Masahiro Koshiba

Association of the glutathione S-transferase M1 homozygous null genotype with susceptibility to Sjögren's syndrome in Japanese individuals

OBJECTIVE To investigate the role of polymorphisms of the glutathione S-transferase M1 (GSTM1) and GSTT1 genes in determining susceptibility to Sjögrens syndrome (SS) and autoantibody production. METHODS Polymorphisms of the GSTM1 and GSTT1 genes in 106 Japanese patients with primary SS and in 143 healthy controls were analyzed by polymerase chain reaction. RESULTS Frequency of the GSTM1 homozygous null genotype was significantly increased in SS patients compared with controls (57.5% versus 44.1%; P = 0.035). Moreover, a significantly greater frequency of SSA antibodies was found among SS patients with the GSTM1 null genotype than among those with the GSTM1 non-null genotype (P = 0.0013). Frequency of the GSTT1 polymorphism was not different between SS patients and controls. CONCLUSION The GSTM1 homozygous null genotype could be a genetic factor that determines susceptibility to SS and may be involved in SSA antibody production.

Oxidative stress mediates cell surface expression of SS-A/Ro antigen on keratinocytes.

Exposure to ultraviolet radiation exacerbates the skin lesions of autoimmune diseases, and is known to induce cell surface expression of SS-A/Ro antigen on keratinocytes in vitro. Following up on recent reports on ultraviolet-B (UVB)-induced oxidative stress, we examined the role of oxidative stress in the surface expression of SS-A/Ro antigen on human keratinocytes. First, the exclusive induction by UVB irradiation of the 52-kDa protein (Ro52) but not of the 60-kDa protein (Ro60) of SS-A/Ro antigen was demonstrated by means of indirect immunofluorescence. The surface expression of Ro52 induced by UVB irradiation was concentration-dependently inhibited by N-acetyl-L-cysteine, an antioxidant. Furthermore, surface expression of Ro52 was similarly induced by diamide, a chemical oxidant. We next used Hoechst 33342 staining and the TUNEL assay to demonstrate that a low dose (20 mJ/cm(2)) of UVB did not induce apoptosis but induced the surface expression of Ro52. Moreover, zVAD-fmk, a pan-caspase inhibitor, did not inhibit UVB-induced surface expression of Ro52 even at a high dose (200 mJ/cm(2)) of UVB, which was sufficient to induce apoptosis in keratinocytes in the absence of zVAD-fmk. Taken together, we concluded that UVB-induced surface expression of Ro52 on keratinocytes is mediated by oxidative stress through a pathway other than apoptosis.

2-Chloroadenosine but not adenosine induces apoptosis in rheumatoid fibroblasts independently of cell surface adenosine receptor signalling.

The apoptotic effect of adenosine and its analogues was studied in fibroblast‐like synoviocytes derived from rheumatoid arthritis patients (RA‐FLSs). Evoked cell death was quantitatively examined by assessing DNA fragmentation using an enzyme‐liked immunosorbent assay and by measuring phosphatidylserine exposure through flow cytometric analysis of annexin V binding. Exposing cells for 24 h to 2‐chloroadenosine (2‐CADO), a nonspecific, adenosine deaminase (ADA)‐resistant, adenosine receptor (AdoR) agonist, induced DNA fragmentation, and thus apoptosis, in RA‐FLSs at concentrations 50 μM. By contrast, incubation with adenosine for up to 72 h did not evoke DNA fragmentation, even in the presence of ADA inhibitor coformycin and nucleoside transporter inhibitor nitrobenzylmercaptopurin (NBMPR). Transcription of all four AdoR isoforms was detected in RA‐FLSs; nevertheless selective AdoR agonists similarly failed to induce DNA fragmentation. DNA fragmentation evoked by 2‐CADO was inhibited by NBMPR and by 5′‐iodotubercidin, an adenosine kinase inhibitor, but not by xanthine amine congener, an A1 and A2 receptor antagonist, or by selective AdoR antagonists. The nonspecific caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp fluoromethyl ketone abolished the apoptotic effect of 2‐CADO. These results suggest that 2‐CADO induces apoptosis in RA‐FLSs independently of AdoR‐mediated signalling. Instead, 2‐CADO, but not adenosine, is taken up into RA‐FLSs via human equilibrative nucleoside transporter‐1, where it is phosphorylated by adenosine kinase. The resultant phospho‐2‐CADO induces DNA fragmentation by activating a caspase pathway.

