Terukatsu Arima

Cloning of a cDNA associated with acute and chronic hepatitis C infection generated from patients serum RNA

SummaryIn order to clone hepatitis C (blood-borne non-A, non-B hepatitis) virus, lambda gt11cDNA library was constructed from RNA extracted from 100 liters serum collected from 1,047 donors with elevated ALT levels and negative for hepatitis B virus-DNA. The library was immunoscreened on Y1090 cells with pooled serum obtained from patients with acute hepatitis C or chronic hepatitis C. By screening 29 clones specific for Japanese hepatitis C infection were isolated. The specificity of these clones for hepatitis C infection was determined by panels constructed in 3 laboratories. Of these, 12 clones were specific for American hepatitis C infection as well. The nucleotide sequence (201 bp) of one of them was determined to be unique compared to known human viruses including hepatitis A virus, hepatitits B virus and hepatitis D virus. Southern blot analysis showed the absence of the sequence of the human genome in the clone. The predicted amino acid sequence is rich in residues of lysine, arginine, glutamic acid and asparagine, while lacking leucine, cysteine and methionine.

Arsenic trioxide and the growth of human T-cell leukemia virus type I infected T-cell lines.

A novel therapeutic potential for acute promyelocytic leukemia using arsenic trioxide (As2O3) has been reported. Recent in vitro studies demonstrated that As2O3 effectively inhibits the growth of some cell lines derived from patients with malignant lymphoma, chronic lymphocytic leukemia and multiple myeloma. Adult T-cell leukemia (ATL) is an aggressive neoplasm of mature T-cell origin caused by human T-cell leukemia virus type-I (HTLV-I) the prognosis of which still remains very poor. A possible role of As2O3 for the treatment of ATL is demonstrated from evidence that As2O3 significantly inhibits the growth of HTLV-I infected T-cell lines and induces apoptosis in fresh ATL cells at clinically achievable concentration of the agent. The growth inhibition of As2O3 treated HTLV-I infected T-cell lines was induced by both apoptosis and G1 phase accumulation. Cleaved bcl-2 protein and an enhanced expression of bak protein in the cells were coincidentally observed during As2O3 treatment. A broad spectrum caspase inhibitor, z-Val-Ala-DL-Asp-fluoromethylketone inhibited the apoptosis induced by As2O3. Increased expression of p53, Cip1/p21 and Kip1/p27, and dephosphorylation of retinoblastoma protein (pRb) were detected in the As2O3 treated cells. In conclusion, As2O3 might become a new therapeutic tool in the treatment of ATL as As2O3 induces apoptosis by destruction of the bcl-2 protein and enhancement of the bak protein production proceeding to activate caspases, and also induces G1 phase accumulation by enhancement of p.53, Cipl/p21, Kipl/p27 and dephosphorylation of pRb to HTLV-I infected T-cell lines.

Expression of ABH and lewis blood group antigens in combined hepatocellular-cholangiocarcinoma. Possible evidence for the hepatocellular origin of combined hepatocellular-cholangiocarcinoma

Expression of ABH, Lewis, and sialyl Lea antigens was studied in five combined hepatocellular‐cholangiocarcinomas. Formalin‐fixed liver tissues were immunostained for those antigens using well‐characterized monoclonal antibodies and an avidin‐biotin‐peroxidase complex (ABC) method. Results were compared with those obtained in normal liver tissues and cholangiocarcinomas, and also with the previous observations of the authors on hepatocellular carcinomas. Although not detected in normal parenchymal liver cells, A, H, Lewis, and sialyl Lea antigens were found in combined hepatocellular‐cholangiocarcinoma cells. Incompatible A antigen also was detected in one blood type O patient. Distribution and intensity of the antigens were similar to those in hepatocellular carcinomas and different from those in cholangiocarcinomas. No preferential accumulation of blood‐group antigens could be found in the area of cholangiocarcinoma‐like differentiation of the combined hepatocellular‐cholangiocarcinoma. The observations suggested that.

Arsenic Trioxide Induces Apoptosis in HTLV-I Infected T-cell Lines and Fresh Adult T-cell Leukemia Cells Through CD95 or Tumor Necrosis Factor α Receptor Independent Caspase Activation

Arsenic trioxide (As2O3) has been reported to induce apoptosis in human T-cell leukemia virus type-I (HTLV-I) infected T-cell lines and fresh adult T-cell leukemia (ATL) cells and to induce G1 phase accumulation in HTLV-I infected T-cell lines. The present study aimed to clarify the pathway of As2O3-induced apoptosis in HTLV-I infected T-cell lines, MT-1 and MT-2, and fresh ATL cells separated from peripheral blood of patients with acute or chronic type ATL. Cells were treated up to 72 h at clinically tolerable concentrations of As2O3 (1–2 µmol/l) shown to be safe in patients with acute promyelocytic leukemia (APL). Activation of caspases 3, 8, and 9, loss of mitochondrial transmembrane potential and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) were observed during As2O3 treatment. Furthermore, prior exposure to a broad-spectrum caspase inhibitor blocked As2O3-induced apoptosis but not G1 phase accumulation. While pre-treatment with a CD95 receptor-blocking antibody (Ab) or a TNF-α neutralizing Ab did not show such inhibitions in these cells. In conclusion, As2O3 induces apoptosis in HTLV-I infected T-cell lines and fresh ATL cells through CD95 or TNF-α receptor independent caspase activation.

