A new method for the measurement of complement hemolytic activity of undiluted sera has been developed by using kinetic assay. The complement hemolytic activity was evaluated by the time which required to cause 50% reduction of the initial turbidity, and expressed as CHT 1/2. It seemed that CHT 1/2 reflected the activities of both complement components, mainly early components, and inhibitors and/or inactivators, while CH 50 reflected mainly the activities of late acting components of complement.
Undiluted sera from guinea pigs showed more prolonged CHT 1/2 than that from human beings, in contrast to possesing higher CH 50. Dilution of guinea pigs sera shortened CHT 1/2, suggesting the existence of large amount of inhibitory proteins. This complement kinetic assay using undiluted sera is a completely new concept, and may provide useful information in patients with various diseases clinically.

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Preparation of dendritic cells were obtained from a relatively small amount of human peripheral blood. These isolates contained from 30 to 60% of dendritic cells proving with the morphological characteristics of human lymphoid dendritic cell. By using monoclonal antibodies, they seemed to possess HLA-DR antigen and C3bi receptor. These preparations were compared with macrophage preparation and B cell preparation as a stimulator in autologous mixed lymphocyte reaction (auto-MLR). Auto-MLR was quantitated by the ³H-thimidine incorporation.
The dendritic cell preparation provided the most potent stimulus for T cell proliferation in auto-MLR. On the other hand, macrophage preparation and B cell preparation were a little or not stimulous for auto-MLR. These data suggested that dendritic cell was major stimulator in auto-MLR.
T cell proliferation in auto-MLR was reduced by adding anti-OKDR monoclonal antibody. Dendritic cell was also stimulatory cell in allogeneic MLR.
Our results would be able to apply the clinical investigation of dendritic cell function in some patients with depressed auto-MLR.

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We studied the opsonizing activity to Streptococcus pneumoniae type I (Sp) in 7 patients with the immunoglobulin replacement therapy. They are one patient with symptomatic IgG2 subclass deficiency and 6 patients with common variable immunodeficiency. Using luminol dependent chemiluminescence (CL), we measured the maximum (peak) cpm of CL with polymorphonuclear leukocytes (PMN) isolated from a single normal adult and stimulated with opsonized Sp's.
The results of CL correlated with the immunoglobulin level of the same serum in cases with normal states but not with infectious state of the respiratory tract and with IgG2 subclass deficiency. And also low CL correlated more closely with infectious states than the immunoglobulin levels of each patients.
Our results suggest that the CL under our condition can measure more directly the antipneumococcal opsonizing antibody in vivo than the semi-quantitative assay of spesific antibody as enzyme-linked immunosolbent assay, and is a useful aid at the screening of IgG2 subclass deficiency.
We suggest that the opsonizing activity to Sp is more useful than the levels of serum immunoglobulin in setting the schedule of replacement therapy, especially to prevent and cure the bacterial respiratory tract infections in the patients with hypogammaglobulinemia.

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Sera from 160 patients with hepatocellular carcinoma (group I) were examined for antinuclear antibodies (ANA) by indirect immunofluorescence using 3 types of cultured cells as substrate: human hepatoma cell (PLC/PRF/5), human larygeal carcinoma cell (HEp-2) and baby hamster kidney cell (BHK). Sixty six sera from patients with non-hepatic cancers (group II), 62 sera from patients with chronic liver disease (group III) and 235 sera of normal subjects (group IV) were tested as controls. The incidences of ANA which was detected in HEp-2 or PLC/PRF/5 but not in BHK were as follows, 36 patients (22.5%) in group I, 19 patients (27.5%) in group II, 12 patients (19.4%) in group III and only 1 case (0.4%) in group IV. Reciprocal titers of ANA that were positive against HEp-2 but negative against BHK in group I or II were higher than in group III or IV. Two sera of patients with hepatocellular carcinoma reacted with nuclei of HEp-2 and PLC/PRF/5 were investigated further for ANA by indirect immunofluorescence using other 4 types of transformed cells and 4 types of non-transformed cells. We observed that the ANA in these two sera reacted only with transformed cells differed from that in sera of patient with SLE. It was suggested that the ANA reacting only with nuclei of transformed cells was present in sera of patients with hepatocellular carcinoma. However, the nature of nuclear antigens against the antibody remains unidentified.

