Reiji Kasukawa

An Ancient Lectin-Dependent Complement System in an Ascidian: Novel Lectin Isolated from the Plasma of the Solitary Ascidian, Halocynthia roretzi

Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway. To elucidate the origin and evolution of MBL, MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose. SDS-PAGE of the eluted proteins revealed a major band of ∼36 kDa (p36). p36 cDNA was cloned from an ascidian hepatopancreas cDNA library. Sequence analysis revealed that the carboxy-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type lectin, but it lacks a collagen-like domain that is present in mammalian MBLs. Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding lectin (GBL). The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin. The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis. These observations strongly suggest that GBL acts as a recognition molecule and that the primitive complement system, consisting of the lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.

Pulmonary Hypertension in Rats 1.

We administered human interleukin (IL)-6 to rats to examine whether it is implicated in the development of pulmonary hypertension (PH). The rats injected with IL-6 developed PH as determined by the weight ratio of the right ventricle to the left ventricle+septum. This ratio decreased with the appearance of IgG anti-human IL-6 antibody. Histologically we observed in the lungs, luminal occlusion of small muscular arteries, capillaries filled with fibrin thrombi, and localized hemorrhage. IL-6 increased the number of platelets, and the number of platelets correlated with the extent of PH. Thus, it seems likely that (IL-6) is involved in the formation of PH in this model.

Immunohistological Study of Endothelin-1 and Endothelin-A and B Receptors in Two Patients with Scleroderma Renal Crisis

Abstract: Scleroderma renal crisis (SRC) developed in two patients with systemic sclerosis (SSc) and they died from respiratory failure. Findings on autopsy revealed congestion and oedema in both lungs and intimal thickening of the small renal arteries in both patients. Immunohistological investigations showed positive staining of anti-human endothelin (ET)-1 in the media of the small renal arteries and ET-B receptor in the medial smooth muscle of the small renal arteries. This observation suggests an important pathophysiological role of ET-1 in the development of SRC in some patients with SSc.

Clinico-Laboratory Characteristics of Patients with Dermatomyositis Accompanied by Rapidly Progressive Interstitial Lung Disease

Abstract: The clinico-laboratory features of 16 patients with dermatomyositis (DM) were compared between patients with accompanying rapidly progressive interstitial lung disease (RP-ILD, n= 7) and those with chronic interstitial lung disease (C-ILD, n= 9), and also between deceased (seven RP-ILD and three C-ILD) and living patients (six C-ILD). The extent of muscle weakness of the extremities and frequency of autoantibody positivity were significantly lower in DM patients with RP-ILD than in DM patients with C-ILD. Furthermore, significantly lower serum ceatine kinase/lactate dehydrogenase levels (0.26 ± 0.27) were found in the 10 patients who died than in the six living patients (1.21 ± 1.09). A higher CD4+/CD8+ T-lymphocyte ratio in the peripheral blood (3.51 ± 2.65) was detected in the four DM patients with RP-ILD who died than in the six living DM patients with C-ILD (1.22 ± 0.49).

Unmethylated oligo-DNA containing CpG motifs aggravates collagen-induced arthritis in mice.

OBJECTIVE To investigate the effects of an intradermal injection of an unmethylated oligodeoxynucleotide (ODN) containing CpG motifs on the severity of collagen-induced arthritis (CIA). METHODS CIA was induced in DBA/1 LacJ mice by immunization with bovine type II collagen (CII) in Freunds complete adjuvant followed 3 weeks later by immunization with CII in Freunds incomplete adjuvant (yielding CIA mice). Unmethylated ODN containing a CpG motif was injected intradermally into DBA/1 LacJ mice at a dosage of 20 microg (yielding CpG-CIA mice) 1 week prior to the first immunization with CII. Unmethylated ODN containing a GpC motif instead of a CpG motif and ODN containing a methylated CpG motif were used to produce controls (GpC-CIA mice and mCpG-CIA mice, respectively). After the second immunization with CII, arthritis scores were measured weekly up to the eighth week. At the eighth week, the mice were killed, histopathologic changes in the ankle joints were examined, and titers of interferon-gamma (IFNgamma) in the supernatants of splenocytes (1 x 10(7)) stimulated in culture by CII for 3 days were determined by enzyme-linked immunosorbent assay. RESULTS CpG-CIA mice had significantly higher arthritis scores than CIA mice. CpG-CIA mice had more severe histopathologic changes than CIA mice and mCpG-CIA mice. Moreover, splenocytes in CpG-CIA mice produced higher IFNgamma titers in response to CII than did splenocytes in CIA mice and mCpG-CIA mice. CONCLUSION Injection of unmethylated oligo-DNA containing CpG motifs aggravated CIA through activation of the Th1-type immune response, suggesting that microbial infection could be one of the mechanisms for aggravation or exacerbation of arthritis or, alternatively, that such infection could be an adjuvant in the induction of arthritis in rheumatoid arthritis.