Association of the TAP2*Bky2 allele with presence of SS-A/Ro and other autoantibodies in Japanese patients with systemic lupus erythematosus

We previously reported that a new allele of transporter associated with antigen processing (TAP) 2 gene, TAP2*Bky2 (Val577), was significantly increased in Japanese patients with Sjögren’s syndrome (SS) and had a strong association with SS-A=Ro antibody production. In the present study, it was investigated whether the association of TAP2*Bky2 with SS-A=Ro antibody production was also found in Japanese patients with systemic lupus erythematosus (SLE). Polymorphisms of the TAP1 and TAP2 genes were determined in 114 Japanese SLE patients by the polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) method. The allele frequencies of the TAP1 and TAP2 genes in SLE patients were not significantly different from those in controls, although the allele frequency of TAP2*Bky2 was slightly higher in SLE patients than in healthy control subjects (9.2% vs 5.5%, P = 0.126). The allele frequency of TAP2*Bky2 was significantly higher in SLE patients with oral ulcers than in those without. It was noteworthy that TAP2*Bky2 was significantly associated with the appearance of not only SS-A=Ro antibody but also SS-B=La, nRNP, and Sm antibodies in the patients. The association of TAP2*Bky2 was found with the antibody production to both 60 and 52 kDa SS-A=Ro antigens. As TAP2*Bky2 had a strong linkage disequilibrium with DRB1*08032, TAP2*Bky2 or its haplotype with DRB1*08032 may be involved in SS-A=Ro antibody production not only in SS but also SLE patients, indicating that TAP2*Bky2 may be a susceptible gene not only to the disease of SS but also to the SS-A=Ro autoantibody production.

Preventive and therapeutic effects of tacrolimus in an interleukin-10-deficient mouse model of colitis

ObjectiveTo investigate the preventive and therapeutic effects of tacrolimus on colonic inflammation in interleukin-10-deficient (IL-10−/−) mice, which spontaneously develop T-cell-mediated colitis.MethodsTacrolimus or prednisolone, an anti-inflammatory glucocorticoid, was administered to IL-10−/− mice with pre- or post-symptomatic colitis. Effects on colonic inflammation were examined by measuring indices of colitis such as colonic weight/length ratio, cell infiltration, and goblet cell depletion. Effects on cytokine production in colonic lamina propria mononuclear cells (LPMCs) isolated from IL-10−/− mice were also examined.ResultsTacrolimus prevented development of colitis and improved already-developed colitis. Prednisolone prevented the development of colitis, but had no effect on already-developed colitis. Tacrolimus completely inhibited IFN-γ and TNF-α production of activated T-cells in LPMCs, but only partially inhibited IFN-γ, TNF-α, and IL-12 production of activated monocytes/macrophages in LPMCs. Prednisolone inhibited cytokine production in both cell types but exhibited greater potency on monocytes/macrophages than on T-cells.ConclusionThese results suggest that the preventive and therapeutic effect of tacrolimus in IL-10−/− mice colitis might be attributed to the inhibition of colonic T-cell activation rather than monocyte/macrophage activation. T-cell immunosuppression may thus be a promising strategy for treating colonic inflammation.

Inhibition of natural killer cytotoxicity in vitro by clinical grade serine protease inhibitors

The effects of clinical grade serine protease inhibitors on natural killer (NK) activity were compared. Cytotoxicity was measured with the Calcein-AM release method, using K562, Raji as a target. There is a significant correlation between measurements of NK activity by the Calcein-AM method and the 51Cr release assay. Cytotoxicity was inhibited with a calcium chelating agent or a perform inhibitor. Although up to 65% of cytotoxicity was inhibited by nafamostat mesilate with an E/T ratio of 10:1, and by 55% by ulinastatin, neither gabexate mesilate nor antithrombin III inhibited any cytotoxicity. None of these agents inhibited lymphokine-activated killer cell activity. In clinical applications, it should be noted that some protease inhibitors have been proven to have immunosuppressive effects.

Long-term effects of irbesartan on plasma aldosterone concentration and left atrial volume in hypertensive patients.

