Toshihiko Namihisa

A multi-center double-blind controlled trial of ursodeoxycholic acid for primary biliary cirrhosis.

SummaryA multi-center double-blind controlled trial of ursodeoxycholic acid (UDCA) for treatment of primary biliary cirrhosis (PBC) was carried out. Twenty two and 23 patients were treated with 600mg/day UDCA and placebo, respectively, for 24 weeks. In UDCA - treated patients, fall of serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and gamma glutamyltranspeptidase activities started within 4 weeks after start of the trial and continued throughout the trial period. The serum IgM level fell in 7 UDCA-treated patients examined but not in 10 placebo-treated patients examined. Serum bilirubin concentration showed no significant change at the end of the study in either of UDCA- and placebotreated group of patients. There was no significant difference between these two groups with respect to the frequency of improvement of pruritus. In UDCA-treated patients, serum bile acid composition changed markedly, though its concentation showed no significant change. The percentage of total bile acid which ursodeoxycholic acid took up increased, whereas those which cholic acid, chenodeoxycholic acid and deoxycholic acid took up were decreased.

Immunocytochemical localization of myosin in normal and phalloidin-treated rat hepatocytes

To clarify the intracellular distribution of myosin in normal rat hepatocytes and its alterations in phal-loidin-treated rat hepatocytes as a morphologic basis for the dysfunction of microfilaments, we performed indirect immunofluorescence using monospecific antibody raised against rat hepatocyte myosin. Cryostat rat liver sections analyzed by the use of this antibody showed a characteristic polygonal staining pattern, indicating that myosin is localized close to the plasma membrane including the region of bile canaliculi. The observed myosin staining pattern of normal liver coincides with the pattern of actin distribution as demonstrated by double-staining on the same liver section with antimyosin antibody and rhodamine-phalloidin. Upon administration of phalloidin to rats, the following changes in the myosin staining pattern were observed. (a) Peripheral fluorescence along the plasma membrane, especially around the bile canaliculi and sinusoids, was greatly enhanced. (b) Numbers of small fluorescent dots appeared in the cytoplasm of hepatocytes. These changes in the localization of myosin are shown to overlap with those of actin filament distribution. Accompanying these changes of localization, cellular myosin content appears to be increased, as the myosin marker-enzyme NH4+(-)ethylenediaminetetraacetic acid-adenosine triphosphatase activity in hepatocyte extracts was elevated threefold after 7 days of phalloidin treatment. This increase of myosin may be due to the previously observed stabilizing effect on microfilaments of phalloidin against cellular proteases. Thus, phalloidin, which primarily alters actin filament distribution, induces the changes in myosin localization and the increase in cellular myosin content without causing dissociation of myosin from actin in the hepatocyte.

Multiplicity of mitochondrial inner membrane antigens from beef heart reacting with antimitochondrial antibodies in sera of patients with primary biliary cirrhosis

Mitochondrial inner membrane proteins extracted from beef heart tissue were examined for reactivity to antimitochondrial antibody (AMA) present in sera of patients with primary biliary cirrhosis (PBC) by an immunoblotting technique. Four proteins, which reacted with AMA, had molecular weights of 70 kDa, 54 kDa, 51 kDa and 45 kDa, as defined by their RF in SDS-PAGE gel. There was no correlation between the number of specificities and the titers of AMA as determined by immunofluorescence analysis. The 70-kDa protein was dissociated into a 36-kDa protein by trypsin digestion which still reacted with AMA. The reactivity to AMA of the 54-, 51- and 45-kDa proteins was abolished by trypsin digestion.

Fluorescent staining of microfilaments with heavy meromyosin labeled with N-(7-dimethylamino-4-methylcoumarinyl) maleimide.

For fluorescent staining of microfilaments in cells, heavy meromyosin (HMM) or subfragment-1 (S-1) was labeled with a novel thiol-directed fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM), instead of the usual dyes, such as fluorescein-isothiocyanate (FITC). DACM-labeled HMM or S-1 gave characteristic fluorescence patterns to a variety of cell types similar to those reported with the use of FITC-labeled HMM or S-1 or with immunofluorescence techniques using anti-actin antibody. The fluorescence of DACM was fairly photoresistant as compared with FITC, so that HMM or S-1 required only 1 mol of the dye per myosin head. Consequently, F-actin need not be used to preserve the actin binding activity of the myosin fragments when labeling with the dye.

Immunocytochemical Localization of Myosin in Rabbit Liver Cell

It has been established that the liver cell possesses its own myosin which resembles other non-muscle myosins in subunit composition and in its dependence of actin-activated Mg2+-ATPase activity on light chain phosphorylation (Ueno T, Sekine T: Biochem Int, 1987;15:1205). We have raised a specific antibody against rabbit liver cell myosin. Immunoblot analysis has shown that the purified antibody reacts only with the heavy chain of liver cell myosin. The antibody did not react with rabbit skeletal muscle myosin or with smooth muscle myosin extracted from rabbit intestinal wall. Cryostat liver sections analyzed by indirect immunofluorescence microscopy showed a characteristic polygonal staining pattern, indicating that myosin is concentrated close to the plasma membrane, particularly in the region of bile canaliculi. Myosin therefore appears to be localized in the area where actin filaments are also abundant.

Guillain-barré syndrome associated with acute hepatitis a

SummaryA case of Guillain-Barré syndrome in a 49-year-old male is described. The syndrome presented as tetraplegia with sensory impairment and the pre-icteric phase of acute hepatitis A. Both viral hepatitis and neurologic disturbances improved, although neurologic changes lasted for 3 months after the onset. HA IgM antibody in cerebrospinal fluid was first manifest with onset of neurologic symptoms. On the basis of this finding and some reports in the literature it seems possible that the Guillain-Barré syndrome may be related to the immunopathogenetic mechanism of hepatitis A virus infection.