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Complement hemolytic activity (CH 50) in the sera from patients with malignancies has been reported to increase with the progression of the disease. However, C_??_ inhibitor (C_??_INH) and other protease inhibitors also increase in such conditions. In this study, we examined the control proteins of the complement system, C_??_INH, C4 binding protein (C4bp), factor I (I) and factor H (H), in 254 sera from patients with various kinds of malignancies in relation to CH 50.
Patients were classified according to the stage of the diseases. CH 50 was measured by the method of Mayer employing sensitized sheep erythrocytes. Protein concentrations of the complement components, C_??_INH, C4bp, I and H were determined by a single radial immuno-diffusion method. Pseudo-cholinesterase (ChE) activity was employed as a marker of protein synthesis, and patients in stage IV+V were divided into two groups according to their ChE levels.
CH 50 gradually increased as the disease progressed and the elevation in stage IV+V was significant from normal subjects. Complement components C4, C3, C5, C7, C8 and C9 were correlated with CH 50. The protein concentrations of C_??_INH, C4bp, I and H also increased as the disease progressed, and all of 4 control proteins showed significant elevations in stage IV+V. There were significant correlation between CH 50 and all these control proteins.
When patients in stage IV+V were subdivided according to their serum ChE levels, the patients with ChE values over 2.00I. U./l showed further elevations of CH 50, C4bp, I and H. On the other hand, patients with ChE values below 2.00I. U./l revealed significantly low CH 50, C4bp, I and H.
Although CH 50 in sera of patients with malignancies increased as the disease progressed, the control proteins of the complement system also increased. It was suggested that the activation of the complement system in vivo could be interrupted by these elevated control proteins, and the state with increased levels of these control proteins seemed to be disadvantageous for the host defense against malignancy.

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The peripheral blood mononuclear cells (MNC) were obtained by Conray-Ficoll gradient sedimentation method from 24 patients with rheumatoid arthritis (RA) and 30 healthy donors as controls. The percentage of Bl antigen positive cells in MNC was examined with mouse monoclonal antibody. And MNC were cultured with or without stimulation of Pokeweed mitogen (PWM, 10μl/ml) for 7 days. After culture, immunoglobulin (Ig) secreting cells (ISC) and the amount of Ig secreted in supernatants were simultaneously measured using reverse hemolytic plaque assay and enzyme-linked immunosorbent assay (ELISA) respectively.
The percentage of B1-positive cells in MNC of RA patients was not significatnly different from controls. The numbers of both spontaneous and PWM-induced ISC in RA patients were significantly lower than controls. The amounts of IgG and IgA secreted in culture supernatants after 7 day-culture with addition of PWM in RA patients (IgG; Mean±SEM=618±108ng/me, n=20: IgA; 382±104) were not significantly different from controls (IgG; 684±93: IgA; 538±119, n=22). On the other hand, the amount of IgM secreted in RA patients (438±92) was significantly lower than controls (868±100), although the amount of total Ig secreted in RA was not different from controls. In addition, when the amount of Ig secreted in culture supernatants was divided by the number of ISC in stimulation of PWM, measured simultaneously, it became clear that the amount of Ig produced by a single ISC was significantly larger in RA patients than controls.

