Advances in the genomics of ANCA-associated vasculitis-a view from East Asia.

Recent genome-wide association studies (GWAS) in populations of European ancestry have identified several susceptibility genes to anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The most significant association was observed in HLA-DP variants in granulomatosis with polyangiitis and proteinase 3 (PR3)-ANCA positive vasculitis, while HLA-DQ variants were strongly associated with microscopic polyangiitis (MPA) and myeloperoxidase (MPO)-ANCA positive vasculitis (MPO-AAV). In non-HLA genes, SERPINA1, PRTN3 and PTPN22 were identified as susceptibility genes to AAV. The observations in GWAS suggested the presence of shared and non-shared susceptibility genes among AAV subsets. Epidemiological features of AAV are strikingly different in the East Asian populations; the proportions of MPO-AAV among total AAV, MPO-ANCA positive patients among GPA, and patients with interstitial lung disease among total AAV are considerably higher in Japan as compared with Europe. Such population differences suggest the critical role for genetic background behind these conditions. Although no GWAS has been reported in the Asian populations so far, the association of HLA-class II alleles with MPA and MPO-AAV was identified. Future genomics studies on AAV, especially from Asian populations, will provide valuable information to elucidate the molecular mechanisms and to identify molecular targets for AAV.

Association of functional (GA)n microsatellite polymorphism in the FLI1 gene with susceptibility to human systemic sclerosis.

OBJECTIVE: Susceptibility genes that can account for characteristic features of SSc such as fibrosis, vasculopathy and autoimmunity remain to be determined. In mice, deficiency of Friend leukaemia integration 1 transcription factor (Fli1) causes SSc-like disease with these features. The human FLI1 gene contains (GA)n microsatellite, which has been shown to be associated with expression level. Because microsatellite polymorphisms are difficult to capture by genome-wide association studies, we directly genotyped FLI1 (GA)n microsatellite and examined its association with SSc. METHODS: Genomic DNA from 639 Japanese SSc patients and 851 healthy controls was genotyped for (GA)n microsatellite using the fragment assay. The cut-off repeat number for susceptibility to SSc was determined by receiver operating characteristics (ROC) analysis. Association with susceptibility and clinical characteristics was examined using logistic regression analysis. FLI1 mRNA levels were determined using quantitative RT-PCR. RESULTS: Based on the ROC analysis, (GA)n alleles with ≥22 repeats were collectively defined as L alleles and alleles with ≤21 repeats as S alleles. (GA)n L alleles were significantly associated with susceptibility to SSc (P = 5.0e-04, odds ratio 1.34, additive model). Significant association was observed both in diffuse cutaneous and limited cutaneous SSc. Among the SSc, (GA)n L alleles were significantly enriched in the patients with a modified Rodnan total skin thickness score ≥10 compared with those with a score <10. FLI1 mRNA levels were significantly decreased in healthy controls carrying (GA)n L alleles as compared with non-carriers. CONCLUSION: Extended repeat alleles of FLI1 (GA)n microsatellite may be associated with lower FLI1 mRNA levels and susceptibility to human SSc.

Association of ETS1 polymorphism with granulomatosis with polyangiitis and proteinase 3-anti-neutrophil cytoplasmic antibody positive vasculitis in a Japanese population.

ETS proto-oncogene 1, transcription factor (ETS1) is involved in various immune responses. Genome-wide association studies on systemic lupus erythematosus in Chinese populations identified the association of ETS1 polymorphism in 3' untranslated region, rs1128334A, which was associated with lower ETS1 expression. In view of substantial sharing of susceptibility genes across multiple autoimmune diseases, we examined whether ETS1 is associated with a rare autoimmune rheumatic disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Association of rs1128334 was tested in 466 Japanese patients with AAV and 1099 healthy controls by logistic regression analysis under the additive model. AAV patients were classified into 285 microscopic polyangiitis (MPA), 92 granulomatosis with polyangiitis (GPA), 56 eosinophilic GPA, and 33 unclassifiable AAV, according to the European Medicines Agency (EMEA) algorithm. Among the patients, 376 were positive for MPO-ANCA and 62 for PR3-ANCA. When the patients were classified according to the EMEA classification, rs1128334A allele was significantly increased in GPA (P = 0.0060, P c = 0.030, odds ratio (OR), 1.54; 95% confidence interval (CI), 1.13-2.10). With respect to the ANCA specificity, significant association was observed in PR3-ANCA positive AAV (P = 0.0042, P c = 0.021, OR, 1.72; 95% CI, 1.19-2.49). In conclusion, ETS1 polymorphism was suggested to be associated with GPA and PR3-ANCA positive AAV in a Japanese population.

