Motoyoshi Yano

Long term effects of phlebotomy on biochemical and histological parameters of chronic hepatitis C

OBJECTIVE:There is considerable evidence that iron is a risk factor for liver injury in chronic hepatitis C. Known as iron reduction therapy, phlebotomy reduces serum ALT activity. This effect might continue with maintenance phlebotomy and result in slower progression of liver fibrosis.METHODS:We examined the biochemical parameters and liver histology of patients with chronic hepatitis C treated by maintenance phlebotomy. For biochemical evaluation, 25 patients were treated by initial phlebotomy to reduce serum ferritin levels to 10 ng/ml or less and then observed for 5 yr with maintenance phlebotomy to maintain the iron-deficient state. For histological evaluation, liver biopsies were performed before and after the study period in 13 of the patients. Thirteen patients who were virological nonresponders to interferon alone and had undergone second liver biopsies after more than 3 yr served as histological controls.RESULTS:Serum aminotransferase levels were decreased significantly by initial phlebotomy and remained at the same levels during the study period (p < 0.05). The grading scores were improved significantly in the study group (p < 0.05) and unchanged in the controls. The staging scores remained unchanged in the study group but were increased in the controls (p < 0.005). Disease progression was significantly different between the two groups (p < 0.05).CONCLUSIONS:These results suggest that phlebotomy with maintenance lowers serum aminotransferase levels, improves liver inflammation, and suppresses the progression of liver fibrosis in chronic hepatitis C.

Mutational analysis of ATP7B and genotype-phenotype correlation in Japanese with Wilson's disease.

The gene ATP7B responsible for Wilsons disease (WD) produces a protein which is predicted to be a copper‐binding P‐type ATPase, homologous to the Menkes disease gene (ATP7A). Various mutations of ATP7B have been identified. This study aimed to detect disease‐causing mutations, to clarify their frequency and distribution, to determine whether genotype correlates with phenotype, and to determine the rate of abnormal findings in heterozygotes for the WD gene. We analyzed 41 unrelated Japanese WD families, including 47 patients. Twenty‐one mutations, including nine novel ones, were identified. 2871delC (15.9%), 1708‐5T→G (11.0%), and Arg778Leu (13.4%) were the most common mutations. 2871delC was detected mainly in eastern Japan and 1708‐5T→G in western Japan. The homozygotes for the 1708‐5T→G, 2871delC, or Arg778Leu mutations did not show a correlation with their phenotypes. Ceruloplasmin and copper levels were abnormally low in 28.6% and 35.0% of heterozygotes, respectively. When patients and their families are screened for WD, a high rate of abnormal laboratory data in heterozygotes must be taken into account. Hum Mutat 15:454–462, 2000.

Production of interleukins 10 and 12 by peripheral blood mononuclear cells (PBMC) in chronic hepatitis C virus (HCV) infection.

We previously reported that interferon‐gamma (IFN‐γ) production by PBMC in response to HCV core protein was increased in patients with type C chronic liver disease. To understand better the pathophysiology of this disease, we evaluated production of IL‐10 and IL‐12 by PBMC from 41 patients with chronic HCV infection, including asymptomatic HCV carriers with persistently normal serum ALT values. IL‐10 is known to inhibit many effector functions of the immune system, suppressing Th1‐type cell development, while IL‐12 stimulates differentiation of Th1‐type cells, facilitating cell‐mediated immunity. IL‐10 production was determined by culturing lymphocytes with concanavalin A (Con A), while IL‐12 was produced by monocytes in the presence of Staphylococcus aureus Cowan 1 (SAC) with or without recombinant HCV core protein, respectively. The cytokine levels in culture supernatants were measured by ELISA. Spontaneous IL‐10 production was greater in patients with chronic hepatitis (CH) (229±119 pg/ml, P<0.01) and liver cirrhosis (LC) (185±88 pg/ml, P<0.05) than in controls (119±27 pg/ml), while it was decreased during IFN treatment (70±25 pg/ml). Both HCV core protein and Con A enhanced IL‐10 production by cells from HCV‐infected patients. IL‐12 was not detectable in medium alone cultures, and SAC‐induced IL‐12 production did not differ between various patient groups and controls. Simultaneous addition of HCV protein resulted in an increase of IL‐12 production in chronic liver disease compared with SAC‐alone cultures. Addition of IL‐10 to the cultures equally suppressed IFN‐γ production for both controls and patient groups, but the enhancing effect of IL‐12 on IFN‐γ production was significantly less in LC than in controls and other patient groups. The findings suggest that secretion of IL‐10/IL‐12 by cells from control individuals and various patient groups may be different, and that the cytokines might show different effects on IFN‐γ production by some cells.