A lambda gt11-cDNA clone specific for chronic hepatitis C generated from pooled serum presumably infected by hepatitis C virus

SummaryA lambda gt11-random-primed-cDNA clone specific for chronic hepatitis C was isolated from pooled serum preumably infected by hepatitis C virus. The translation product of the clone detect 50% of patients with chronic hepatitis C in 4 test panels but none of patients with acute hepatitis C, other liver diseases or normal controls was positive for the peptide. The nucleotide sequence of the cDNA clone, the size of which is 66 bp, has no homology to the complete sequences of known human viruses such as adenovirus, coxsackievirus, rhinovirus, immunodeficiency virus type 1, Epstein-Barr virus, polioma virus, poliovirus, papilloma virus, parvovirus, papovavirus, varicella-zoster virus, yellow fever virus, endogenous retrovirus, T-cell lymphotropic virus types I, II, and III Japanese encephalitis virus, and hepatitis A, B, and D viruses. Probably only one or two epitopes are present on the molecule encoded by the clone as the peptide consists of only 22 amino acid residues.

N- and C-terminal amino acid sequences of a γ-heavy chain disease protein YOK

Abstract The N- and C-terminal peptides of a γ-heavy chain disease protein YOK were isolated by cyanogen bromide cleavage of the protein followed by reduction, carboxymethylation and gelfiltration, and their sequences were determined by the conventional methods. Protein YOK consisted of Fc-fragments of IgG 1 and had heterogeneous N-termini, serine, threonine and histidine, which corresponded to positions 215, 223 and 224 respectively, from the N-terminal end of the gamma chain of immunoglobulin EU. A new amino acid substitution was found in position 431, in which YOK contained glycine in contrast to alanine in corresponding position of known human γ 1 -chains, suggesting one structural correlation with a genetic marker. These results were compared with those of other γ-heavy chain disease proteins and discussed for the genetic implications of the sequence of this protein.

A cDNA clone encoding a peptide highly specific for hepatitis C infection

SummaryA random primed lambda gtll-cDNA library was constructed from donors plasma presumably infected by blood-borne non-A, non-B hepatitis (hepatitis C:HC) agent and immunoscreened with serum pooled from patients with acute or chronic HC. Twelve lambda gtll-cDNA clones encoding antigens associated with HC infection in Japan as well as in the USA were isolated. Of these one clone consisting of 114 nucleotides and showing a discrete band on an immunoblot analysis, was extensively studied. The clone is not derived from the host DNA encoding one polypeptide specific and highly sensitive for serum from patients with HC and has no homology to the nucleotide sequences of known human viruses including hepatitis A, B and D viruses, Ebstein-Barr virus, coxsackievirus, immunodeficiency virus type 1 or Japanese encephalitis virus. These results suggest that this clone is derived from the genome of HC agent.

Natural killer activity in patients with chronic hepatitis treated with OK432, interferon, adenine arabinoside and glycyrrhizin.

SummaryNatural killer (NK) activity in the peripheral blood of patients with chronic liver disease was measured using51Cr labeled K562 cells1) as target cells. NK activity was elevated but not significantly in patients with chronic hepatitis compared with healthy controls and significantly lower in the patients with hepatocellular carcinoma. The activity decreased in the order of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Although the level of NK activity in patients with chronic hepatitis did not correlate with the level of alanine aminotransferase (ALT), it tended to be elevated in association with elevation of ALT in patients treated with OK432, interferon-β, giycyrrhizin or adenine arabinoside. In chronic liver disease, phytohemmagglutinin (PHA) skin test showed a positive correlation with NK activity. In all patients who were treated with the immunopotentiator, OK432, and whose HBeAg became negative, NK activity was elevated during the treatment. These results suggest that the NK activity in peripheral blood may be related to hepatocytic injury even if this is not the effector mechanism of the injury.

Serum glycoproteins in the liver diseases. VIII. Desialylated glycoproteins in the liver cirrhosis.

SummarySerum desialylated glycoprotein level of cirrhotic patients was determined and a diagnostically significant elevation of these proteins was observed. The level of these patients was usually 2–10 times of that seen in normal subjects and the elevation was significant (p<0.001) when compared to the level in patients with chronic aggressive hepatitis, severe (2B). Serial determinations of these proteins in the cirrhotic patients showed no correlation between them and SGPT as a whole but in several cases in which SGPT fluctuated the former associated with the latter. In patients with decompensated cirrhotic liver these proteins returned nearly to the level of compensated patients when it was improved.The level of these proteins in cirrhotic patients correlated, not always, with serum albumin (r=-0.46, p<0.02) and indocyanine green clearnace rate (r=-0.43, p<0.05), but not with SGPT as well as the other liver function tests.Serum desialylated glycoprotein level of cirrhotic patients was determined and a diagnostically significant elevation of these proteins was observed. The level of these patients was usually 2–10 times of that seen in normal subjects and the elevation was significant (p<0.001) when compared to the level in patients with chronic aggressive hepatitis, severe (2B). Serial determinations of these proteins in the cirrhotic patients showed no correlation between them and SGPT as a whole but in several cases in which SGPT fluctuated the former associated with the latter. In patients with decompensated cirrhotic liver these proteins returned nearly to the level of compensated patients when it was improved. The level of these proteins in cirrhotic patients correlated, not always, with serum albumin (r=-0.46, p<0.02) and indocyanine green clearnace rate (r=-0.43, p<0.05), but not with SGPT as well as the other liver function tests.

Serum glycoproteins in the liver diseases. III. Desialylated glycoproteins in the acute hepatitis.

SummaryCirculating desialylated glycoprotein level in acute hepatitis was studied by using the competitive binding assay reported by us. Statistically significant differences of the level among acute hepatitis in the peak of illness, fulminating hepatitis and normal subjects were observed. The desialylated glycoprotein level in acute hepatitis was elevated associating with S-GPT and serum bilirubin levels, and it returned to the normal range before S-GPT and serum bilirubin were normalized. The desialylated glycoprotein in a fulminant hepatitis was increasing associated with bilirubin even when S-GPT was decreasing.