Transethnic meta-analysis identifies GSDMA and PRDM1 as susceptibility genes to systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease characterised by skin and systemic fibrosis culminating in organ damage. Previous genetic studies including genome-wide association studies (GWAS) have identified 12 susceptibility loci satisfying genome-wide significance. Transethnic meta-analyses have successfully expanded the list of susceptibility genes and deepened biological insights for other autoimmune diseases. METHODS: We performed transethnic meta-analysis of GWAS in the Japanese and European populations, followed by a two-staged replication study comprising a total of 4436 cases and 14 751 controls. Associations between significant single nuclear polymorphisms (SNPs) and neighbouring genes were evaluated. Enrichment analysis of H3K4Me3, a representative histone mark for active promoter was conducted with an expanded list of SSc susceptibility genes. RESULTS: We identified two significant SNP in two loci, GSDMA and PRDM1, both of which are related to immune functions and associated with other autoimmune diseases (p=1.4×10-10 and 6.6×10-10, respectively). GSDMA also showed a significant association with limited cutaneous SSc. We also replicated the associations of previously reported loci including a non-GWAS locus, TNFAIP3. PRDM1 encodes BLIMP1, a transcription factor regulating T-cell proliferation and plasma cell differentiation. The top SNP in GSDMA was a missense variant and correlated with gene expression of neighbouring genes, and this could explain the association in this locus. We found different human leukocyte antigen (HLA) association patterns between the two populations. Enrichment analysis suggested the importance of CD4-naïve primary T cell. CONCLUSIONS: GSDMA and PRDM1 are associated with SSc. These findings provide enhanced insight into the genetic and biological basis of SSc.

Association of HLA-G 3' Untranslated Region Polymorphisms with Systemic Lupus Erythematosus in a Japanese Population: A Case-Control Association Study.

HLA-G plays a role in fetal-maternal tolerance as well as immunoregulation, and has been suggested to be involved in autoimmune diseases and cancers. HLA-G encodes two potentially functional polymorphisms in the 3' untranslated region, 14bp insertion/deletion (14bp indel, rs371194629) and a single nucleotide polymorphism rs1063320, previously reported to affect HLA-G expression level or splicing isoform and to be associated with susceptibility to systemic lupus erythematosus (SLE). However, the results of SLE association studies are inconsistent, probably due to the small sample size of each study and lack of consideration of linkage disequilibrium (LD) with HLA-class II haplotypes in each population. In this study, we performed association studies of these polymorphisms on 843 patients with SLE and 778 healthy controls in a Japanese population, in many of whom HLA-DRB1 alleles have been genotyped at the four-digit level. LD was detected between DRB1*13:02, protective against multiple autoimmune diseases in the Japanese, and the rs1063320 G (D' = 0.86, r2 = 0.02) and with 14bp del (D' = 0.62, r2 = 0.01), but not between SLE-susceptible DRB1*15:01 and HLA-G. Although significant association with overall SLE was not detected, 14bp ins allele was significantly associated with SLE with the age of onset <20 years, when compared with healthy controls (P = 0.0067, PFDR = 0.039, OR 1.44, additive model) or with SLE patients with the age of onset ≥20 (P = 0.033, PFDR = 0.0495, OR 2.09, additive model). This association remained significant after conditioning on DRB1*13:02 or DRB1*15:01. On the other hand, significant association was detected between rs1063320 C and anti-RNP antibody and anti-Sm antibody positive SLE, which was dependent on negative LD with DRB1*13:02. eQTL analysis showed reduced HLA-G mRNA level in 14bp ins/ins individuals. In conclusion, our observations showed that HLA-G 14bp ins allele represents a genetic contribution on early-onset SLE independent of DRB1.

