Okayama University Medical School

To investigate cellular interactions between human T and B lymphocytes in various diseases, we established a technique to prove terminal differentiation of B lymphocytes into immunoglobulin synthesizing and secreting cells. We also established a double antibody radioimmunoassay to measure the amount of IgG, IgA and IgM synthesized and secreted in culture supernatants. Purified immunoglobulins were obtained from sera of patients with myeloma or macroglobulinemia. The peripheral blood lymphocytes from 25 normal individuals had the geometric mean synthetic rates of 1886 ng for IgG, 1607 ng for IgA and 1173 ng for IgM per 1 X 10(6) cells when cultured for nine days in the presence of pokeweed mitogen. The method is simple and sensitive, and is thought to be useful for examining human lymphocyte function in vitro.

Masugi nephritis was induced in rats by a single intravenous injection of rabbit anti-rat kidney serum, and studied with a scanning electron microscope. A characteristic finding was the presence of white cells, probably polymorphonuclear leukocytes, with many microspikes which penetrated through degenerated glomerular endothelial cells to be in direct contact with the glomerular basement membrane. This finding confirms the pathogenic role of leukocytes in glomerulonephritis induced by anti-glomerular basement membrane antibody.

Thirty-seven consecutive cases of mitral valve replacement have been retrospectively reviewed. The prognostic significance of preoperative clinical, hemodynamic and quantitative angiographic factors for survival has been evaluated. In the Mitral stenosis (MS) group, all of the patients who showed small Stroke volume index (SVI) (less than 45 ml/m2) with pulmonary hypertension died from the low output syndrome. The prognosis was poor in patients who had large cardiothoracic ratio (CTR) in the MS group. Aortic valve replacement must be considered when moderate aortic regurgitation is associated with mitral valve disease. In the MR factors for predicting the survival. The eccentricity ratio is also a sensitive parameter for recognizing a patient who will have a poor prognosis after mitral valve replacement. The main mode of death was found to be heart failure due to myocardial impairment.

Insulin and human erythrocyte cell membrane interactions were studied with respect to binding and dissociation. The per cent of specific binding of 125I-labeled insulin to erythrocytes was directly proportional to the cell concentration. The optimum pH for binding was 8.1. The initial binding rate was directly proportional to, and the steady state insulin binding was reversely proportional to, the incubation temperature. The per cent of specific binding of 125I-labeled insulin was 12.10 +/- 1.13 per cent (mean +/- SD)/4 X 10(9) cells (n = 10) at 0.8 ng/ml insulin. Native insulin competed with 125I-labeled insulin for binding and showed almost complete inhibition at 10(4) ng/ml. The Scatchard plots were upward concave. Maximum binding capacity was 230 binding sites per cell. The average affinity constant decreased as the per cent of fractional occupancy increased. Affinity constants for the empty and filled sites were 1.49 and 0.16 X 10(8) M-1 respectively. Bound insulin was displaced by native insulin. The dissociation rate by "dilution + native insulin" was higher than that by "dilution only". The dissociation rate was accelerated even by the physiological concentration of insulin and maximum at 100 ng/ml. It is concluded that human erythrocytes have insulin binding sites which are indistinguishable from insulin receptors on the target tissues for insulin.

A permeable cell system has been developed by treatment with saponin for studying in vitro replication of DNA and chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in saponin-treated permeable cells was found to be more efficient than that in digitonin-treated permeable cells. Autoradiography of the agarose-gel revealed that [alpha-32P]dCTP was incorporated into SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated complete replication of SV40 DNA and chromatin with a full number of nucleosomes. The saponin-treated permeable cell system will serve as a useful system for studying in vitro replication of DNA and chromatin in eukaryotic cells.

