Expression of fas/fas ligand (fasL) and its involvement in infiltrating lymphocytes in hepatocellular carcinoma (HCC).

BACKGROUND: This study was conducted to examine the expression of Fas/Fas ligand (FasL), to elucidate its relationship with tumor-infiltrating lymphocytes (TILs), and to detect possible gene mutation of Fas/FasL in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). METHODS: Indirect immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections of liver biopsy and surgery specimens from five normal livers, and from the livers of 30 patients with HCC. Fas/FasL mRNA-expressing cells and apoptotic cells were detected by in situ hybridization and DNA nick end labeling (TUNEL), respectively. We also performed polymerase chain reaction (PCR)-amplifying and direct sequencing for the Fas/FasL gene. RESULTS: Fas/FasL and its mRNA were localized on the membrane or in the cytoplasm in some HCC cells, as well as hepatocytes. Their expression was enhanced in areas with infiltrating inflammatory cells in the noncancerous regions of liver tissue and on the margins of the cancerous tissue. The positivity rate for TUNEL was elevated along these margins. The labeling index of Fas/FasL was lower in the cancerous liver tissue than in the surrounding noncancerous region (P < 0.01), and tended to decrease in proportion to the malignancy of tumor cells; Fas/FasL expression was not found on poorly differentiated type cancer cells. Fas(-)/FasL(+), FasL-mRNA(+) HCC cells were seen in one specimen of moderately differentiated type. Some CD8+T lymphocytes were TUNEL-positive around the cancerous region. In this study, cancerous and noncancerous tissues in HCC revealed no genetic mutations in any exons of Fas/FasL. CONCLUSIONS: These findings suggest that Fas/FasL expression was decreased in proportion to the malignancy of tumor cells, and that infiltrating CD8+ T lymphocytes play a role in apoptosis in HCC. The apoptosis in HCC could be regulated by the suppression of Fas/FasL expression, or, sometimes, by the enhancement of FasL expression.

Decreased function of peripheral blood dendritic cells in patients with hepatocellular carcinoma with hepatitis B and C virus infection.

BACKGROUND: Tumour immunity does not seem to be induced effectively in tumour-bearing hosts, including in patients with hepatocellular carcinoma (HCC). One possible reason is that function of dendritic cells (DC) is decreased in such hosts. METHODS: We evaluated T cell stimulatory activity and interleukin (IL)-12 production of DC and interferon (IFN)-gamma and IL-10 production of T cells of peripheral blood from 12 control individuals and 21 patients with chronic hepatitis C virus (HCV) infection (six with chronic hepatitis (CH), eight with liver cirrhosis (LC) and 13 with HCC). Five hepatitis B virus (HBV)-infected patients with HCC were included as a disease control group. The DC were prepared by the culture of T cell-depleted populations of peripheral blood mononuclear cells in the presence of granulocyte-macrophage colony stimulating factor and IL-4 for a total of 11-12 days. The cytokine levels were assayed by ELISA. To test the stimulatory function of DC in T cell proliferation, mytomycin C-treated DC were cultured with allogeneic T cells from a control. RESULTS: When the T cell-stimulatory activity of DC was expressed as stimulation index value of [3H]-thymidine incorporation of T cells, the values were lower in HCV-infected HCC (2.6 +/- 1.8, P < 0.01) than in controls (5.5 +/- 2.0) and CH (5.0 +/- 1.3). Staphylococcus aureus Cowan 1-induced IL-12 production of DC was decreased in HCV-infected HCC (P < 0.001, P < 0.01 and P < 0.05, respectively) compared with controls, CH and LC, while similar amounts of IL-10 were produced in patients and controls. Interleukin-10 and IFN-gamma production of T cells in response to anti-CD3 antibody or IL-12 were equivalent between patient groups and controls, respectively. Similarly decreased DC function and normal T cell response were observed in HBV-infected HCC patients. CONCLUSIONS: These findings suggest that the depressed function of DC is associated with pathogenesis of HCC with HBV or HCV infection.

Gene mutation of transforming growth factor beta1 type II receptor in hepatocellular carcinoma.

