Recently, monoclonal antibodies and immunofluorescence analysis by flow cytometry provide a highly sensitive and specific approach to phenotypic analysis of human mononuclear cell subsets. On this point of view, flow cytometric analysis using monoclonal antibodies to various cell surface antigen have been usefull for immunomonitoring of various autoimmune disorders.
In this report, clinical utility of lymphocyte subsets such as CD4⁺, CD8⁺ T cells, Ia⁺ T cells, Leu-1 B cells and CR 1⁺ cells were described. For example: the CD4⁺ subset contains helper/inducer T lymphocytes, and the CD8⁺ subset contains suppressor/ cytotoxic T lymphocytes: Ia⁺ T cells contains activated T cells: Leu-1⁺ B cells have been usually found in B-CLL and these B cell subsets are elevated in RA and secrete IgM-RF: The CR 1⁺ cells reflect diverse complement levels and kinetics: Examination of these cell subset may give rise to beneficial mean to estimate immunological kinetics of autoimmune disorders.

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The serum SOD values were measured by using enzyme-linked immunosorbent assay and immunohistological investigation of SOD was evaluated in alcoholic liver injury (ALI) .
Serum Cu, Zn-SOD and Mn-SOD exhibited an inclination to increase as the releasing enzymes, revealing especially high values for ALI. Mn-SOD proved more useful for clinical diagnosis of liver disease than Cu, Zn-SOD due to its significantly higher values.
The localization modes of hepatic SOD were mainly the liver cytoplasm diffusion type in ALI. Immunohistological frequency of Cu, Zn-and Mn-SOD localization modes were as follows: Corresponding values of Cu, Zn-SOD and Mn-SOD were 63.2% and 52.7% in cytoplasm diffusion type, 42% and 0% in nuclear diffusion type, 42.1% and 0% in vacuolated membrane type, 15.8% and 0% in small granulosum type respectively.
A close relationship was observed between ALI and immunohistological frequency of SOD in these studies, a particular protective physiological role of SOD against activated oxygen was strongly postulated.

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We treated the noncancerous and cancerous patients with severe infections using the COG-78 and antibiotics, and determined the absolute numbers of neutrophils, lymphocytes, lymphocyte subpopulations and serum concentrations of immunoglobulins. (1) In the noncancerous patients, the numbers of neutrophils and the N/L ratios decreased to normal ranges, in contrast, in the cancerous patients, these parameters increased throughout the test periods. (2) The OKT₄/T₈ ratios of the noncancerous patients were always higher than those of the cancerous patients. (3) In both noncancerous and cancerous patients, the numbers of lymphocytes decreased at the sixth day after the start of treatment but the numbers of active T cells increased throughout the test periods. (4) The cancerous patients who manifested poor prognosis showed markedly increased numbers of neutrophils and high N/L ratios. (5) In the noncancerous patients, the serum concentrations of IgG increased after administration of COG-78, on the contrary, in the cancerous patients, the concentrations of IgG decreased.

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Interleukin-1 (IL-1) mediate immunological, physiological, and metabolic changes associated with inflammation and host defense system. Measurement of IL-1 activity can vary with the bioassay employed and substances have been described in several bioassay systems that either inhibit or mimic IL-1 activity. We report a sensitive and specific enzyme-linked immunoassay (ELISA) for human IL-1α and IL-1β. IL-1 monoclonal and polyclonal antibodies were used to develop sensitive sandwich ELISA assays. The IL-1 ELISA assay detects 10 pg/ml of recombinant human IL-1α and 100 pg/ml of recombinant human IL-1β. We have used this assay to measure IL-1 in a cell supernatant of stimulated human blood mononuclear cells which contained substances which interfere with in vitro and/or in vivo IL-1 biological assay. The IL-1 ELISA assay could discriminate between IL-1α and IL-1β, which the bioassay can not, and were faster and easier to perform than bioassay. Sensitive IL-1 ELISA assay can be used to identify and quantitate the amount of IL-1 synthesized under a variety of clinical and experimental conditions in the presence of other cytokines.

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Antigen-induced arthritis (AIA) is produced by intra-articular injection of antigen in previously immunized animals. To study the role of allergic reactions of chronic synovitis in rheumatoid arthritis (RA), AIA was induced in mice using heat-killed E. coli 0: 14 (E. coli group) and egg albumin antigen (ovalbumin group), and immunological comparative studies between both groups were undertaken using mouse monoclonal antibodies including anti-macrophage (Mac-1), anti-Lyt-1, and anti-Lyt-2. Within 1 day after challenging injection in both groups immunological tissue damages by immune complex had occurred and exudative inflammations were observed suggesting Arthus reaction. At three days after challenging injection a local delayed hypersensitivity reaction recognized by the method of macrophage migration test and immunohistological examinations, and subsequent proliferative synovitis was observed in only E. coli group but not seen in ovalbumin group. Thereafter, persisting chronic synovitis with pannus formation was seen. These studies suggest that cytokines play an important role for establishment of persistent chronic arthritis like RA.