Serum specimens from 12 patients with type A hepatitis were analyzed for immunoglobulin M-type antibody to hepatitis A virus (IgM anti-HA). A recently developed solid-phase radioimmunoassay kit for IgM anti-HA (HAVAB-M, Abbott Laboratories) and a competitive binding radioimmunoassay kit (HAVAB, Abbott Laboratories) with or without 2-mercaptoethanol treatment, as modified by Yano et al. (Acta Hepatol. Jpn. 21, 704-712, 1980) were used to obtain an M-index. All specimens obtained within 60 days of the onset of illness and specimens from 2 of 4 patients later than 60 days after the onset were positive with the HAVAB-M test. This test gave negative results to sera which were positive for anti-HA by a standard HAVAB test in the following: 3 patients with type B hepatitis; 5 with non-A, non-B hepatitis; 11 healthy adults; and 10 sera strongly positive for rheumatoid factor. The M-index for type A hepatitis in sera within 30 days of the onset (mean value of the M-index, m, = 1.52; standard deviation, SD, = 0.25) was significantly higher than that for non-A hepatitis (m = 1.05; SD = 0.15) and for healthy adults (m = 1.02; SD = 0.10). The simplicity and usefulness of the HAVAB-M test in diagnosis of acute type A hepatitis over those measuring the M-index by HAVAB tests were shown by direct comparison of the results.

<p>A female patient who died of apparent postradiation sarcoma in the inguinal region after irradiating a metastatic squamous cell carcinoma of the same site was reported. For approximately 20 months, the patient had received a total of 6,600 and 9,600 Roentgen to the right para-aortic and inguinal areas, respectively. About 10 years later, she developed a sarcoma, namely a malignant fibrous histiocytoma. Sputum cytology demonstrated numerous giant cells with bizarre nuclei; subsequent chest films also presented apparent metastatic tumor shadows. The cellular characteristics and also rather low incidence of detection of nonepithelial malignant tumor by sputum cytology were briefly discussed, and ways of enhancing cytodiagnostic accuracy were proposed.</p>

Using a direct immunofluorescent method, histological locations of immunoglobulins (IgG, IgM, IgA and IgD of heavy chain, and kappa and lambda of light chain) and complement components (C3 and C4) were studied in 78 brain tumors, which included 24 astrocytomas, 6 metastatic tumors, 5 medulloblastomas, 4 malignant lymphomas, 15 meningiomas, 8 schwannomas, 8 pituitary adenomas, and 8 other miscellaneous brain tumors. IgG-positive cells were observed in the perivascular regions of astrocytomas, but were more marked in those of high grade, metastatic tumors and meningiomas. Malignant lymphomas demonstrated IgG and IgM-positive cells accompanied by either kappa of lambda light chains. C3 and C4 were much less evident in these tumors. Pituitary adenomas showed slight positive stains for both immunoglobulins and complement components on the blood vessel walls, Immune reactions against brain tumors were discussed including the clinical application of autologous lymphocyte infusion in malignant gliomas and combination chemotherapy in intracranial malignant lymphomas.

<p>The kinetics in tumor cells and various factors affecting the tumor accumulation of 67Ga-citrate and 201Tl-chloride were studied in vitro. 67Ga was taken up gradually by tumor cells and its excretion from the cells decreased with time. 201Tl was taken up rapidly by tumor cells. Its excretion was very rapid, indicating that the two nuclides had entirely different kinetics in tumor cells. The uptake of 201Tl by culture cells correlated with that of 42KCl and was inhibited by Ouabain. 201Tl was hardly taken up by nonviable tumor cells. These facts indicate that active transport involving Na-K ATPase is involved in the tumor accumulation of 201Tl. The uptake of 67Ga and 201Tl by tumor cells was not affected by the administration of anticancer agents. The uptake of 67Ga by tumor cells was dependent upon the concentration of transferrin in the medium, which apparently plays a role as one of the pathways of tumor accumulation of 67Ga.</p>

Effect of nicomol on high density lipoprotein (HDL) subfractions, HDL2e and HDL3e, separated by electrophoresis.

Partial excess of chromosome 1 (q25-q32) was noted in malignant cells from all of 10 patients who had disorders such as non-African Burkitt's lymphoma, adult T-cell leukemia, myelofibrosis, malignant lymphoma, chronic lymphocytic leukemia or chronic myelocytic leukemia in blast crisis. The break points on chromosome 1 were at centromere, q12, q21, q23, q25 and q32. Variations in the specific region of the long arm of chromosome 1, q25-q32, were thought to be important in the evolution of malignant cell proliferation.

