We analyzed the molecular basis of the pathogenesis of vascular disease.
Firstly, we cloned three types of smooth muscle myosin heavy chain genes, SM1, SM2 and SMemb from rabbit cDNA library. Their expression during vascular development is differentially regulated at RNA levels. SM1 is constitutively expressed and SM2 appears after birth. Contrarily, SMemb is predominantly expressed in embryonic and perinatal stage. Interestingly, SMemb was reexpressed in proliferating smooth muscle cells of arteriosclerotic neointimas. These results suggest that smooth muscle proliferation is coupled to the expression of SMemb and that dedifferentiation of smooth muscles toward the embryonic phenotype is involved in the mechanisms underlying atherosclerosis.
Secondly, we have also cloned macrophage scavenger receptor genes and determined their primary structures. The macrophage scavenger receptors showed unusual ligandbinding properties indicating higher binding affinity to modified LDL, such as oxidized and acetylated LDL rather than to LDL. These scavenger receptors were expressed in foam cells of the atherosclerotic neointima. An understanding of the structure and function of this family of receptors will provide us new insights into the mechanism of the development of atherosclerosis.
Finally, we investigated cell-mediated autoimmunity in Takayasu's disease. We found that the dominant populations of the infiltrating cells in the arterial tissues obtained from patients with Takayasu's disease were large granular lymphocytes which contained a lot of perform. Our results suggested that cell-mediated cytotoxicity plays an important role in the development of arterial tissue damage involved in Takayasu's disease.

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Interleukin 8 (IL-8) stimulated an increase in cytoplasmic free Ca²⁺ ( [Ca²⁺] _i) and intracellular pH (pH_i) in parallel at low concentrations (0.5-5 ng/ml), and stimulated O^-₂ release and membrane depolarization in parallel at high concentrations (50-5, 000 ng/ml) . IL-8-induced O^-₂ release was potentiated by tumornecrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in a dose-dependent manner. These characteristics and the time-courses of the responses stimulated by IL-8 were similar to those stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP), except that the cells stimulated by IL-8 showed shorter duration and less magnitude in some responses. In addition, IL-8 was found to be a potent priming agent and to enhance O^-₂ release stimulated by FMLP. Priming effect of IL-8 was very rapid and was maximal within 5 min of preincubation. The dose-response curves for priming was identical to those for triggering of an increase in [Ca²⁺] _i and pH_i. These findings suggest that IL-8 stimulates or primes human neutrophils according to its concentrations and cross-talks with TNF, GM-CSF, G-CSF or FMLP at the inflammatory sites.

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cDNA clones encoding antimicrobial guinea pig neutrophil cationic peptides GNCP-1 and GNCP-2 were isolated from a bone marrow cell cDNA library. Analysis of these clones indicated that both GNCPs were produced as precursor proteins com-prising 93 amino acid residues, which were composed of signal sequences (N-terminal 19 residues), pro-peptide sequences (43 residues) and mature GNCP sequences (31 residues) . The deduced amino acid sequences showed that there were only two amino acid differences between GNCP-1 and GNCP-2, one in the pro-peptide region and another in the mature peptide region. Interestingly, Northern blot analysis and transcription run-off assay revealed that the expression of GNCP mRNA and the transcription of GNCP gene was observed in bone marrow cells but not in mature neutrophils.
These observations suggest that GNCP is synthe-sized by maturing neutrophils in the bone marrow.

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The neutrophilic alkaline phosphatase (NAP) is a well-known marker of the hematological disease. We know the NAP as a clinical phenomenon, however we don't know clearly its function. Then, we studied the measurement of NAP, and assumed its function. In normal volunteer, there is a difference between membranous NAP (we called free activity) and whole cell NAP (we called total activity) . Between male and female, there is a difference. Between smoker and non-smoker, or among the ovulation period, there is a difference, too. But free/total ratio has no differ-ence in each group.

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A study was designed to clarify the role of inter-actions among polymorphonuclear leukocytes (PMNs), platelets, and cytokines in Behçet's disease. PMNs were obtained from 67 patients with Behçet's disease attending the department of ophthalmology in Osaka University Hospital. The effect of thrombin-activated platelets on the release of superoxide anion by PMNs preincubated with or without granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied. The results were compared with those of PMNs obtained from 35 normal volunteers.
Thrombin-activated platelets induced Superoxide release from PMNs preincubated without GM-CSF in 48/67 of patien+s with Behçet's disease and in 23/ 35 of normal volunteers. The frequency was not statistically different between the two groups (X² test) .
Superoxide release from PMNs primed with GM-CSF was increased by thrombin-activated platelets in 48/67 of patients with Behçet's disease and in 34/35 of normal volunteers. Statistical analysis performed with X² test showed that the frequency was significantly decreased in patients with Behçet's disease (P<0.01) . This suggests that PMNs are desensitized to GM-CSF and activated platelets in some patients with Behçet's disease. The interaction of PMNs and platelets may be implicated in the pathogenesis of Behçet's disease.

