Anti-fibrogenic effect of an angiotensin converting enzyme inhibitor on chronic carbon tetrachloride-induced hepatic fibrosis in rats.

The tissue renin-angiotensin system has recently been demonstrated to reduce fibrogenesis in various organs. However, little has been clarified regarding its role in hepatic fibrosis. The purpose of this study was to investigate the effect of angiotensin-converting enzyme inhibitors on liver fibrogenesis induced in rats by low-dose chronic carbon tetrachloride administration. We used lisinopril that is absorbed in its active form and not metabolized in the liver to avoid any influence by the administration of the chemical. Carbon tetrachloride was administered twice a week i.p. Twelve and 24 weeks after the start of treatment, expanded periportal fibrosis or portal-portal bridgings and severe fat deposition were observed in the rats treated with carbon tetrachloride alone, and these findings were significantly reduced with the simultaneous treatment with lisinopril. The hydroxyproline content of the liver was significantly lower in the lisinopril-treated group. Angiotensin II up-regulated mRNA of pro alpha (I) collagen and transforming growth factor-beta in isolated hepatic stellate cells. These results suggest that the local tissue renin-angiotensin system plays a role in rat hepatic fibrogenesis induced by chronic carbon tetrachloride administration and that hepatic fibrogenesis is significantly reduced by ACE inhibitors.

Th1 and Th2 cytokines differentially regulate the transformation of Kupffer cells into multinucleated giant cells but similarly enhance the Kupffer cell-induced hepatic stellate cell proliferation.

To investigate the effects of T-helper cytokines on Kupffer cells (KCs), the effects of interferon-gamma (IFN-gamma; Th1 cytokine) and interleukin-4 (IL-4; Th2 cytokine) on KC morphology and their role in modulating the growth of hepatic stellate cells (HSCs) were examined. Fluorescence microscopic and electron microscopic data demonstrated that IL-4 transforms rat KCs into multinucleated giant cells (MGCs) in vitro. This transformation was inhibited by the addition of anti-ICAM-1 and anti-CD18 monoclonal antibodies. In addition, IL-4-induced KC transformation was suppressed by the presence of IFN-gamma. The formation of mouse hepatic MGCs was also demonstrated in vivo by the intraperitoneal injection of recombinant mouse IL-4. Although the presence of MGCs was found in all five out of five livers from IL-4-treated Th2-dominant BALB/c mice, but it was in only two out of five livers from IL-4-treated Th1-dominant C57BL/6 mice. In addition, fewer MGCs were found in the liver of C57BL/6 mice. In contrast, IFN-gamma treatment did not form hepatic MGCs in mice at all. The growth of HSCs in vitro was significantly increased by the addition of culture supernatant from lipopolysaccharide-stimulated rat KCs. Pretreatment of the KCs with either IFN-gamma or IL-4 further enhanced the growth stimulation. These results suggest that IFN-gamma and IL-4 affect KC morphology differently, but that both Th1 and Th2 cytokines play a similar role in the modulation of HSC growth by Kupffer cells in the presence of lipopolysaccharide.

[A case of multiple sclerosis with recurrent episodes of psychiatric symptoms].

We reported a 19-year-old woman with multiple sclerosis (MS) with recurrent episodes of psychiatric symptoms at the age of 10 and 19, as the first and 11th relapse, respectively. Her first episode of MS resembled acute encephalitis at the age of 9 and she relapsed 12 times. The psychiatric symptoms were not mood disorders but altered states of consciousness, such as the delirous state. She was treated with high doses of intravenous methylpredonisolone and her symptoms were almost resolved, but her anxiety neurosis remained. Her MRI showed demyelination in the white matter of the frontal lobe and the hippocampus, which suggested that psychiatric symptoms of MS would be related to the frontal lobe lesion.

Up-regulation of E-cadherin and I-catenin in human hepatocellular carcinoma cell lines by sodium butyrate and interferon-alpha.

Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-alpha. E-cadherin expression was only up-regulated by butyrate and interferon-alpha (IFN-alpha) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, beta-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-alpha. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that alpha- and y-catenin expression was not changed, while only the expression of beta-catenin increased. Beta-catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type beta-catenin are potentially modulated by butyrate and IFN-alpha, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.

[Epileptogenesis of acute encephalitis and acute encephalopathy: epilepsy with its onset in the acute phase and without a latent period].