Protective Role of HLA-DRB1*13:02 against Microscopic Polyangiitis and MPO-ANCA-Positive Vasculitides in a Japanese Population: A Case-Control Study.

Among antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), granulomatosis with polyangiitis (GPA) and proteinase 3-ANCA-positive AAV (PR3-AAV) are prevalent in European populations, while microscopic polyangiitis (MPA) and myeloperoxidase-ANCA-positive AAV (MPO-AAV) are predominant in the Japanese. We previously demonstrated association of DRB1*09:01-DQB1*03:03 haplotype, a haplotype common in East Asians but rare in the European populations, with MPA/MPO-AAV, suggesting that a population difference in HLA-class II plays a role in the epidemiology of this disease. To gain further insights, we increased the sample size and performed an extended association study of DRB1 and DPB1 with AAV subsets in 468 Japanese patients with AAV classified according to the European Medicines Agency algorithm (MPA: 285, GPA: 92, eosinophilic GPA [EGPA]: 56, unclassifiable: 35) and 596 healthy controls. Among these patients, 377 were positive for MPO-ANCA and 62 for PR3-ANCA. The significance level was set at α = 3.3x10-4 by applying Bonferroni correction. The association of DRB1*09:01 with MPO-AAV was confirmed (allele model, P = 2.1x10-4, odds ratio [OR] = 1.57). Protective association of DRB1*13:02 was detected against MPO-AAV (allele model, P = 2.3x10-5, OR = 0.42) and MPA (dominant model, P = 2.7x10-4, OR = 0.43). A trend toward increased frequency of DPB1*04:01, the risk allele for GPA in European populations, was observed among Japanese patients with PR3-AAV when conditioned on DRB1*13:02 (Padjusted = 0.0021, ORadjusted = 3.48). In contrast, the frequency of DPB1*04:01 was decreased among Japanese patients with MPO-AAV, and this effect lost significance when conditioned on DRB1*13:02 (Padjusted = 0.16), suggesting that DRB1*13:02 or other allele(s) in linkage disequilibrium may be responsible for the protection. The differential association of DPB1*04:01 with PR3-AAV and MPO-AAV and difference in DPB1*04:01 allele frequencies between populations supported the hypothesis that the HLA-class II population difference may account in part for these epidemiologic characteristics. Furthermore, taken together with our previous observations, the haplotype carrying DRB1*13:02 was suggested to be a shared protective factor against multiple autoimmune diseases.

Identification of secreted phosphoprotein 1 gene as a new rheumatoid arthritis susceptibility gene.

OBJECTIVE: To evaluate the contribution of the SPP1 rs11439060 and rs9138 polymorphisms, previously reported as autoimmune risk variants, in the rheumatoid arthritis (RA) genetic background according to anti-citrullinated protein antibodies (ACPAs) status of RA individuals. METHODS: We analysed a total of 11,715 RA cases and 26,493 controls from nine independent cohorts; all individuals were genotyped or had imputed genotypes for SPP1 rs11439060 and rs9138. The effect of the SPP1 rs11439060 and rs9138 risk-allele combination on osteopontin (OPN) expression in macrophages and OPN serum levels was investigated. RESULTS: We provide evidence for a distinct contribution of SPP1 to RA susceptibility according to ACPA status: the combination of ≥3 SPP1 rs11439060 and rs9138 common alleles was associated mainly with ACPA negativity (p=1.29×10(-5), ORACPA-negative 1.257 (1.135 to 1.394)) and less with ACPA positivity (p=0.0148, ORACPA-positive 1.072 (1.014 to 1.134)). The ORs between these subgroups (ie, ACPA-positive and ACPA-negative) significantly differed (p=7.33×10(-3)). Expression quantitative trait locus analysis revealed an association of the SPP1 risk-allele combination with decreased SPP1 expression in peripheral macrophages from 599 individuals. To corroborate these findings, we found an association of the SPP1 risk-allele combination and low serum level of secreted OPN (p=0.0157), as well as serum level of secreted OPN correlated positively with ACPA production (p=0.005; r=0.483). CONCLUSIONS: We demonstrate a significant contribution of the combination of SPP1 rs11439060 and rs9138 frequent alleles to risk of RA, the magnitude of the association being greater in patients negative for ACPAs.

