Jiro Kudo

Purification and immunological determination of α2-macroglobulin in serum from injured rats

Abstract Rat α 2 -macroglobulin was isolated and purified from the pooled sera of turpentine-injected rats by sequential use of dextran sulphate, DEAE-cellulose, and gel filtration chromatography. The final protein product obtained by this procedure proved to be α 2 -macroglobulin of a high degree of purity based on electrophoretic, immunologic and centrifugal analysis. The α 2 -macroglobulin preparation also binds stoichiometrically to trypsin preventing subsequent inhibition by protein trypsin inhibitors. SDS-polyacrylamide gel electrophoresis of rat α 2 -macroglobulin after incubation with trypsin suggested that there are at least two susceptible peptide bonds in the 170 000-dalton α 2 -macroglobulin subunit. The concentration of α 2 -macroglobulin in the sera of rats was measured by electroimmuno assay using a monospecific antiserum against α 2 -macroglobulin. Purified α 2 -macroglobulin was used as a standard. Sera from normal male rats contained 32 ± 4 μ g of α 2 -macroglobulin per ml. To determine the time course of response of α 2 -macroglobulin to inflammation, rats were subjected to either laparotomy or subcutaneous injection of turpentine. After either type of injury, the concentration of α 2 -macroglobulin increased rapidly, reaching a maximum value of 110–140 times that of the control value by 24 h. Little difference was noted in responsiveness between the two sexes.

Fulminant hepatic failure during perinatal period in a pregnant woman with Wilson's disease.

SummaryWilson’s disease associated with hepatic failure is not common and the underlying mechanism triggering the event is not known at present. We treated a 28-year-old Japanese woman with Wilson’s disease who developed hepatic failure associated with hemolytic crisis just after delivery. She was diagnosed as having Wilson’s disease at 12 years of age, at which time she started taking D-penicillamine. She had previously delivered two children without difficulty. When she found out she was pregnant this time, she stopped taking D-penicillamine in contrast to taking it faithfully during her first two pregnancies. On the day of delivery of her full-term baby, jaundice developed accompanied with severe hemolytic crisis. Plasma exchanges and blood transfusion were performed and D-penicillamine administration was started again. She gradually recovered and apparently was following a good clinical course. However, on day 30 the second hemolytic crisis occurred and subsequent liver failure led her to death on day 50. At autopsy her liver was cirrhotic and showed massive necrosis. Prophylactic oral administration of D-penicillamine and careful observation are therefore recommended to prevent hemolytic crisis during the perinatal period.

Kupffer cells modulate natural killer cell activity in vitro by producing prostaglandins

The effect of Kupffer cells on natural killer (NK) cell-mediated cytotoxicity was examined. Kupffer cells prepared from rat liver suppressed NK activity against K562 cells and other tumor cell lines through a soluble factor secreted into the culture supernatant. When human peripheral blood mononuclear cells were incubated with the Kupffer cell-culture supernatant, a significant reduction of the cytotoxic activity was observed in the 6-hr chromium-release assay. This activity was dose dependent and was evident at various effector/target cell ratios. Lipopolysaccharide stimulated generation of the suppressive factor released from Kupffer cells in a dose-dependent manner. Suppression of the NK activity was observed when the Kupffer cell-culture supernatant was present in the assay system, whereas pretreatment of effector/target cells with the supernatant had minimal inhibitory effects. Autologous monocytes in human peripheral mononuclear cells were not related to this suppression. The suppressive factor in the fraction had a molecular weight below 10,000. Indomethacin, an inhibitor of prostaglandin synthesis, ameliorated the suppressive effects. These results suggest that Kupffer cells may modulate NK activity by producing PGs (E1, E2, and F2 alpha).

Rotenone, a mitochondrial NADH dehydrogenase inhibitor, induces cell surface expression of CD 13 and CD38 and apoptosis in HL-60 cells

We previously demonstrated that the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene was overexpressed in human acute myelogenous leukemia (AML) cells. Since this finding suggested that ND2 gene expression was related to myeloid differentiation, we here investigated the effects of rotenone, a specific NADH dehydrogenase inhibitor, on HL-60 cell growth, differentiation and death. Fifty nM rotenone inhibited the growth of HL-60 cells and caused an increase in the cell population in the G(2) +M phase. In the quantitative comparison of myeloid antigen, the expression of CD13 and CD38 were relatively increased in the rotenone-treated cells. These findings suggest that the inhibition of NADH dehydrogenase changes the cell cycle and induces some specific surface antigens of HL-60 cells. On the other hand, the expression of ND2 gene remained unchanged after the rotenone treatment, suggesting the rotenone-mediated mitochondrial inhibition did not affect the mitochondrial gene expression. Five mu M rotenone strongly inhibited the cellular proliferation. Electron microscopy and an electrophoretic analysis of DNA showed that the majority of the HL-60 cells were induced into typical apoptosis within 24-48 hours. On the basis of this and other studies, we believe that mitochondrial function is directly involved in both cellular differentiation and apoptotic cell death.