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Anti-DNA antibodies were released in the culture supernatant of spleen cells from BALB/c and autoimmune NZB/W F₁ mice by the addition of LPS. The authors investigated the effect of several immunomodulators and immunosuppressants on the in vitro production of anti-DNA antibody. It was demonstrated that the anti-DNA antibody production in both mouse strain was not reduced by levamisole (LMS), bucillamine (SA96), and lobenzarit disodium (CCA), all which augmented the in vitro anti-SRBC PFC response. On the contrary, mizoribine, salazosulfapyridine (SASP), and platonin (NK-19) inhibited both anti-DNA antibody production and anti-SRBC PFC response. Mizoribine and SASP reduced the viability of cultured splenocytes, but NK-19 didn't. The results shows that every anti-rheumatic drug tested had a diffent action on the anti-DNA antibody production, suggesting the importance of further studies for their clinical application.

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We investigated the inter-relationships of superoxide (O^-₂) release, membrane depolarization and an increase in cytoplasmic free Ca²⁺, [Ca²⁺] i, in human granulocytes stimulated by various agonists. When concanavalin A (Con A) or the Ca²⁺ ionophore ionomycin was used as stimulus, an increase in [Ca²⁺] i clearly preceded the onset of membrane depolarization, which was followed by O^-₂ release. O^-₂ release and membrane depolarization stimulated by Con A, N-formyl-methionyl-leucyl-phenylalanine or ionomycin were markedly potentiated in parallel by pretreatment of cells with a low concentration of Phorbol myristate acetate (PMA, 0.25 ng/ml), whereas an increase in [Ca²⁺] i was not affected or minimally potentiated. The lag time between addition of the stimulus (Con A or ionomycin) and onset of membrane depolarization or O^-₂ release was significantly reduced by pretreatment of cells with PMA, whereas the lag time between addition of Con A and onset of the increase in [Ca²⁺] i was not affected. These findings suggest that (a) an increase in [Ca²⁺] i stimulates membrane depolarization indirectly; (b) a low concentration of PMA potentiates membrane depolarization and O^-₂ release by acting primarily at the post-receptor level, in particular, at the level distal to an increase in [Ca²⁺] i, but not by augmenting an increase in [Ca²⁺] i.

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Scleroderma (PSS) is characterized by diffuse accumulation of collagen and vascular change in the skin and in a variety of internal organs. We planned to evaluate the role of monokine in the pathogenesis of fibrosis in scleroderma. Monokine, the supernate of LPS-stimulated plastic dish adherent mononuclear cells, stimulated the proliferation of fibroblast detected by ³H-thymidine incorporation. This activity of scleroderma monokine was greater than that of normal monokine.
Collagen secretion determined by the method of Peterkofsky and Diegelmann was inhibited by monokines. This inhibitory activity of scleroderma monokine was lower than normal one. These results may indicate that monokine contributes to the increased collagen accumulation observed in scleroderma.
Based on these findings, the immunomoregulatory drugs were used to regulate fibrosis of scleroderma. D-penicillamine was most frequently used for patients with severe fibrotic features. Actions of this drug may be suppressive effects on mononuclear cells (activated T cell), fibroblast and collagen cross linking.

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H₂O₂ production by neutrophils was measured by single cell analysis using flow cytometry (Epics-C) . This method is very useful for clinical evaluation of patients especially in pediatrics, because it requires only a little volume of blood (about 0.1 ml) and time less than an hour.
Cells were preincubated for 15 min with 5μM 2', 7'-dichlorofluorescin diacetate, which diffused into cells and were trapped within the cells. The cells, added stimulant such as phorbol myristate acetate, were incubated another 30 min. After lysing red blood cells by hypotonic shock, cells were washed and resuspended into PBS, and introduced to flow cytometry. The fluorescent intensity was partially correlated with H₂O₂ production by each neutrophil.
In case of carrier of chronic granulomatous disease there were two subpopulations of neutrophils, one was normal responding cells and the other was non-responding. Cells from septic patients revealed populations of neutrophils with increased oxidative responses.

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Electron spin resonance (ESR) is regarded as the least ambiguous method for the detection of free radicals. Using spin trapping technique with 5, 5-dimethyl-pyrroline-N-oxide (DMPO) we measured superoxide generated by stimulated polymorphonulcear leukocytes (PMN) . The results were compared to those by chemiluminescence study with Cypridina luciferin analog (CLA) which is highly sensetive to superoxide. ESR spectrum was obtained by JEOL-JES-FE2XG ESR spectrometer. The intensity of the signal of DMPO-OOH adduct was measured as ratio to the intensity of Mn²⁺ signal. Phorbol myristate acetate or opsonized zymosan were used as stimulants of PMN. The ESR signal of DMPO-OOH was completely inhibited by superoxide dismutase, but not affected by catalase or sodium azide. The relative intensity of DMPO-OOH signal and the maximal increase of CLA-dependent chemiluminescence were increased in proportion to the xanthine oxidase unit in hypoxanthine +xanthine oxidase system, and to the cell concentration in PMN system. There were positive correlation between the relative intensity of the ESR signal of DMPO-OOH and CLA-dependent chemiluminescence by stimulatd PMN from patients. By ESR assay, increased generation of superoxide by PMN in patients with fairly controlled diabetes was shown.