Hepatic transport of serum bilirubin, bromsulfophthalein, and indocyanine green in patients with congenital non-hemolytic hyperbilirubinemia and patients with constitutional indocyanine green excretory defect.

We carried out a retrospective study of 71 patients with congenital non-hemolytic hyperbilirubinemia who had been treated at our institution over the 25 years from 1965 to 1990. Twenty patients had Gilberts syndrome, 1 had Crigler-Najjar syndrome, 1 had new type unconjugated hyperbilirubinemia, 21 had Dubin-Johnson syndrome, and 28 had Rotors syndrome. We also reviewed 20 patients with constitutional indocyanine green (ICG) excretory defect. The study focused on the hepatic transport of serum bilirubin, bromsulfophthalein (BSP), and ICG. In Dubin-Johnson syndrome, a defect appeared in late-stage transport, while uptake and storage capacity were normal. In Rotors syndrome, defects were found in the early stage, and storage capacity was reduced, while excretion into bile was slightly suppressed. A secondary rise in serum ICG was seen in 5 of the 10 patients with Dubin-Johnson syndrome. The transport defect in Gilberts syndrome was unclear. It could not be considered to be homogeneous, but it may exist at multiple sites, from the conjugation with serum proteins to excretion into bile. Following phenobarbital administration, the ICG secondary rise in the 5 patients with Dubin-Johnson syndrome disappeared, and ICG was rapidly cleared from blood. However, in patients with Dubin-Johnson syndrome, BSP clearance in serum did not show any change before and after phenobarbital administration. ICG excretion in patients with constitutional ICG excretory defect was due only to the impairment o ICG transport, and the defect was suggested to be hepatic uptake. These results indicate that studies of the hepatic transport of bilirubin, BSP, and ICG are useful for determining the etiological factors involved in congenital hyperbilirubinemia and constitutional ICG excretory defect.

N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM): an alternative label for fluorescence tracing

N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), a fluorescent thiol reagent, has a high molar extinction coefficient and a high quantum yield when it reacts with protein sulphhydryl bonds. DACM proved to be effective as a fluorescent probe when used for labelling rabbit immunoglobulin G anti-human gamma-chain (IgG). DACM-labelled rabbit immunoglobulin G anti-human gamma-chain conjugates (DACM-IgG) can be purified efficiently by Sephadex G-50 gel chromatography. The immunological properties of the IgG are not altered appreciably by conjugation. As a label DACM has advantages with regard to ease of preparation, specific immunofluorescence and strong photoresistance.

Hepatic Dye Transport in Constitutional Hyperbilirubinemia and Constitutional ICG Excretory Defect

体質性黄疸35例,体質性ICG排泄異常症16例の色素移送動態を検討した.Gilbert病とD-J症候群ではビリルビン移送異常が,BSP, ICGより優位であり,Rotor型とICG異常症ではBSPとICG移送異常がビリルビンより優位であった.Gilbert病のBSP消失曲線は,初期消失遅延型と後期消失遅延型の2群に,ICGは,後期消失が正常,遅延,中間の3群に分けられた.D-J症候群で,BSP消失曲線の再上昇は全例にみられ,いずれも一定のパターンを示したが,ICGの再上昇は7例中4例で認め,そのパターンは不定であった.Rotor型は排泄障害のほかに摂取障害が著しく,D-Jの移送障害とは異なり,また,ICG異常症の消失曲線はRotor型に似ているが,20～25分のステップ形成が特異的で,Rotor型の様な肝から胆汁中への色素排泄障害は認めなかった.フェノバルビタールによる影響も,色素と疾患により様々で,各疾患は,ビリルビン,BSP, ICGを軸とした3次元の空間にそれぞれ独自の位置を占めていた.

Compartmenal analysis of indocyanine green transport in normal subjects and patients with hepatic dysfunction

SummaryThe plasma disappearance curve following a single intravenous injection of Indocyanine Green (ICG) was analyzed by an established technique and ICG kinetics evaluated for 12 normal control subjects and 57 patients with various hepatic disorders. The two-compartment model was used in this analysis. Three fractional transport rates were determined for the two-pool system: (1) fractional hepatic removal rate; (2) fractional hepatic plasma reflux rate; (3) fractional biliary secretory rate. The mean values and their standard deviation for the three respective rate in normal subjects are: (1) 0.2168 ±0.0199 min-1; (2) 0.0136 ±0.0089 min-1; (3) 0.0642 ±0.0327 min-1. The respective mean rates for 25 patients with biopsy proved hepatic cirrhosis are: (1) 0.0777 ±0.0388 min-1; (2) 0.0127 ±0.0081 min-1; (3) 0.0434 ±0.0188 min-1. The respective means for 7 patients in the acute phase of acute viral hepatitis are: (1) 0.1066 ±0.0498 min-1; (2) 0.0078 ±0.0046 min-1; (3) 0.0218 ±0.0141 min-1. The respective means for 6 patient with almost complete biliary obstruction are: (1) 0.0503 ±0.0211min-1; (2) 0.0717 ±0.0103 min-1; (3) 0.0422 ±0.0190 min-1. Changes in these parameters were dtermined in acute phase and convalescent phase of acute viral hepatitis. This kinetic approach was compared with previous evaluation of sulfobromophthalein (BSP) transport in man and similar results were obtained for the two dyes in normal subjects and in cirrhotic patients.