Epileptogenesis was evaluated in 60 patients with acute encephalitis and in 10 patients with acute encephalopathy. Forty-seven patients have been seizure-free during for more than three years' follow-up (Group III). On the other hand, 23 patients developed epilepsy. Among them, 18 patients developed epilepsy after a latent period of 1 month to 2 3/12 years (Group I). In Group I, a younger age of the onset, a long period of disturbed consciousness and a high activity of CSF neuron-specific enolase (NSE) was associated with refractory epilepsy. The other five patients had continuous seizures from the acute phase of encephalitis without a latent period (Group II). They had more than 2 types of partial motor seizures which occurred frequently during the acute phase of encephalitis. The NSE activity in the CSF of patients in Group II was less than 50 ng/ml, being similar to those in Group III. The epilepsy in Group II, however, was the most refractory. The reason for the development of this continuous refractory epilepsy remained obscure.

Reduced in vitro immunoglobulin secretion from peripheral blood mononuclear cells in responders to high-dose interferon-alpha 2b treatment for chronic hepatitis C.

BACKGROUND/AIMS: Immunological status has been considered to correlate to the response to interferon therapy for chronic hepatitis C. The aim of this study was to evaluate the correlation between humoral immunity and long-term response to interferon treatment for chronic hepatitis C. METHODOLOGY: Seventy-one patients with chronic hepatitis C received 10 million units of interferon-alpha 2b three times a week for 24 weeks. Peripheral blood mononuclear cells were obtained before interferon-alpha 2b was administered and were cultured for 7 days. Immunoglobulin concentration in the culture supernatants was measured by enzyme-linked immunosorbent assay and correlation with the response to the therapy was evaluated. RESULTS: Serum ALT levels normalized in 51.4% and hepatitis C virus RNA disappeared in 35.7% six months after the end of therapy. Immunoglobulin production was significantly lower in the patients in whom serum ALT levels normalized than those in whom serum ALT levels remained elevated. The similar result was obtained when efficacy was evaluated on the basis of hepatitis C virus RNA disappearance. CONCLUSIONS: These results suggest that the less humoral immunity, the better response to interferon will be obtained in patients with chronic hepatitis C, meaning that the balance in T-helper function is one of key factors in the response to interferon treatment.

Induction of hepatic stellate cell proliferation by LPS-stimulated peripheral blood mononuclear cells from patients with liver cirrhosis.

We studied hepatic stellate cell proliferation in vitro. Peripheral blood mononuclear cells (PBMC) from patients with chronic active hepatitis C (CAH) and liver cirrhosis (LC) were cultured for 24h in the presence or absence of Escherichia coli lipopolysaccharides (LPS). Hepatic stellate cell proliferation induced by the culture supernatants was measured, and interleukin-1 (IL-1) and IL-6 levels in the culture supernatants were quantified. Culture supernatants of LPS-stimulated PBMC from LC patients induced rat hepatic stellate cell proliferation by almost 2.8-fold (stimulation index, 2.83 +/- 1.41) compared with when the cells were cultured without addition of PBMC culture supernatants. Production of IL-1beta was significantly higher in the culture supernatants of both CAH and LC patients than in those of ten healthy controls (P < 0.01 and P < 0.05, respectively). But there was no significant correlation between IL-1 production and the induction of hepatic stellate cell proliferation by the culture supernatants. Although there were no significant differences in IL-6 production by LPS-stimulated PBMC among healthy controls and CAH and LC patients, we observed a significant correlation between IL-6 production and the induction of hepatic stellate cell proliferation in the culture supernatants of LC patients. Rat hepatic stellate cells themselves produced IL-6, and treatment with IL-6 antisense oligodeoxynucleotides suppressed the cell proliferation, suggesting that IL-6 is an autocrine growth factor for hepatic stellate cells. The addition of human recombinant IL-6 (hrIL-6) augmented rat hepatic stellate cell proliferation, indicating that excessive IL-6 may further facilitate cell proliferation. These findings suggest that a cytokine cascade including IL-6 may participate in hepatic stellate cell proliferation in LC patients when they are exposed to endotoxin.

Disappearance of serum hepatitis C virus RNA within two days after one dose interferon administration is predictive for response to high-dose interferon-alpha 2b treatment for chronic hepatitis C. Keio Interferon-alpha 2b Study Group.

