous TGF-beta under a low level of Smad 7 could be involved in the progression of liver fibrosis.