PLD4 as a novel susceptibility gene for systemic sclerosis in a Japanese population.

OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune disease for which multiple susceptibility genes have been reported. Genome-wide association studies have shown that large numbers of susceptibility genes are shared among autoimmune diseases. Recently, our group identified 9 novel susceptibility genes associated with rheumatoid arthritis (RA) in a Japanese population. The aim of this study was to elucidate whether the 18 genes that displayed associations or suggestive associations for RA in our previous study are associated with SSc in Japanese. METHODS: We performed an association study that included 415 patients with SSc and 16,891 control subjects, followed by a replication study that included 315 patients and 21,054 control subjects. The 18 markers reported to display association with RA were analyzed for their associations with SSc in the first study, and 5 markers were further analyzed in the replication study. The inverse variance method was used to evaluate the associations of these markers with SSc in a combined study. RESULTS: In the phospholipase D4 gene (PLD4), rs2841277 displayed a significant association with SSc in Japanese patients (P = 0.00017). We observed that rs2841280 in exon 2 of PLD4 was in strong linkage disequilibrium with rs2841277 and introduced an amino acid alteration. We also observed associations between SSc and rs6932056 in TNFAIP3 and rs2280381 in IRF8 (P = 0.0000095 and P = 0.0030, respectively), both of which displayed associations with SSc in a European population. CONCLUSION: We determined that PLD4 is a novel susceptibility gene for SSc in Japanese, thus confirming the involvement of PLD4 in autoimmunity. Associations between SSc and TNFAIP3 or IRF8 were also detected in our Japanese population. SSc and RA appear to share relatively large proportions of their genetic backgrounds.

Effects of APRIL (TNFSF13) polymorphisms and splicing isoforms on the secretion of soluble APRIL.

Functional APRIL (TNFSF13) is a secreted trimer generated by furin protease cleavage. We previously reported the association of APRIL haplotypes formed by two nonsynonymous polymorphisms, Gly67Arg and Asn96Ser, with systemic lupus erythematosus. Here, we tested the hypothesis that polymorphisms and/or alternative splicing may influence the generation of soluble APRIL (sAPRIL). HEK 293T cells were transfected with plasmids containing one of the six combinations of splicing isoforms (α or ß) and haplotypes (susceptible, neutral, or protective). APRIL concentrations were quantitated in the cell lysates and supernatants using an enzyme-linked immunosorbent assay (ELISA). The association between splicing efficiency and polymorphisms was analyzed using quantitative reverse transcription polymerase chain reaction (RT-PCR). The efficiency of cleavage by furin protease was analyzed using western blotting. Although both splicing isoforms were cleaved by furin protease, sAPRIL was not detected in the supernatant of the cells transfected with the ß isoform, regardless of the haplotype. This suggested that, similarly to B-cell activating factor (BAFF), one of the major APRIL splicing isoforms may not be secreted as a functional molecule. Furthermore, the secretion of sAPRIL was decreased in the transfectants expressing the protective haplotype. An association between the polymorphisms and splicing efficiency or furin cleavage efficiency was not detected. In conclusion, these observations suggested that both alternative splicing and polymorphisms may affect the generation of functional sAPRIL.

Association of a functional polymorphism in the 3'-untranslated region of SPI1 with systemic lupus erythematosus.