Iron accumulation in the liver of male patients with Wilson's disease

OBJECTIVES:There is accumulating evidence that ceruloplasmin, a copper protein with ferroxidase activity, plays an important role in iron metabolism. The genetic disorder, aceruloplasminemia, can lead to tissue storage of iron as in hemochromatosis. Because most patients with Wilsons disease, a genetic copper toxicosis, have hypoceruloplasminemia, some could be affected by iron overload.METHODS:Four male patients with Wilsons disease were enrolled in this study of pre- and post-treatment iron metabolism.RESULTS:Pretreatment copper contents of the liver were high in all four male patients studied as diagnostic of Wilsons disease. Genetic analysis supported their clinical diagnosis of Wilsons disease without a background of hemochromatosis. Pretreatment serum ceruloplasmin levels were <20 mg/dl in all four patients. A standard penicillamine treatment for 3–8.5 yr further decreased their serum ceruloplasmin levels. Post-treatment serum ferroxidase activity was low as was the serum ceruloplasmin protein. Copper contents in the liver decreased after treatment in all subjects. In contrast, nonheme iron in the liver increased during treatment. Pretreatment liver specimens were positive for histochemical iron in two patients, and post-treatment specimens were positive in all four patients. In two patients, serum aminotransferase levels rebounded with elevation of serum ferritin concentration during the treatment period. Subsequent iron reduction by phlebotomy ameliorated their biochemical liver damage.CONCLUSION:Iron overload related to hypoceruloplasminemia may be clinically important, particularly in male patients with Wilsons disease.

A significant reduction in serum alanine aminotransferase levels after 3-month iron reduction therapy for chronic hepatitis C: a multicenter, prospective, randomized, controlled trial in Japan

BackgroundIncreasing evidence indicates that iron cytotoxicity plays an important role in the pathogenesis of chronic hepatitis C (CHC). However, the biochemical effects of iron reduction therapy on CHC remain to be confirmed in a controlled study. This study aimed to test whether iron removal by repeated phlebotomy improves serum alanine aminotransferase (ALT) levels in patients with CHC.MethodsPatients were randomly assigned to an iron reduction therapy or control group. The patients in the treatment group received 3-month iron reduction therapy by biweekly phlebotomy, while the patients in the control group were followed up for 3 months with regular blood tests alone.ResultsThirty-three patients completed the 3-month treatment, while 29 patients received the complete follow-up. The serum ALT levels were reduced from 118 ± 79 to 73 ± 39 IU/L in the treatment group, but did not change in the control group (106 ± 45 versus 107 ± 48 IU/L). Posttreatment enzyme activity was decreased significantly from the baseline. Furthermore, it was significantly lower than the 3-month control level. Although 5 patients withdrew from the study, none was affected by any side effects of repeated phlebotomy that required them to discontinue the treatment.ConclusionsThis short-term controlled trial demonstrated the biochemical efficacy and safety of iron reduction therapy for patients with CHC.

Serum aminotransferase levels as an indicator of the effectiveness of venesection for chronic hepatitis C

BACKGROUND/AIMS Iron cytotoxicity may play an important role in chronic hepatitis C. The effects of venesection suggest that a slight iron overload contributes to hepatic injury in subjects infected with hepatitis C virus. A better indication of the efficacy of venesection was studied in patients with and without overt iron overloading. METHODS All 40 patients had chronic hepatitis C but none had hemochromatosis of a known etiology. A serum ferritin level of 10 ng/ml or less was chosen as the treatment goal. A mean blood volume of 2400 +/- 1100 ml was removed during treatments lasting 5 +/- 3 months. RESULTS Treatment significantly reduced the mean serum levels of alanine aminotransferase activity from 128 +/- 74 to 63 +/- 28 IU/l (p < 0.01). The baseline enzyme activity was highly correlated with reduction in activity after treatment (r = 0.94, p < 0.01), but the baseline levels of ferritin and histochemistry for iron showed poor correlations with the reduction in enzyme activity (r = 0.63 with p < 0.01 and r = 0.38 with p < 0.05, respectively). CONCLUSIONS Thus, serum levels of aminotransferases were a more important indicator for venesection than conventional indices of iron overload, probably because cytotoxic iron includes some reactive iron species rather than stored iron alone.

Compound overload of copper and iron in patients with Wilson's disease.

This review of the copper–iron interaction in Wilsons disease was mainly based on ten patients (three females and seven males) studied in our institutes because the genetic tests of ATP7B for Wilsons disease of primary copper toxicosis and HFE for hemochromatosis, the biochemical parameters of copper and iron, and morphological studies on biopsied liver specimens were complete. All patients had hypoceruloplasminemia and hepatic lesions compatible with Wilsons disease. One patient was homozygous and nine patients were compound heterozygous for the mutations in ATP7B, and all patients were free from the major mutation, C282Y, of HFE. The biochemical parameters of iron metabolism were not specific, except for serum ferritin concentration. Judging from the traditional criteria, seven patients had hyperferritinemia. Histochemical iron was stained in the livers of seven patients and histochemical copper was found in nine patients. Microanalysis was more sensitive than histochemistry, detecting copper and iron accumulation in the hepatocellular lipofuscin particles of all patients. Using an improved fixative, intralipofuscin distribution was found to be different between cuprothionein and iron complexes. Iron overload in Wilsons disease might be worsened after treatment because of the close relation to hypoceruloplasminemia, in which the iron efflux from the liver to the circulation is disturbed.