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To evaluate the proportion of peripheral lymphocyte subsets we investigated 49 patients with Behçet's disease and 17 healthy controls. The proportion of CD4⁺, CD45RA⁺, CD45RO⁺ cells was determined by double-labelling flow cytometry. The percentage of CD4⁺CD45RA⁺ cells which might correspond to naive cells decreased significantly (P<0.001) in Behçet patients (mean 10.3%) as compared with healthy controls (mean 19.6%) . Low proportion of CD4⁺ CD45RA⁺ cells was observed in the patients with complete type, and especially remarkable was a group with longer disease courses.
Behçet patients with central nervous system (CNS) lesion showed a much lower proportion of CD4⁺ cells (mean 28.6%) compared with non-CNS-involved cases (mean 39.9%) and healthy controls (mean 43.8 %) . It was also demonstrated in the patients with CNS lesion that significant correlation existed between CD4⁺ and CD4⁺CD45RA⁺ cell populations.
These data suggested quantitative defect of naive cells might play an important role in the immunoregulatory disturbance in Behçet's disease.

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We investigated the immunological effects of bucil-lamine which has been known as an immunomodula-ting drug with no anti-inflammatory effects, in 80 rheumatoid arthritis (RA) patients by using the flowcytometric analysis. All patients treated with bucillamine have been followed up for 12 months or over. Long term administration of bucillamine markedly improved arthritis related to the decrease titer of RAHA. Moreover, T cell subsets in the peripheral blood showed that the cell constituent ratio of CD4⁺ CD45RA⁺, characteristically increased by administration of bucillamine. On the other hand, CD4⁺CD 45RO⁺, CD4⁺HLA-DR⁺, CD8⁺HLA-DR⁺ and CD 5⁺CD20⁺ significantly decreased in accordance with the activity of RA. These results suggested that bucillamine play a role of an alterative lymphocyte subsets and improving the severity of RA.

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The inhibitory influences of indomethacin (IND) and proglumetacin maleate (PGM) on prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) activities in synovial fluid in osteoarthritis (OA) were investigated. Clinically, no significant difference was observed in final global improvement rate between IND and PGM groups. PGM group showed an improvement higher than IND group in joint effusion. IND showed selective inhibition of PGE₂ formation, but caused a increase in LTB₄ formation. On the other hand, PGM showed a almost same inhibitory effects both on PGE₂ and LTB₄ formation.
These results indicated that PGM, so called dual inhibitor, had a dual inhibitory effects on cyclooxy-genase and lipoxygenase in vivo.

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The effect of clindamycin (CLDM) at subminimal inhibitory concentrations (sub-MIC), i.e. one-tenth MIC, on the production of human neutrophil chemotactic factors in comedonal bacteria, Propionibacte-rium acnes (P. acnes) strains and coagulase-negative staphylococci (CNS) strains, was assayed using Boyden chamber method. Sub-MIC of CLDM markedly inhibited the production of neutrophil chemotactic factors in all strains of P. acnes and CNS. Our results seem to suggest that sub-MIC of CLDM has an anti-inflammatory effect presumably due to the inhibition of neutrophil chemotactic factors production in comedonal bacteria on the inflammatory process of acne.

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The effect of human recombinant interleukin-1β (IL-1β) on the contractile responses of canine airway smooth muscle to acetylcholine (ACh) was studied under isometric conditions in vitro. Exposure of bronchial rings to IL-1β for 4h but not for 15 min decreased the ACh-induced contraction in a dose-dependent fashion. This inhibitory effect was not altered by indomethacin or propranolol, but was eliminated by pretreatment with cycloheximide. These results suggest that IL-1β inhibits airway smooth muscle contraction through protein synthesis.

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We examined the effects of histamine on interle-ukin-6 (IL-6) production in human peripheral blood lymphocytes (PBL) .
PBL obtained from healthy volunteers were cultured in RPMI 1640 with 1% FCS for 72 hours. The effects of histamine, 2-pyridylethylamine (H₁ receptor agonist) and dimaprit (H₂ receptor agonist) on phyto-hemagglutinin (PHA) -driven IL-6 production and intra-cellular cyclic AMP (cAMP) accumulation were analyzed with time course.
The production of IL-6 in PBL was gradually increased by 24 hours with reaching plateau after PHA activation. Both histamine and dimaprit significantly reduced the production of IL-6 in PBL, but 2-pyridylethylamine did not. The content of intra-cellular cAMP in PBL was rapidly increased after incubation with both histamine and dimaprit.
These results indicated that histamine increased intra-cellular cAMP and then reduced IL-6 production in PBL through H₂ receptor.

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The effect of immunotherapeutic agents on influenza virus B/USSR haemagglutinin (B/HA) induced delayed-type hypersensitivity (DTH) in BALB/c mice was investigated. Until now we have studied in this experiment by using influenza virus A/Kumamoto haemagglutinin (A/HA) . In this study, B/HA induced the DTH reaction same as A/HA did. In immune complex type hypersensitivity, mizoribine (MZR) inhibited the footpad swelling but cyclosporine A (CyA) and salicylazosulphapyridine (SASP) had no effect on the reaction. MZR and CyA inhibited DTH reaction but SASP had no effect on the HA induced footpad swelling which was augmented by cyclophosphamide pretreatment. These findings suggest that immunotherapeutic agensts have various effects respectively.

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