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A rapid and automated method of an enzyme linked immunosorbent assay (EIA) was devised for the detection of antibodies to adult T-cell leukemia associated antigens (ATLA, HTLV-I), by the combined use of HTLV-I coated microcup E-0773 (Eisai) and Behring ELISA Processor II (Hoechst). Comparing to the ordinary method of EIA using the Corty series (Toshiba Co. Ltd.), the results were correlated well with “Five times” of washing program.
The absorbance of twenty sera of SLE, ten sera of ATL and ten normal sera were elevated variously by the inactivation with heat treatment (56°C, 30min.), however, only ATL sera were inhibited specifically by the absorption test with virus antigen. Furthermore, eighteen out of 1354 sera which were inactivated by heat treatment were also inhibited specifically with the inhibition rate over 1.0 O.D. in all of them.
HTLV-I carriers were 1.6% of all patients examined and were detected highly in Parkinsonism, polyneuritis, hepatoma, esophageal cancer, polyarthritis, and blood diseases. An appearance of atypical lymphocytes (50%), and myelocytes (48%) in the peripheral blood were remarkable in these carriers. By the Western blot method P-40 were detected in five cases.

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The effects of sera from 20 patients with acquired immunodeficiency syndrome (AIDS) and 33 patients with AIDS-related complex (ARC) on human immunodeficiency virus (HIV-1) reverse transcriptase (RT) were studied. The results of RT assays showed strong inhibiting and enhancing effects of some of these sera on HIV-1 RT activity in vitro. The modulating effects were not observed in assays containing sera taken from HIV-1 uninfected homosexuals, suggesting that the inhibitory and enhancing phenomena are associated with HIV-1 infection. The present study has focused on the inhibitory function of the sera. Kinetic enzymatic study showed that the inhibitory sera contained one or more heat-resistent (56°C, 30min) inhibitors, which display a high affinity for protein A. These results suggest that the inhibitor (s) is probably an anti-HIV-1 RT antibody that inhibit RT activity. Further, sera were tested for the presence of neutralizing activity against HIV-1 infection. For the neutralizing activity assay, HIV-1 infection of human T cell line (H 9) was assessed by monitoring DNA synthesis of H 9 cells, viable cell counts, HIV-1 gag protein expression, and RT activity in the supenatant. The RT inhibitory antibody seems to contribute partially to the sera's neutralizing activity against HIV-1. Sera from AIDS subjects that displayed RT inhibiting effects showed significantly greater neutralizing activity that sera displaying RT enhancing effects or no effects. The existence of an antibody that inhibits RT activity suggests a novel approach to the development of antiviral agents, and its apparent correlation with neutralizing capacity may present new possibilities for anti-HIV-1 vaccines. In addition, the recognition of this inhibitory phenomenon brings to light potential methodological problems involving both clinical and experimental uses of RT assays.

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Rat mast cells purified according to a Percoll gradient method were challenged with compound 48/80 or calcium ionophore A 23187 and the effect of Staurosporine, a novel protein kinase inhibitor, on histamine release from the cells was investigated.
Mast cells were treated with Staurosporine for 5min prior to the challenge at 37°C and the mixture was further incubated for 1min. After stopping the reaction, the amount of histamine in the supernatant was measured with the fluorometric assay.
Histamine release induced by compound 48/80 or calcium ionophore A 23187 was inhibited by Staurosporine in a concentration-dependent manner at 0.01 to 1μM and 0.01 to 1μM, respectively. Staurosporine at the concentrations of 0.1 and 1μM significantly inhibited compound 48/80 and calcium ionophore A 23187-induced histamine release.
K-252 a, an another protein kinase inhibitor, also inhibited compound 48/80 and calcium ionophore A 23187-induced histamine release from the cells. The inhibitory effect of K-252a was significantly stronger than that of Staurosporine on calcium ionophore A 23187-induced histamine release.
These results suggest that protein kinase may play some role in the Sp process during mediator release from rat mast cells.