Ursodeoxycholic acid induces glutathione synthesis through activation of PI3K/Akt pathway in HepG2 cells

Ursodeoxycholic acid (UDCA) is widely recognized as an effective compound in the treatment of chronic hepatitis and is known to modulate the redox state of the liver accompanied by an increase of GSH. In the present study, to access the antioxidative effect of UDCA and to clarify the molecular basis of the action on GSH level, we evaluated its effects in HepG2 cells exposed to excessive iron. UDCA inhibited both a decrease in the GSH level and an increase in the reactive oxygen species caused by excessive iron in the cells. UDCA increased the gene expression of the catalytic- and modifier-units of glutamine-cysteine ligase (GCL), which is a key enzyme in GSH synthesis. We further investigated the effect of UDCA on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and obtained results showing that UDCA-induced increase in the GSH level was prevented by LY294002, a PI3K inhibitor. In addition, Western blot analysis of Akt showed that, while the total Akt level remained unchanged, the phosphorylated Akt level was increased by UDCA, and this increase was also prevented by LY294002. Moreover, UDCA promoted the translocation of a transcription factor, nuclear factor-E2-related factor-2 (Nrf2), into the nucleus, and this action was inhibited by LY294002. From these results, it was indicated that UDCA increased the GSH synthesis through an activation of the PI3K/Akt/Nrf2 pathway. This may be a primary mechanism of antioxidative action of UDCA concerned with its therapeutic effectiveness in chronic hepatitis.

Mutational analysis of the MRP2 gene and long-term follow-up of Dubin-Johnson syndrome in Japan.

BackgroundRecent studies have indicated that dysfunction or loss of the multidrug resistance protein 2 (MRP2) is the molecular basis of Dubin-Johnson syndrome (DJS). To clarify the genetic basis of the disease and the long-term stability of serum bilirubin levels, we conducted a mutational analysis of the MRP2 gene and followed up serum bilirubin levels in Japanese DJS patients 30 years after they were originally diagnosed, based on traditional criteria.MethodsPatients were interviewed by telephone, and blood tests, including a genetic analysis of MRP2, were performed on the patients and family members who gave informed consent.ResultsOver the 30 years, hyperbilirubinemia remained unchanged in four of the five patients studied, while it worsened in 1 patient whose DJS was complicated by chronic hepatitis C. From an MRP2 gene mutational analysis, six mutations, including the novel mutation 1177C>T, were found. Three patients of a consanguineous family were homozygotes for three mutations (298C>T, 1967+2T>C, and 2439+2T>C). Two patients were compound heterozygotes (1177C>T/2302C>T and 1967+2T>C/2026G>C). A familial study showed no difference in serum bilirubin levels between mutant/wild heterozygotes and wild/wild homozygotes.ConclusionsThe hyperbilirubinemia of four Japanese patients with DJS, one of whom had a novel mutation, 1177C>T, of the MRP2 gene, had not worsened with aging.

TT virus infection in hemodialysis patients

OBJECTIVE:Recently, TT virus (TTV), associated with posttransfusion hepatitis, was discovered. Prevalence of TTV infection in maintenance hemodialysis (HD) units and its pathogenicity to liver was investigated.METHODS:A total of 115 patients on HD were assessed for presence of serum TTV. DNA was purified from sera, and nested polymerase chain reaction was done for the detection of TTV DNA.RESULTS:TTV was detected in 59 patients on HD (51.3%), as compared with healthy blood donors (15 of 91 [16.5%], p < 0.0001). Serum HCV RNA and HBs antigen were positive in 16 and three patients, respectively. The prevalence rate of TTV was already 58.3% in the patients on HD for only 1 yr, and did not change according to the duration of HD until 15 yr on HD. TTV was positive in 51.2% (43 of 84) of the patients with history of blood transfusion, and in 51.6% (16 of 31) of those without it. In HCV-negative patients, alanine aminotransferase (ALT) levels of TTV-positive patients were similar to those of TTV-negative patients. Contrarily, in HCV-positive patients, ALT levels were more frequently ≥15 IU/L in TTV-positive patients (14 of 18) than in TTV-negative patients (five of 15) (p < 0.05).CONCLUSIONS:TTV infection is remarkably prevalent in patients on HD and in healthy blood donors. It is suggested that TTV generally does not cause liver disease by itself, but there remains the possibility that TTV may aggravate liver disease caused by HCV.