Interleukin-6 induces proliferation of rat hepatocytes in vivo

Abstract Background/Aims: The aim of this study was to assess the effect of interleukin-6 (IL-6) on the proliferation of hepatocytes and to study the interaction between IL-6 and hepatocyte growth factor (HGF) in vivo . Methods: IL-6 was injected at a dose of 200 μg/mg subcutaneously into rats every day for 14 days. Liver and blood samples were obtained at 1, 3, 7 and 14 days during IL-6 administration. Hepatocyte proliferative activity of sera was measured using 3 H-thymidine incorporation into cultured rat hepatocytes. To evaluate the proliferative activity of the hepatocytes in tissue sections, hepatic DNA content and immunostaining of the liver tissue sections for proliferating cell nuclear antigen (PCNA) were performed. Plasma HGF levels were measured using specific EIA. In addition, total RNA was extracted from the liver and expression of HGF mRNA was detected by RT-PCR. Results: The DNA contents of liver taken from IL-6-treated rats were increased during IL-6 administration compared with untreated rats. Sera taken from IL-6-treated rats at various intervals during administration also significantly increased 3 H-thymidine incorporation by cultured rat hepatocytes compared with sera from untreated rats, suppressing 3 H-thymidine incorporation at day 1 and 3 by anti-HGF antibody. IL-6 itself did not increase 3 H-thymidine incorporation. Increased expression of PCNA in these hepatocytes was noted from 1 day after IL-6 administration, and at 14 days, the number of PCNA-positive cells was sevenfold greater than in the livers of untreated rats. However, plasma HGF levels showed a peak at day 1, decreased gradually from day 3, and became undetectable by day 14. HGF mRNA expression in livers of IL-6-treated rats was suppressed from day 3 to day 14 of IL-6 administration. Conclusions: These data show that IL-6 induces an early phase of liver cell growth in vivo and suggest that an increase level of HGF mediates this effect.

Anticardiolipin Antibodies Are Associated with Pulmonary Hypertension in Patients with Mixed Connective Tissue Disease or Systemic Lupus Erythematosus

Anticardiolipin antibodies (aCL) were studied in relation to pulmonary hypertension (PH) in 22 patients with mixed connective tissue disease (MCTD) or systemic lupus erythematosus (SLE). The mean pulmonary arterial pressure (mPAP) values were similar in the 12 MCTD and 10 SLE patients: 26 +/- 11 and 25 +/- 11 mm Hg, respectively. However, the frequency of PH was higher in SLE (60%) than in MCTD patients (33%). The titers of aCL were significantly higher in SLE (38 +/- 27 IU/ml) than in MCTD (17 +/- 7 IU/ml; p < 0.02). Two SLE patients with high titers of aCL had multiple cerebral infarction and transverse myelitis, and deep vein thrombosis, respectively. A significant correlation between the titers of aCL and mPAP was observed in patients with MCTD (p < 0.05), but not in patients with SLE.

Protective effect of beraprost sodium, a stable prostacyclin analogue, in development of monocrotaline-induced pulmonary hypertension.

Experimental pulmonary hypertension (PH) was induced by a single injection of monocrotaline (MCT), a pyrrolizidine alkaloid extracted from Crotalaria spectabilis. The effect of beraprost sodium, a stable prostacyclin analogue, on the development of MCT-induced PH in rats was studied. Chronic administration of beraprost sodium at a dose of 30 micrograms/kg/day initiated on the same day as MCT injection decreased the degree of PH determined by weight ratio of right ventricular free wall to that of left ventricle plus septum depending on the duration of administration. Although the injection of prostaglandin E1 (PGE1) at a dose of 200 micrograms/kg/day initiated 1 week after MCT injection did not decrease the degree of PH significantly, beraprost sodium administration at doses of 30 and 100 micrograms/kg/day decreased the degree of PH significantly. The cytoprotective effect of beraprost sodium against endothelial cell (EC) damage is believed to be involved in inhibiting development of PH in MCT-injected rats. The amounts of cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) produced by alveolar macrophages decreased in accordance with the inhibiting effect of beraprost sodium on development of PH, indicating that beraprost sodium inhibited the development of PH in MCT-injected rats not only through its effect of vasodilation and anti-platelet aggregation in pulmonary circulation but also through its antiinflammatory effects.

Soluble P-selectin in the plasma of patients with connective tissue diseases

We measured the soluble P-selectin (sP-selectin) in plasma of 54 patients with connective tissue diseases and 12 normal controls by a 2-step sandwich enzyme immunoassay. Our purpose was 2-fold: to determine (1) whether the level of sP-selectin of such patients is higher than normal, and (2), if it is, whether it correlates with any of the laboratory data currently available. The mean levels in patients with systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD) and rheumatoid arthritis (RA) were 306, 1,048 and 844 ng/ml, respectively, compared with 220 ng/ml for controls. The mean levels in patients with SLE and nephropathy, MCTD and either nephropathy or thrombosis, and malignant RA were 351, 1,116 and 1,721 ng/ml, respectively. No correlation was found between the levels of sP-selectin and other laboratory data (WBC, CRP, ESR, antinuclear antibody, RF, aCL) except the number of platelets (y = 0.057, r = 0.37). In the clinical course of patients with lupus nephritis and MCTD with nephropathy, sP-selectin became a sensitive parameter. Thus, the level of sP-selectin is higher than normal in patients with connective tissue diseases, especially when complications exist, and it does not correlate with any of the laboratory data currently available except the number of platelets. Measurement of sP-selectin levels should be included in the laboratory tests of patients with connective tissue diseases, especially when complicated by nephropathy or thrombosis.

Role of para‐esophageal collateral veins in patients with portal hypertension based on the results of endoscopic ultrasonography and liver scintigraphy analysis

Background and Aims: Para‐esophageal collateral veins (para‐ECV) are observed by endoscopic ultrasonography (EUS) in patients with portal hypertension. However, the role of para‐ECV in the portal venous system is not clear. To verify the role of para‐ECV in the portal venous system, we investigated the relationship between the development of para‐ECV as determined by EUS, and the portal blood flow ratio (PBFR) as determined by liver scintigraphy using 99mTc‐phytate.