We have reported the Keio multicenter randomized trial of interferon-alpha 2b treatment for chronic hepatitis C, hypothesizing that disappearance of serum hepatitis C virus (HCV) ribonucleic acid (RNA) during the first 2 days by one does administration of interferon is a predictive factor of final response to the high-dose interferon treatment. In this study we quantified HCV RNA by multicyclic reverse transcription-polymerase chain reaction in the stored sera of the same patients with our previous study. The multivariate analysis confirmed that the pretreatment HCV RNA levels and HCV genotype were significantly correlated with the response to the 6-month course interferon treatment. Although the relationship between decreased HCV RNA titers within the first 2 days after one dose administration of interferon and the efficacy of the therapy was not obtained, the cases in which HCV RNA disappeared within 2 days significantly responded to the 6-month course treatment (73.7% vs 12.5% in other cases, p < 0.01). The present study has confirmed the hypothesis suggested in the previous study that clearance of HCV RNA during the first 2 days has a predictive value for the final outcome.

Kupffer cell-mediated cytotoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18.

Rat Kupffer cell (KC)-mediated cytotoxicity against both the syngeneic hepatoma cell line AH70 and hepatocytes was evaluated by changes in mitochondrial function, and the possible role of ICAM-1/CD18 in the interaction between the cells was studied. Rhodamine 123 fluorescence, a marker of the mitochondrial membrane potential, decreased in AH70 cells after co-culture with CK, while that in hepatocytes was unchanged by co-culture. This decrease was blocked by anti-ICAM-1 anti-CD18 and the inhibition of nitric oxide synthesis. Cytometric studies demonstrated that ICAM-1 expression on AH70 cells increased after addition of IFN-gamma, IL-1beta, tumor necrosis factor (TNF)-alpha or KC, while in hepatocytes ICAM-1 was not increased. Anti-ICAM-1 pretreatment inhibited the increase in ICAM-1 expression and the decrease in rhodamine 123 fluorescence on AH70 cells after co-culture with KC. CD18 on KC was increased only after co-culture with AH70. TNF-alpha but not IFN-gamma was detected in the supernatant of co-culture between KC and AH70 cells, and this production was partially inhibited by anti-ICAM-1 and anti-CD18. The activity of inducible nitric oxide synthase in Kupffer cells and the levels of nitrites and nitrates in the co-culture supernatant increased over time, and this increase was attenuated either by addition of NO synthesis inhibitors, anti-ICAM-1 or anti-CD18. These results indicate that the rat KC causes mitochondrial dysfunction in cancer cells via the production of NO and cell-to-cell adhesion via ICAM-1/CD18 has an important role in this cytotoxic process.

Cloning of variable regions of an antibody that reacts with the soluble fraction of human liver cells and its possible value in chronic liver disease.

A gene encoding the variable regions of the heavy and light chains of a mouse monoclonal antibody designated H2, which specifically reacts with human liver cells, was cloned into a phagemid vector. The clone of the variable region was designed to be expressed as a separate protein, the structure of which is the same as that of the mouse antibody. The cloned phage protein specifically reacted with anti-idiotypic antibodies produced in rabbits against the original mouse antibody, and this reaction was specifically blocked by the original antibody. The soluble protein, expressed as a fusion protein, was detected as a single 30-kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and specifically bound to an anti-H2 idiotypic antibody as determined by Western blot analysis. Sera of patients with various diseases were assayed for antibodies to anti-H2 by sandwich enzyme-linked immunosorbent assay (ELISA). Only sera from patients with chronic liver disease reacted strongly. This binding was specifically blocked by the cloned soluble protein. The nucleotide sequences of the variable regions were determined by the dideoxy chain-termination method, and the sequences were approximately 95% identical to those of other mouse immunoglobulins. These findings suggest that a human antibody with the same idiotype as a mouse monoclonal antibody that reacts with human liver cells, can be detected in patients with chronic liver disease, suggesting that autoimmunity may be partly responsible for these diseases.

Reduction of LAK-sensitivity and changes in antigen expression on hepatoma cells by sodium butyrate.

We demonstrated that sodium butyrate (SB) induced differentiation of functions in human hepatocellular carcinoma (HCC) cell lines. To investigate relationship between the sensitivity for cellular cytotoxicity and the cellular differentiation of HCC cells, the effect of SB on lymphokine-activated killer (LAK) sensitivity and antigen expression of a human HCC cells were studied. SB induced LAK-resistance of human HCC cell lines, HCC-T and HCC-M, time-dependently. A flowcytometric analysis of cell surface antigens revealed that SB markedly reduced the expression of laminin and fibronectin and increased the expression of liver-specific antigen defined by a mouse monoclonal antibody time-dependently, but did not modify that of major histocompatibility complex antigens, intercellular adhesion molecule (ICAM)-1, or CEA. Leukocyte function-associated antigen (LFA)-3 expression on HCC-T was reduced slightly by SB treatment. LAK sensitivity was inhibited by anti-laminin, but not with anti-beta 2-microglobulin, anti-HLA DR, anti-ICAM-1, anti-fibronectin, or anti-CEA. Anti-LFA-3 reduced LAK sensitivity of HCC-T, but not HCC-M, although the reduction was less than that obtained by anti-laminin treatment. These results provided evidence that SB induced LAK-resistance of human HCC cells according to cellular differentiation and extracellular matrix functionality played an important role in this LAK-mediated cell killing. Moreover, the structure expressed on HCC cells, which contributed to LAK cytolysis, was different for each HCC cell.