Selective IgA Deficiency: Analysis of Ig Production in Vitro

The cellular basis of the pathogenesis of selective IgA deficiency (SIgAD) was investigated by examining surface immunoglobulin (SmIg) andin vitro pokeweed mitogen (PWM)-stimulated immunoglobulin (Ig) synthesis and by assaying in combination the counterpart lymphocytes from individuals with SIgAD and healthy donors. Peripheral blood lymphocytes (PBL) from 14 individuals with SIgAD synthesized normal amounts of IgG and IgM but did not synthesize normal amounts of IgA. Functional defects of lymphocytes for IgA synthesis were classified into four types: (i) B-lymphocyte dysfunction, (ii) increased function of suppressor T lymphocytes (Ts), (iii) decreased function of helper T lymphocytes (Th), and (iv) B-lymphocyte dysfunction and increased Ts function. The cells bearing SmIgG, SmIgM, and SmIgD were demonstrated at normal percentage ratios in all cases by immunofluorescent staining. The cells bearing SmIgA were at normal percentage ratios in the cases of T-lymphocyte dysfunction, while in the cases of B-lymphocyte defect SmIgA-bearing cells were reduced.

Alpha1-antitrypsin synthesis by human lymphocytes

Abstract De novo synthesis of alpha1-antitrypsin (α1AT) by human peripheral lymphocytes has been demonstrated in the present study. Treatment of the mononuclear cells with concanavalin A(Con A) resulted in a triple increase in the amount of α1AT synthesized by the untreated cells. A small amount of α1AT, equivalent to that synthesized by the unstimulated mononuclear cells, was observed in cultures of monocyte-depleted lymphocytes, with or without Con A stimulation. Monocytes treated with or without Con A scarcely synthesized α1AT. Conditioned media derived from monocyte enriched mononuclear cells treated with Con A enhanced about threefold α1AT synthesis by the Con A-stimulated lymphocytes. α1AT is suggested to be synthesized by lymphocytes assisted by monocytes.

Influence of acute-phase proteins on the activity of natural killer cells

The effects ofα1-antitrypsin (α1,-AT),α1,-acid glycoprotein (α1AGP), and haptoglobin (Hp), the main constituents ofα-globulin and which belong to acute phase proteins, on NK activity were examined using K562 cells as the NK target cells. Among the three proteins,α1,-AT andα1AGP had inhibitory effects on NK activity for “fast target” K562 cells. Theα,-AT preparations having the same protein concentration and a different trypsin inhibitory capacity (TIC) had an equal effect. Althoughα1AT andα1,-AGP equally reduced the NK activity, the mechanism involved in the reduction differed, in that the effect ofα1,-AT directed toward NK cells reduced their binding capacity with the target cells,α1,-AGP probably interacts with a cytotoxic factor secreted from NK cells following effector-target interaction. These studies suggest that each of the acute-phase proteins, which increase following inflammation, inhibits NK cell function by two distinct mechanisms.

Spontaneous remission from acute exacerbation of chronic adult T-cell leukemia

SummarySpontaneous remission without any anti-cancer therapy in a 57-year-old woman with adult T-cell leukemia (ATL) is reported. The patient was referred to our department because of persistent cough and appearance of abnormal lymphocytes in the peripheral blood, and she was diagnosed as having chronic ATL. Eight months later, she was re-admitted because of cystitis, watery diarrhea and worsening of respiratory symptoms with an increase of ATL cells (WBC 31 × 109/l with 56% ATL cells). Acute exacerbation of ATL was diagnosed. Interestingly, antibiotic therapy for the pulmonary and urinary tract infections brought about spontaneous reduction of the ATL cell count. Spontaneous remission of ATL continued for one year without chemotherapy. The role of infection as a trigger of acute exacerbation and spontaneous remission of ATL is discussed.

Nodular regenerative hyperplasia of the liver with portal vein thrombosis and hyperplasia of the adrenal gland

SummaryNodular regenerative hyperplasia (NRH) of the liver is an uncommon entity of unknown origin and pathogenesis. We report here a case of NRH of the liver which was associated with portal vein thrombosis and adrenal hyperplasia. A 48-year-old man who was admitted for further examination of portal hypertension and splenomegaly, died of acute myocardial infarction. Autopsy revealed an enlarged heart with occluded coronary arteries and fresh multiple necrosis in the myocardium. The spleen and the liver were enlarged. In the liver diffusely distributed nodules of regenerative hyperplasia were noted. Organized thrombi of the portal vein and adenomatous hyperplasia of the left adrenal gland were also noted. Antinuclear antibody was positive in the serum.

Expression pattern of mitochondrial ND2 gene in human leukemia and in HL60 cells during growth and differentiation

We differentially screened 5,000 clones from a cDNA library of acute myelogenous leukemia (AML) cell line HL60 using cDNA probes derived from normal granulocytes or from acute myelomonocytic leukemia cells, the objective being to identify genes preferentially expressed in myeloid lineage leukemic cells. One clone, corresponding to a mitochondrial DNA fragment, including NADH dehydrogenase subunit 2 (ND2) gene, was selected for use as a probe. We examined expression of the ND2 gene in various leukemic cell populations and in normal peripheral blood cells. DNA-RNA hybridization studies revealed that ND2 messenger RNA (mRNA) was more markedly expressed in AML cells than in other leukemic cells and normal peripheral blood granulocytes. The expression of ND2 mRNA decreased in HL60 cells several hours after treatment with phorbol myristate acetate (PMA), or dimethyl sulfoxide (DMSO). However, the ND2 gene expression did not depend on the growth-state of HL60 cells because the steady-state level of its expression was observed during transitions of growth. These results suggest that ND2 mRNA is involved in the maturation of myeloid cells and in cellular differentiation, in a lineage-preferential manner. A comparison of the nucleotide sequence of this clone with the documented human mitochondrial DNA sequence revealed several single-base substitutions, insertions and a 39-bases insertion.