Alteration of transforming growth factor beta1 (TGF-beta1) type II receptor (RII) appears to cause unresponsiveness to TGF-beta1 in tumorigenic cells. Defect in the mononucleotide repeat sequence, i.e., poly A region of TGF-beta1RII gene has been reported to be related to replication error-positive cancer cells. We examined if there is any TGF-beta1RII mutation in a coding microsatellite in hepatocellular carcinoma (HCC). Genomic DNAs were extracted from formalin-fixed, paraffin-embedded liver tissues obtained at surgery or autopsy in 3 normal individuals and 96 patients with hepatitis C virus-induced chronic liver disease; 3 with chronic hepatitis, 20 with liver cirrhosis and 73 with HCC. The DNA was PCR-amplified at 2 segments of TGF-beta1RII: poly A region which includes the (A)10 microsatellite sequence, and poly GT region. PCR products were directly sequenced. DNA from normal and patients with chronic liver disease contained the 10 wild-type adenines but 3 cases with liver cirrhosis in whom there were only 9 adenines within poly A tract. This microdeletion of one A resulted in a frameshift and truncated a predicted length of amino acids. In HCC lesions, the same deletion was noted in 4 cases (25%) of well-differentiated type, 10 (40%) of moderately differentiated type, 18 (53%) of poorly differentiated type. None of the lesions had mutations within the GT region. Our findings indicate that one adenine deletion of poly A microsatellite tract within TGF-beta1RII is frequently detected in patients with HCC, and the mutation may cause the abrogation of the function of TGF-beta1RII gene.

[A case of recurrent multiple HCC after surgical resection showing regression by two TAEs using 5-FU and zinostatin stimalamer].

A-68-year-old man had hepatocellular carcinoma (HCC) in the area of S6 segment which was resected surgically. Three months after surgery, multiple recurrent lesions were found in both lobes of the liver. A transcatheter arterial embolization (TAE) with 5-FU and zinostatin stimalamer (SMANCS) was done. After TAE, the tumor mass disappeared in the right lobe and reduced to 10% in the left lobe. Although mass lesions remained unchanged for 3 months, the increased of AFP (143.9 ng/ml) was noticed after 4 months. Ten months after the second TAE with 5-FU and SMANCS, the disappearance of tumor masses was confirmed by diagnostic images.

Clinical usefulness of serum 1,5-anhydroglucitol in monitoring glycaemic control.

BACKGROUND: To evaluate prospectively the clinical value of measuring serum concentrations of 1,5-anhydroglucitol (1,5AG) in monitoring glycaemia in patients with newly diagnosed non-insulin-dependent diabetes mellitus (NIDDM), we measured serum 1,5AG in 56 such patients. METHODS: 28 patients (group A) were started on, and continuously received, an oral hypoglycaemic agent for at least 6 weeks. The other 28 patients (group B) were given such agents for 4 weeks, and then stopped taking them for at least 2 weeks. All patients were then followed for an additional 10 weeks. Serum 1,5AG, fructosamine, glycated haemoglobin (HbA1c), and self-monitoring of blood glucose were monitored every 14 days for 16 weeks. FINDINGS: When sudden worsening of glycaemia occurred within 2 weeks, entailing withdrawal of oral treatment, 1,5AG accurately detected the slight change in glycaemia whereas HbA1c and fructosamine both failed to detect it. Although the change was detected by measurement of fasting plasma glucose (FPG) concentrations, FPG was less sensitive than 1,5AG. In patients with "near-normoglycaemia" (HbA1c about 6.5%) in the preceding 8 weeks, those who showed a lower concentration of 1,5AG (<10.0 micrograms/mL) manifested a higher mean daily plasma glucose concentration even though HbA1c measurement suggested good control of glycaemia. Results of 1,5AG were correlated more strongly with the FPG (r=0.790) and mean daily plasma glucose (r=-0.835) estimated on the same day than those estimaoffted in the preceding 2, 4 and 8 weeks, and with a fall in the Spearman correlation coefficient at any preceding time interval. INTERPRETATION: Because 1,5AG accurately detected a slight change in glycaemia without delay, it is suitable for use in monitoring for strict control of glycaemia, an important clinical goal.

Role of transforming growth factor beta 1 on hepatic regeneration and apoptosis in liver diseases.

AIMS: To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on regeneration and induction of apoptosis of liver cell and bile duct in various liver diseases. METHODS: Formalin fixed paraffin wax sections of 18 liver tissue samples were obtained by needle biopsy, surgery, or necropsy; these included six liver cirrhosis, three obstructive jaundice; five fulminant hepatitis, one subacute hepatitis, and three normal liver. Expression of TGF-beta 1, apoptosis related Le(y) antigen, Fas antigen, a receptor for tumour necrosis factor, and biotin nick end labelling with terminal deoxynucleotidyl transferase mediated dUTP (TUNEL) for locating DNA fragmentation, was investigated histochemically. RESULTS: TGF-beta 1 was expressed in areas of atypical bile duct proliferation, where bile duct continuously proliferated from liver cells. In occlusive jaundice and fulminant hepatitis, TUNEL was positive in nuclei and cytoplasm of metaplastic cells which formed incomplete bile ducts, and these cells appeared to extend from TGF-beta 1 expressing liver cells. Fas antigen was found only on the cell membrane of proliferated bile duct in fulminant hepatitis, which differed from TGF-beta 1 and TUNEL positive areas. Le(y) antigen was expressed in liver cell and bile duct at the areas with atypical bile duct proliferation, but its coexpression with TUNEL was rare. CONCLUSIONS: TGF-beta 1 plays a role in the arrest of liver cell regeneration and atypical bile duct proliferation, and in areas of rapidly progressing atypical bile duct proliferation, such as in fulminant hepatitis or bile retention. Apoptosis appears to be induced by TGF-beta 1. This phenomenon may account for the inadequate hepatic regeneration that occurs with liver disease.