Enhancing effect of the liver extract and flavin adenin dinucleotide mixture on anti-viral efficacy of interferon in patients with chronic hepatitis C.

A combined preparation of liver extract and flavin adenin dinucleotide (FAD) (Adelavin) has been widely used in patients with chronic liver diseases in Japan. One milliliter of this agent contains 15 microliters of phenol-soluble phase of liver nucleic acid fraction and 10 mg of FAD. To examine the advantages of using this preparation in the elimination of hepatitis C virus (HCV) from patients with chronic hepatitis (CH)-C receiving interferon (IFN), 2 ml of this preparation was intravenously (n = 9) or intramuscularly (n = 8) administered daily for 5 days before 6 million units of IFN-alpha was intramuscularly injected once. Before and 48 hours after the injection of IFN, serum ALT, 2'5'-oligoadenylate synthetase (2'5-AS) activity, and HCV RNA levels were measured. The daily administration of this preparation alone for 5 days did not significantly change serum ALT, 2'5-AS activities, and HCV RNA levels. The 2'5-AS activities were significantly increased by IFN after the intravenous injection of this preparation (p < 0.01), while an injection of IFN alone of this dose did not change its activities (n = 10). HCV RNA levels were significantly decreased by IFN only after the administration of the preparation (intramuscular, p < 0.01; intravenous, p < 0.01). The effect of intravenous injection of this preparation was also elicited in patients with HCV genotype II and with HCV more than 10(5) copies/ml. These results suggest that this preparation may enhance the 2'5-AS production by IFN as a result of the increase in mitochondrial adenosin triphosphate production and may be a potent agent to enhance the anti-viral efficacy of IFN in patients with CH-C.

[Epilepsy in patient with structural autosomal abnormality].

Few cases have been reported on the structural autosomal abnormality (SAA) focusing on epilepsy excluding those of Down syndrome and Klinefelter syndrome. We investigated patients who had SAA with special reference to epilepsy. Various types of epilepsy were observed in its severity in our cases as well as previously reported cases. There was no correlation between the degree of mental retardation, motor dysfunction, brain damage on CT scan, and severity of epilepsy. Some cases had brain dysplasia, such as agenesis of corpus callosum, pachygyria, and mega cisterna magna. No correlation was found between these brain dysplasia and severity of epilepsy. It is important for a pediatrician to find a common epileptic syndrome or EEG abnormality in a SAA. An observation of symptoms in patients with the same chromosomal deletion or duplication will lead to identification of responsible gene for an epileptic symptom.

[Clinical consideration of patients with neonatal bilateral basal ganglia-thalamic lesion due to hypoxic ischemic encephalopathy].

We describe herein the clinical symptoms, clinical course and results of investigation of 7 patients with bilateral basal ganglia-thalamic lesions (BBTL). All patients had spastic quadriplegia with rigidity. They were unable to sit and turn over. They could follow objects, turn head towards a sound and recognize parents to some degree. They were all evaluated as having the most severe degree of disability (Oshima's classification 1). They all had dysphagia and 2 patients had a episode of bradycardia and hypothermia, which might be evidences of brain stem disorders. Muscle hypertonia, vomiting, hematemesis and obstructive respiration, which were the major complications for the patients, worsened with age. High percentage of histories of birth asphyxia and poor feeding in the neonatal period suggested that perinatal brain insults might be one of the important factors for developing BBTL. It seemed to be difficult to explain that such diffuse brain injuries in our cases were caused by only the insults during parturition. Brain insults during parturition as well as prenatal factors probably participate in developing BBTL. Although the cerebrum of the patients seem to be relatively preserved in the images of head CT-scan, MRI of the patients revealed diffuse brain lesions. All of five patients tested had an abnormal auditory brain stem response (ABR). These investigations demonstrated that patients with BBTL have diffuse brain damage including brain stem. Further observation is needed to verify the mechanisms of development and the time of onset of BBTL.