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We evaluated the optimal conditions for stimulating leukotriene B₄ (LTB₄) production in whole blood by zymosan. And the production of LTB₄, LTB₄ isomers and LTB₄ metabolite in 19 healthy donors was compared between the stimulated whole blood method by zymosan or Ca ionophore A 23187 and the separated polymorphonuclear (PMN) method by Ca ionophore A 23187.
(1) For an optimal stimulation in whole blood, EC 50 value for zymosan was 200μg/ml (whole blood), reaction time was about 20 minutes.
(2) Whole blood method was characterized by lower amount of LTB₄ isomers production (about 10% of the amount of LTB₄ production), compared with separated PMN method.
(3) The maximal amount of LTB₄ produced in whole blood method by zymosan was much higher than that in separated PMN method by zymosan (from literature) .
(4) There was a poor correlation between the LTB₄ production (ng/10⁶ PMN) in whole blood by zymosan or Ca ionophore A 23187 and that in separated PMN method by Ca ionophore A 23187, although there was a moderate correlation between the LTB₄ production (ng/10⁶ PMN) in whole blood method by zymosan and that in whole blood method by Ca ionophore A 23187.
(5) There was almost no correlation between the LTB₄ production (ng/ml) in whole blood by zymosan or Ca ionophore A 23187 and the PMN numbers in whole blood.

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We examed the peripheral leukocytes of an 18 year old female case of cytochrome c oxidase (CCO) deficiency, a type of mytochondria myopathy. As neutrophil function test, chemotaxis (Table 2), exocytic release of myeloperoxidase (Table 3), yeast phagocytosis (Table 4), chemiluminescence (Fig. 1) and superoxide generation (Table 5) were measured. Almost all of neutrophil function test of patient were normal. But chemotaxis was better than control. We tested also quantative nitroblue tetrazolium (NBT) reduction test by Baehner using peripheral neutrophils. NBT reduction of patient's neutrophil was ca. 50% of control. This value is almost same as the carrier of chronic granulomatous disease (CGD) (Fig. 2) . By the way, we tried to diagnose CCO deficiency using peripheral leukocytes, instead of the biopsied muscle. CCO activity of isolated lymphocytes from peripheral blood of patient showed only ca. 5% of control (Table 1) . We conclude that it will be possible to diagnose CCO deficiency using peripheral leukocytes.

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The effect of one of Kampo prescriptions, Tsumura Juzentaihoto (TJ-48), on phagocytes was investigated in BALB/c mice.
The oral administration of TJ-48 for 3 to 5 days resulted in the enhanced phagocytic activity of peritoneal exudate cells (PEC) and bone marrow cells (BMC) and the maximum increase was observed at 2 g/kg/day of TJ-48. The enhanced phagocytosis was maintained for 5 days after the withdrawal of TJ-48 and returned to the basal level on day 7. A large increase of chemiluminescence was obtained in PEG from mice given TJ-48 orally for 7 days. Moreover, TJ-48 showed the direct activatingeffect on the phagocytosis of PEC and BMC in in vitro assay.

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The distribution and characteristics of renal prostaglandin E₂ (PGE₂) binding sites were examined in the normal and aminonucleoside-induced nephrotic rats by Scatchard analysis.
PGE₂ binding site density in the medulla of normal rats was 2.7-fold higher than that in the cortex, but there was no significant difference in the dissociation constants (Kd) between cortex and medulla.
The Kd in the kidney medulla of the nephrotic rats were significantly higher (P<0.05) than that of normal rats, while the densities remained without changes.
These results indicate that PGE₂ binding sites of the normal rat kidney are distributed in the medulla more than in the cortex, and the affinities of PGE₂ binding sites in the nephrotic rat kidney are lower than those in normal rat kidney.

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We have already reported an efficient method for simultaneous and quantitative determination of plasma PGs from bronchial asthmatic patients using reversed-phase HPLC. In the present study, the method was arranged and more sensitive determinations were obtained. Standard PGs were incubated with 9-anthryldiazomethane for one night, loaded on ODS column, and fluorescence was measured by the fluorometer. The addition of acetate into the eluate sharpened the peaks of TXB₂ and 6-keto PGF_1α. To separate residual interferences from PGs, samples were eluted with 75% (v/v) methanol/25% (v/v) water in the presence of 1.5% (w/v) acetate. The quantitation of 10 pg PGE₁, PGE₂, PGF_2α, 20pg 6-keto PGF_1α, 50pg TXB₂, and 60pg PGA₂ became possible with these methods.

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Myofibroblasts (MFb) were isolated from scar tissue in rat oral mucoperiosteum and cultured in vitro. Collagen synthesizing activity in the MFb was compared with that in fibroblasts (Fb) derived from normal tissue. In the present study, collagen synthesizing activity in MFb was higher than that in Fb. On the other hand, no significant difference was found in non-collagen protein synthesizing activity between MFb and Fb. As a result, the ratio of the synthesizing activity of collagen to that of total protein was higher in MFb than Fb. Collagen type analysis showed that Type III/Type I ratio was higher in MFb than in Fb.

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[in Japanese]

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