Isao Matsuo

The Multidrug Resistance‐associated Protein Gene Confers Drug Resistance in Human Gastric and Colon Cancers

To determine the expression of multidrug resistance‐associated protein (MRP) gene and its role in gastric and colon cancers, we analyzed 10 gastric and 10 colon non‐drug‐selected cell lines and a similar number of tissue samples of these cancers. We compared the expression of MRP and mdr1 mRNA in cell lines and tissues using reverse‐transcriptase polymerase chain reaction. In mdr1‐negative cells, the relationship between the level of MRP gene expression and sensitivity to anticancer drugs was examined. The effect of verapamil, an MRP‐modulating agent, was also examined in these cells. The expression of MRP gene in gastric cancer cell lines varied from a low to a high level, but mdr1 was not detected in any of these cell lines. Colon cancer cell lines expressed low to intermediate levels of MRP gene, and half of the cells co‐expressed low to high levels of mdr1. In tissue samples, the expression pattern of the two multidrug resistance (MDR) genes was broadly similar to that described for the cell lines, except that most of the gastric cancer tissue samples did express low levels of mdr1. No significant correlation was observed between the level of MRP gene expression and sensitivity to anticancer drugs in gastric and colon cell lines. However, verapamil significantly increased the sensitivity to etoposide, doxorubicin and vincristine in cells highly expressing MRP gene. Our results indicate that MRP gene may be important in conferring MDR in gastric and colon cancer cells.

Autoimmune cholangitis syndrome with a bias towards primary biliary cirrhosis

&NA; The apparent coexistence of primary biliary cirrhosis (PBC) and autoimmune hepatitis in the same patient raises unresolved problems for nosology and therapy. These are exemplified by a 45‐year‐old Japanese woman with overlapping clinical, serological and histological features of autoimmune cholangitis and autoimmune hepatitis. The classical serological test for PBC, antimitochondrial antibody (AMA) by immunofluorescence, was atypical. By immunoblotting there was reactivity with one of the enzymes of the 2‐oxo‐acid dehydrogenase complex (2‐OADC) family, now recognized as autoantigens responsible for AMA reactivity. Also there was reactivity by immunofluorescence for antinuclear antibodies (ANA), one showing the typical speckled pattern of anti‐Sp‐100 and the other the peripheral pattern of antinuclear membrane antibody, both with titres > 106. There was also a positive result to the lupus erythematosus (LE) cell test. Treatment with ursodeoxycholic acid was beneficial. Thus while the clinical presentation suggested the overlapping syndrome of autoimmune hepatitis and PBC, PBC eventually proved to be the likely diagnosis. We suggest that apparent cases of overlapping PBC‐autoimmune cholangitis‐hepatitis syndromes, after detailed testing, will mostly align with PBC.

Frequency of IgG, IgM, and IgA class autoantibodies against 2-oxo-acid dehydrogenase complex in 102 Japanese patients with primary biliary cirrhosis

Abstract We investigated the proportion of serum samples containing IgG, IgM, and IgA class autoantibodies against 2-oxo-acid dehydrogenase complex (2-OADC) tested by sensitive immunoblotting with enhanced chemiluminescence in 102 Japanese patients with primary biliary cirrhosis (PBC). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy in our assay condition were 99, 86, 89, 99 and 93%, respectively. Among these patients, 13 were seronegative for antimitochondrial antibodies (AMA) by indirect immunofluorescence (AMA-negative group). Of the 102 serum samples, 98 (96%), 85 (83%), and 72 (71%) contained IgG, IgM, or IgA class antibodies against at least one of the 2-OADC enzymes, respectively. All of the 13 AMA-negative sera contained IgG, IgM, or IgA class antibodies against at least one of the 2-OADC enzymes. In contrast, only one serum sample positive for AMA by immunofluorescence at a titer of 1:20 did not contain any IgG, IgM, or IgA class antibodies. Although the proportion of samples containing IgG class autoantibodies against E2 subunit of branched chain oxo-acid dehydrogenase complex (BCOADC-E2) (77%) was similar to that against E2 subunit of pyruvate dehydrogenase complex (PDC-E2) (74%), IgM and IgA class antibodies against BCOADC-E2 were not frequently detected compared with those against PDC-E2. Overall, our results indicated that IgG class antibodies against PDC-E2 and BCOADC-E2 showed the most dominant reactivities both in AMA-positive and -negative groups of PBC.