Cholestasis with htperbilirubinemia was induced in female, but not male, Sprague-Dawley rats by daily treatment with phalloidin for 7 days. Increases in serum direct bilirubin level and alkaline phosphatase (Al-Pase) activity were observed concomitantpy with diminished bile flow and a decreased output of bile acid and cholesterol. Kidht microscope findings of the liver revealed proliferated bile ductules and enhanced mitosis of hepatocytes.

Alpha-getoprotein (AFP) was purified from fetal rat serum by a combined technique of affinity chromatography with Affi-Gel Blue and disc electrophoresis followed by extraction of AFP from the gel. The purified AFP was immunologically identical with the original AFP in fetal rat serum.

Most of the common organic solvents are excreted into urine as metabolites. A correlation exists for several organic solvents between the amount taken in and the amount of metabolites excreted. Many methods have been developed for the measurement of these urinary metabolites. The methods were classified into three group: colorimetry, gas chromatography and high performance liquid chromatography. The characteristics and availability of these methods in a laboratory for routine work were reviewed. The correction equation for the amounts of metabolites in spot urine is discussed.

Cholestasis with hyperbilirubinemia was induced in female, but not male, Sprague-Dawley rats by daily treatment with phalloidin for 7 days. Increases in serum direct bilirubin level and alkaline phosphatase (Al-Pase) activity were observed concomitantly with diminished bile flow and a decreased output of bile acid and cholesterol. Light microscope findings of the liver revealed proliferated bile ductules and enhanced mitosis of hepatocytes.

Papular acrodermatitis of childhood (PAC) has recently been reported to be associated with hepatitis B surface antigen (HBsAg) subtype ayw. Between September, 1978, and June, 1979, we saw 14 patients with PAC in a small epidemic occurring in Iwakuni City, Japan. HBsAg was detected in sera from all patients. Subtyping of HBsAg in 11 patients showed that 8 had a determinant adr and 3 had no detectable determinant because of low antigen titers. The result suggests that factors other than the specific HBsAg subtype contribute to the development of PAC.

In rats, microinjection of FeCl3 solution into the left sensorimotor cortex was performed to induce a chronic epileptic focus. One month or more after the microinjection, electrocutaneous stimuli were applied to part of the wrist joint and 50 consecutive somatosensory evoked potentials (SEPs) were averaged. SEP from the left cortex showed only an initial negative monophasic deflection while SEP from the contralateral cortex showed a normal configuration with initial positive-negative biphasic deflection in the majority of experimental animals.

The usefulness of liposomes (in neutral, positively and negatively charged forms) as a carrier for adriamycin (ADM) was studied by examining the distribution of ADM and related fluorescent compounds in Ehrlich solid tumor-bearing mice. The mice were given free or liposome-entrapped ADM intraperitoneally. The distribution of ADM and related fluorescent compounds between the administration of the free form and liposome-entrapped form was measured by high performance liquid chromatography : The distribution was dependent on the form of the liposomes. The amounts of ADM and its metabolites in the mouse serum 20 min after administration of neutral-liposome-entrapped ADM were 10 times those after the administration of free ADM, 6 times those after the administration of a negatively charged form, and 3.5 times those in the administration of positively charged form. There was no marked difference in the concentrations of these compounds 5 h after administration. The concentration of these compounds in the liver 60 min after administration of each liposome-entrapped form of ADM were in inverse correlation with the concentrations in the serum obtained at 20 min after administration. Total concentrations of ADM and its metabolites in the tumors 20 min after administration of each entrapped form of ADM were 4-5 times that in administration of free ADM after 20 min. There were no marked differences in the concentration of ADM for administration of the various liposome forms. Statistically significant decreases in mean tumor weight were seen in the groups given neutral, positively and negatively charged liposome-entrapped forms compared to corresponding control groups given with free ADM.

To investigate the action of motilin on the sphincter of Oddi, the flow rate of the perfusate (FRP) discharged into the duodenal lumen through the orifice of the common bile duct was measured by means of an electric drop counter in decerebrated dogs. Motilin in doses above 0.5 micrograms/kg i.v. reduced or stopped the FRP. The fifty percent recovery time of FRP was 20 min and full recovery time was 30 min. The reduction of FRP induced by motilin was unaffected by denervation and atropinization. These results suggest that motilin caused an increase in tone of the sphincter of Oddi by acting on the sphincter muscle.