BACKGROUND Plasma aldosterone concentration (PAC) is related to cardiac remodeling in patients with hypertension. However, we do not know the detailed relationship between changes in PAC and regression of left atrial (LA) volume following long-term treatment with angiotensin II receptor blocker (ARB) or calcium-channel blocker (CCB). OBJECTIVE The aim of this study was to investigate the effects of anti-hypertensive monotherapy, an ARB irbesartan or a CCB amlodipine, on PAC and LA reverse remodeling in hypertensive patients. METHODS A total of 48 patients with untreated hypertension were randomly assigned to irbesartan (ARB group, n=26) and amlodipine (CCB group, n=22). We examined the correlation between LA volume index (LAVI) and other echocardiographic parameters or PAC (n=40) at the baseline and after 12 months of treatment. RESULTS After 12 months, blood pressure (BP) decreased similarly in both groups. LAVI and PAC significantly decreased in the ARB group, but not in the CCB group (-16±8% vs. 22±9%, p<0.01, -16±9% vs. 11±9%, p<0.05). Larger %-decrease in PAC was associated with larger %-reduction of LAVI in the ARB group (r=0.54, p<0.05), but not in the CCB group. CONCLUSIONS While BP reduction was similar between the two groups, decrease in LA volume was larger in the ARB group than in the CCB group. Decrease in LA volume was larger in patients with a greater decrease in PAC than in those with smaller decrease in PAC. ARB may facilitate reverse remodeling of LA through decreases in PAC in hypertensive patients.

Modification of Cytokine Milieu by A2A Adenosine Receptor Signaling–Possible Application for Inflammatory Diseases

Pro‐inflammatory cytokine TNF‐alpha (TNF) production from in vitro lipopolysaccharide (LPS)‐stimulated human peripheral blood CD14+ cells (PB‐CD14) was inhibited by A2A adenosine receptor (AdoR) (A2AR) or ß2 adrenergic receptor (ADR) (ß2R) signaling in a concentration‐dependent manner. These inhibitory effects were presumably mediated by the increase in intracellular cAMP. Furthermore A2AR agonist and ß2R agonist synergistically inhibited the TNF production of LPS‐stimulated PB‐CD14 cells. These results suggest that the anti‐inflammatory effect of extracellular adenosine is, at least in part, due to the modification of the cytokine milieu via A2A signaling, and that the targeting of both A2AR and ß2R may have strong therapeutic potential for the inflammatory diseases.

4. CMAP derived from proximal and distal muscle by stimulating Erb point and CMCT in cervical spondylotic amyotrophy

The usefulness of the electrophysiological tests on the differentiation of the affected regions (either the nerve root or the anterior horn of the spinal cord) of cervical spondylotic amyotrophy (CSA) was examined on 14 patients with proximal type and 3 patients with distal type CSA. The compound muscle action potentials (CMAP) stimulated at supramaximal Erb point were recorded from the proximal (deltoid and biceps) and distal muscles (extensor digitorum and abductor digiti minimi), and central motor conduction time (CMCT) was calculated from motor evoked potentials and F wave. CMAP was correlated with the manual muscle testing. In distal type CSA patients, decrease in CMAP was observed from both of the distal and the proximal muscles. The delayed CMCT and F wave abnormalities, which suggests the anterior horn legions, were found in 5 proximal and all distal type CSA patients. Furthermore 4 patients with poor prognosis out of 8 operated cases showed the delayed CMCT. Thus, these electrophysiological tests appear to be useful not only to differentiate the affected regions in CSA patients but to predict the prognosis after operation.

Expression of Granulysin mRNA in the Human Megakaryoblastic Leukemia Cell Line CMK

Granulysin is a newly reported cytolytic molecule and colocalizes with perforin and granzymes in the granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this study, we found that the megakaryoblastic leukemia cell line CMK, established from a patient with Down’s syndrome, expressed granulysin mRNA. CMK was positive for CD13 and CD41 and negative for CD56. CMK also expressed CD2 and CD7. However, no rearrangement of the T-cell receptor β-chain gene, an early marker of T-cell lineage, was found in CMK cells. Thus, CMK is assumed to originate from the clonal evolution at the immature cell level. The expression of granulysin in CMK cells suggests that granulysin is occasionally present in immature multilineage cells or may be characteristic of leukemic cells obtained from Down’s syndrome patients. CMK has been reported to be capable of differentiating to mature megakaryocytes and produce platelets with normal function. It therefore seems to be possible that granulysin is also present in normal platelets. Unfortunately, we were not able to obtain evidence that normal platelets contain granulysin mRNA and its antigen.