Autoimmune reactivity of sera to hepatocyte plasma membrane in type 1 autoimmune hepatitis.

Abstract: Type 1 autoimmune hepatitis (AIH-1) is an organ-specific autoimmune liver disease for which no tissue-specific autoantigen has yet been identified. We examined the reactivity by sensitive immunoblotting with enhanced chemiluminescence (IB-ECL) of 43 sera from patients with AIH-1 and 182 sera from patients with other diseases on hepatocyte plasma membrane derived from rat or human liver (RHPM, HHPM) and separated by aqueous two-phase partition. The sera studied were from patients with AIH-1, primary biliary cirrhosis, chronic viral hepatitis, and systemic lupus erythematosus (SLE); and from normal subjects. Specificity of reactivity by IB-ECL was sought: (i) by testing sera on human or rat liver membrane; (ii) by testing sera on liver or kidney membrane; (iii) by serial titration of reactive sera; and (iv) by testing reactive sera from AIH-1 before and after successful treatment with prednisolone. The results were that in AIH-1 there were multiple reactive components which were not species-specific, since they were detected with both RHPM and HHPM, but were mostly tissue-specific for liver. There was no significant correlation between antinuclear antibodies (ANA) titer and the frequencies of sera reactivities against RHPM. Most of these reactive components were demonstrable at a lesser frequency in other liver diseases and in SLE. There was a striking decrease in reactivity by IB-ECL of AIH-1 sera with liver membrane after clinical remission, further suggesting that differences between AIH-1 and other inflammatory liver diseases and SLE are predominantly quantitative rather than qualitative. However, our study did point to candidate liver membrane antigens with molecular sizes of 136, 116, 81, and 49 kDa, additional to components previously described by others. The molecular identification of these prominent reactants with AIH-1 sera could prove informative for ascertaining pathogenesis.

Changes in titers of antimitochondrial and antinuclear antibodies during the course of primary biliary cirrhosis

Abstract A case of primary biliary cirrhosis (PBC) in whom a complete biochemical (serum bilirubin, transaminases and alkaline phosphatase) remission was noted after combination treatment with ursodeoxycholic acid (UDCA) and corticosteroid is reported. The antimitochondrial antibody (AMA) detected by indirect immunofluorescence was initially positive, and the antinuclear antibody (ANA) was negative, but these two antibodies subsequently fluctuated independently (AMA‐positive/ANA‐negative, AMA‐negative/ANA‐negative, AMA‐negative/ANA‐positive, AMA‐positive/ANA‐positive, and again AMA‐negative/ANA‐positive) in spite of a lack of histopathological improvement in the liver after treatment. The clinical presentation in our case suggests that in some cases the diagnosis of PBC or so‐called autoimmune cholangitis (AIC) might depend on the ‘phase’ of the same disease. Our results also suggest that detailed immunoreactive profiles against 2‐oxo‐acid dehydrogenase complex (2‐OADC) enzymes by using immunoblotting, together with a serial histological examination, should provide more precise information for a diagnosis of PBC.

Automated enzymatic mitochondrial antibody assay for the diagnosis of primary biliary cirrhosis: applications of a routine diagnostic tool for the detection of antimitochondrial antibodies.

Background and Aims: An automated enzymatic mitochondrial antibody assay (EMA) kit for the diagnosis of primary biliary cirrhosis (PBC) has become commercially available recently. The aim of this study was to assess the clinical utility of the enzyme inhibition assay using this EMA kit for the diagnosis of PBC.

Serial changes in enzyme inhibitory antibody to pyruvate dehydrogenase complex during the course of primary biliary cirrhosis