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We describe a 15 year-old girl with autoimmune hemolytic anemia and thrombocytopenic purpura (Evans syndrome) associated with subclinical Sjögren's syndrome.
On admission, she was anemic (Hb 7.1g/dl, reticulocytes 144‰) and had jaundice (T. bil. 3.2mg/dl, D. bil. 0.2mg/dl). Petechial hemorrhages were demonstrated on her lower extremities and chest wall. Platelet count was 2.0×10⁴/μl, and serum LDH was increased (798U/l). Increased megakaryocytes and erythroid hyperplasia were identified in the bone marrow. Serum Fe was 77μg/dl, and haptoglobin was markedly decreased (5mg/dl). Both direct and indirect Coombs' tests were continuously negative. ANA was positive at 1:80 (speakled pattern), but anti-DNA antibody and anti-ENA antibody were negative. Anti SS-A antibody was possitive at 1:16, but anti SS-B antibody was negative. Ham test, sugar water test and Donath-Landsteiner antibodies were all negative. Osmotic fragility test, RBC enzymes and Hemoglobin analysis were normal. RBC life span (⁵¹Cr-method) was 7.3days (normal: 25_??_35days). Parotid sialography revealed diffuse sialectasis, and both lacrimal and salivary secretions determined by Schirmer test or Gum test were decreased. Keratoconjunctivitis sicca was demonstrated by the rose-bengal test.
She was diagnosed as Evans syndrome with Sjögren's syndrome, and treated with prednisolone, 40mg daily. Both hemolytic anemia and thrombocytopenia responded to the treatment.
This is the first case report of Coombs' test-negative Evans syndrome associated with Sjögren's syndrome.

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The lymphocytes obtained from a patient with chronic T-cell lymphocytosis were analyzed. The patient has no clinical symptoms or sings during our 8-year observation. The lymphocytes showed the typical feature of large granular lymphocytes (LGLs) morphologically. They had peculiar phenotype different from that of LGLs previously reported. The surface markers of these lymphocytes were CD2⁺, CD3⁺, CD4⁺, CD5⁺, CD8^-, CD11⁺, Leu7⁺, CD16^- and FcIgG^-, but these cells had neither helper nor suppressor action on IgG production by normal B lymphocytes. Natural killer activity of freshly isolated cells could not be detected even after augmentation with interleukin 2 (IL2). Lymphokine activated killer activity could not be detected, either. These cells were able to proliferate upon stimulation with such mitogens as PHA and PWM, and to produce IL2. But they were not induced to proliferate by IL2 even if stimulated with ConA beforehand. Almost all cells were in the resting state according to the cell cycle analysis.
The LGLs of this case are a novel subset of LGLs in both phenotype and immunological function. Only a few cases of CD4⁺ LGL lymphocytosis or leukemia have been reported previously, but the further phenotype and the function of these cases varied. The results show that, even in CD4⁺ LGLs, there may be heterogeneous subpopulation.

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A rare case with systemic lupus erythematosus (SLE) accompanied by normal pressure hydrocephalus (NPH) and an unruptured aneurysm was reported. The relationship between clinical features and complications were discussed.
A 43-year-old female with a 11-year history of SLE was admitted to our hospital for evaluation of transient ischemic attack in December 1982. When she was admitted, she had butterfly rash, oral ulcer, nephritis, leukocytopenia and hypocomplementemia. The titers of antinuclear antibodies and anti-DNA antibodies elevated. There were no neurological abnormalities. The lumber puncture yielded clear, colorless cerebrospinal fluid under an opening pressure 130mmH₂O; the fluid contained normal protein, glucose and cell values; antinuclear antibodies and anti-DNA antibodies were not detected. The electroencephalogram findings showed a mixture of slow waves. The computed tomography revealed marked ventricular enlargement. RI cisternography showed abnormal reflux to the ventricle and a greatly prolonged clearance time. Cerebral angiography demonstrated a saccular aneurysm at the junction of the right median and the striate arteries. A neck clipping operation was performed in February 1983. There was no evidence of thickned leptomeninges or fibrosis of the arachnoid villi. The biopsy tissue demonstrated the degeneration of nerve cells and proliferation of oligodendrocytes, which may be due to ischemia. There was no evidence of angitis with deposition of immune complexes. The operative therapy for the hydrocephalus was not performed and the patient is under careful observation.

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