Biochemical improvement of chronic hepatitis C after gastrointestinal bleeding.

Although chronic hepatitis C is frequently complicated by iron overload, it remains unclear whether iron cytotoxicity is involved in the disease process. Five patients with chronic hepatitis C showed rapid reduction of serum aminotransferase activity after gastrointestinal bleeding. Posthemorrhagic reduction of liver enzyme levels lasted for more than one week. Anemia was associated with a reduction of serum ferritin concentration. Considering the short half-lives of circulating liver enzymes, reduced release of enzymes, that is inactivation of cell lysis, is the likely cause of the improved biochemical indices. Reactive iron, which is cytotoxic for patients infected by HCV, may be rapidly incorporated into hemoglobin when erythropoiesis is stimulated. Our observation also suggests that intensive iron removal by phlebotomy is a safe, economic treatment for patients with chronic hepatitis C.

A germ line mutation within the coding sequence for the putative 5-phosphoribosyl-1-pyrophosphate binding site of hypoxanthine-guanine phosphoribosyltransferase (HPRT) in a Lesch-Nyhan patient: missense mutations within a functionally important region probably cause disease.

Lesch-Nyhan syndrome caused by a complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) is the result of a heterogeneous group of germ line mutations. Identification of each mutant gene provides valuable information as to the type of mutation that occurs spontaneously. We report here a newly identified HPRT mutation in a Japanese patient with Lesch-Nyhan syndrome. This gene, designated HPRT Tokyo, had a single nucleotide change from G to A, as identified by sequencing cDNA amplified by the polymerase chain reaction. Allele specific oligonucleotide hybridization analysis using amplified genomic DNA showed that the mutant gene was transmitted from the maternal germ line. This mutation would lead to an amino acid substitution of Asp for Gly at the amino acid position 140 located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP) binding region. Missense mutations in human HPRT deficient patients thus far reported tend to accumulate in this functionally active region. However, a comparison of the data suggested that both missense and synonymous mutations can occur at any coding sequence of the human germ line HPRT gene, but that a limited percentage of all the missense mutations cause disease. The probability that a mutation will cause disease tends to be higher when the missense mutation is within a functionally important sequence.

Relationship between superoxide dismutase (SOD) and viral liver diseases.

Immunohistochemical evaluation of Cu, Zn- and Mn-superoxide dismutase (SOD) activity in various viral liver diseases was performed by the peroxidase-conjugated antibody indirect method. Anti-human Cu, Zn-SOD (rabbit) and anti-human Mn-SOD (guinea-pig) derived and purified from SOD of human erythrocytes and placentas were used to determine SOD distribution in liver tissues. SOD in the liver tissues was detected in 68 inpatients of our unit. They consisted of 23 cases with chronic hepatitis caused by hepatitis B virus (13) and hepatitis C virus (10), 24 with liver cirrhosis caused by hepatitis B virus (5) and hepatitis C virus (19) (15: compensatory, 9: decompensatory) and 21 with hepatocellular carcinoma caused by hepatitis B virus (2) and hepatitis C virus (18) complicated of liver cirrhosis. In viral liver diseases, SODs in the liver tissues were distributed to hepatocytes mainly in the pattern of cytoplasmic diffusion. The incidence of immunohistochemical Cu, Zn-SOD and Mn-SOD were 47.8% and 56.5% in chronic hepatitis, 93.3% and 86.7% in compensated liver cirrhosis, 11.1% and 22.2% in decompensated liver cirrhosis, respectively. The aggression of viral liver disease was accompanied with the decrease of SOD concentration in the liver tissues. Hepatocellular carcinoma cells were negative for Mn-SOD in all cases, and weakly positive for Cu, Zn-SOD in 2 out of 21 cases. Comparatively strongly positive SOD findings were obtained from normal regions neighboring carcinomas. A close relationship between the depletion of SOD in liver tissues and carcinogenesis in viral liver diseases was observed.