To assess the usefulness of enzyme inhibition assay for the diagnosis of primary biliary cirrhosis (PBC), we determined the serial changes in enzymatic inhibitory antibody to pyruvate dehydrogenase complex (PDC) in patients with PBC, and compared the results to those of immunofluorescence and immunoblotting. Forty‐nine sera from 19 patients with PBC who were followed‐up for at least 16 months were tested for antimitochondrial antibodies (AMA) by indirect immunofluorescence, immunoblotting on bovine heart mitochondria, and enzyme inhibition assay using commercially available TRACE Enzymatic Mitochondrial Antibody (M2) Assay (EMA) kit. Of the 49 sera, 39 (80%), 35 (71%), 38 (78%), 31 (63%), and 36 (73%) were positive for AMA by immunofluorescence, for immunoglobulin G (IgG), IgM, and IgA class antibody against E2 subunit of PDC (PDC‐E2) by immunoblotting, and for enzymatic inhibitory antibody to PDC by EMA, respectively. AMA titers determined by immunofluorescence did not change in 9 patients (47%), increased in 4 (21%), decreased in 3 (16%), and fluctuated in 3 (16%) during follow‐up. The number of anti‐M2 bands by immunoblotting did not change in 9 (47%), increased in 6 (32%), decreased in 2 (11%), and fluctuated in 2 (11%). Units of PDC activity by EMA did not change markedly in 16 (84%), increased in 2 (11%), and fluctuated in 1 (5%). Positive EMA results were common in cases with high levels of serum alkaline phosphatase and IgM, and the units of PDC activity by EMA correlated significantly and inversely with AMA titers by immunofluorescence, and serum reactivity to PDC‐E2 by immunoblotting, respectively. There was no correlation between serial changes in biochemical data and units of PDC activity by EMA. In three patients who showed a decrease in AMA titers, AMA titers correlated more with EMA results than immunoblotting. Moreover, in a patient with fluctuating AMA titers, the units of PDC activity by EMA paralleled AMA titers. Our results suggest that EMA is useful for the diagnosis of AMA‐positive PBC, and also could be used for monitoring the disease course in PBC. J. Clin. Lab. Anal. 14:208–213, 2000.

Elevation of serum gamma-glutamyl transpeptidase precedes that of alkaline phosphatase in the early stages of primary biliary cirrhosis

Abstract To determine the biochemical profile during the early stages of primary biliary cirrhosis (PBC), the serum concentrations of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase (GGTP), alkaline phosphatase (ALP), leucine aminopeptidase, total bilirubin and zinc turbidity test were examined retrospectively, 13 months to 19 years (median 9 years) prior to the subsequent histologic diagnosis of PBC in nine patients. Of these, serum GGTP and ALP were elevated before the diagnosis of PBC in most patients, whereas no clear increase in other tests was noted. Moreover, increased concentrations of serum GGTP occurred 1.1–6.6 years earlier than that of serum ALP in three patients and serum GGTP was exclusively elevated in one patient, while only one patient showed the rise in ALP to precede that of GGTP. Serum ALP and GGTP were increased simultaneously or were already high on the first examination in the other three patients. Our results suggest that elevation of serum GGTP may precede that of ALP in the early stages of PBC.

CASE REPORT: Primary Sclerosing Cholangitis Associated with Lupus Nephritis: A Rare Association

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown etiology characterized by inflammation, obliterative fibrosis, and segmental dilatation of the intrahepatic and extrahepatic bile ducts (1, 2). Immunopathogenic mechanisms seem likely to play a major etiologic role based on the presence of autoantibodies, including anti-neutrophil cytoplasmic antibody (ANCA), and a close relationship with other autoimmune diseases. However, immunosuppressive therapy has had minimal impact on disease progression (3). Therefore, other undefined etiologic mechanisms can be speculated upon. In the present report, we describe the novel association of PSC with lupus nephritis in a patient in whom lupus anticoagulant test was positive.

Synchronized disappearance of serum HCV-RNA, anti-U1 RNP, anti-La/SS-B, and anti-Scl-70 in a patient with chronic hepatitis

The authors report a rare case of chronic hepatitis in whom normalization of serum aminotransferases was associated with disappearance of serum hepatitic C virus (HCV)-ribonucleic acid (RNA), anti-U1 RNP, anti-La/SS-B, and anti-Scl-70 antibodies without treatment of interferon or corticosteroids. A 27-year-old Japanese woman was diagnosed with chronic hepatitis C, with positive anti-nuclear antibody, anti-U1 RNP, anti-La/SS-B, and anti-Scl-70 antibodies. Histopathologic examination of a liver biopsy specimen showed a periportal interface hepatitis with a predominantly lymphoplasmacytic necroinflammatory infiltrate and lobular hepatitis. After two-year treatment with ursodeoxycholic acid (UDCA), serum aminotransferases normalized and serum HCV-RNA, anti-U1 RNP, anti-La/SS-B, and anti-Scl-70 antibodies disappeared. It was unclear whether disappearance of HCV-RNA was spontaneous, due to some immunomodulating effects of UDCA, or other unknown mechanism, but host immune response may be associated with HCV elimination.