OBJECTIVE: SPI1, also referred to as PU.1, is an Ets family transcription factor that interacts with IRF2, IRF4, and IRF8. In view of the significance of the type I interferon pathway in systemic lupus erythematosus (SLE), this study was undertaken to investigate a possible association between SPI1 polymorphisms and SLE. METHODS: A case-control association study was performed using 6 tag single-nucleotide polymorphisms (SNPs), as well as a SNP located upstream of SPI1 previously found to be associated with acute myelogenous leukemia, in 400 Japanese patients with SLE and 450 healthy controls. Resequencing of all exons and known regulatory regions was performed to identify functional polymorphisms. Association of genotype and SPI1 expression was examined using the GENEVAR database and reporter assays. RESULTS: A significant association was detected in 2 SNPs in intron 2 (rs10769258 and rs4752829) (P = 0.005 and P = 0.008, respectively, under the dominant model). The association was stronger in patients with nephropathy. Resequencing identified a potentially functional polymorphism in the 3'-untranslated region (3'-UTR), rs1057233, which was in strong linkage disequilibrium with the SNPs in intron 2. The number of risk alleles at rs1057233 was strongly correlated with SPI1 messenger RNA (mRNA) level in the database analysis (P = 0.0002), and was confirmed by a reporter assay. Interestingly, rs1057233 alters a target sequence for microRNA hsa-miR-569 (miR-569). Transfection experiments demonstrated that miR-569 inhibits expression of a reporter construct with the 3'-UTR sequence containing the nonrisk allele but not the risk allele. CONCLUSION: Our findings indicate that a SNP in the 3'-UTR of SPI1 is associated with elevated SPI1 mRNA level and with susceptibility to SLE.

Association of TNFAIP3 interacting protein 1, TNIP1 with systemic lupus erythematosus in a Japanese population: a case-control association study.

INTRODUCTION: TNFAIP3 interacting protein 1, TNIP1 (ABIN-1) is involved in inhibition of nuclear factor-κB (NF-κB) activation by interacting with TNF alpha-induced protein 3, A20 (TNFAIP3), an established susceptibility gene to systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Recent genome-wide association studies revealed association of TNIP1 with SLE in the Caucasian and Chinese populations. In this study, we investigated whether the association of TNIP1 with SLE was replicated in a Japanese population. In addition, association of TNIP1 with RA was also examined. METHODS: A case-control association study was conducted on the TNIP1 single nucleotide polymorphism (SNP) rs7708392 in 364 Japanese SLE patients, 553 RA patients and 513 healthy controls. RESULTS: Association of TNIP1 rs7708392C was replicated in Japanese SLE (allele frequency in SLE: 76.5%, control: 69.9%, P = 0.0022, odds ratio [OR] 1.40, 95% confidence interval [CI] 1.13-1.74). Notably, the risk allele frequency in the healthy controls was considerably greater in Japanese (69.9%) than in Caucasians (24.3%). A tendency of stronger association was observed in the SLE patients with renal disorder (P = 0.00065, OR 1.60 [95%CI 1.22-2.10]) than in all SLE patients (P = 0.0022, OR 1.40 [95%CI 1.13-1.74]). Significant association with RA was not observed, regardless of the carriage of human leukocyte antigen DR ß1 (HLA-DRB1) shared epitope. Significant gene-gene interaction between TNIP1 and TNFAIP3 was detected neither in SLE nor RA. CONCLUSIONS: Association of TNIP1 with SLE was confirmed in a Japanese population. TNIP1 is a shared SLE susceptibility gene in the Caucasian and Asian populations, but the genetic contribution appeared to be greater in the Japanese and Chinese populations because of the higher risk allele frequency. Taken together with the association of TNFAIP3, these observations underscore the crucial role of NF-κB regulation in the pathogenesis of SLE.

Association of TNFAIP3 polymorphism with susceptibility to systemic lupus erythematosus in a Japanese population.

Recent genome-wide association studies demonstrated association of single nucleotide polymorphisms (SNPs) in the TNFAIP3 region at 6q23 with systemic lupus erythematosus (SLE) in European-American populations. In this study, we investigated whether SNPs in the TNFAIP3 region are associated with SLE also in a Japanese population. A case-control association study was performed on the SNPs rs13192841, rs2230926, and rs6922466 in 318 Japanese SLE patients and 444 healthy controls. Association of rs2230926 G allele with SLE was replicated in Japanese (allelic association P = .033, odds ratio [OR] 1.47, recessive model P = .023, OR 8.52). The association was preferentially observed in the SLE patients with nephritis. When the TNFAIP3 mRNA levels of the HapMap samples were examined using GENEVAR database, the presence of TNFAIP3 rs2230926 G allele was associated with lower mRNA expression of TNFAIP3 (P = .013). These results indicated that TNFAIP3 is a susceptibility gene to SLE both in the Caucasian and Asian populations.

Combining effects of polymorphism of tumor necrosis factor alpha 5'-flanking region and HLA-DRB1 on radiological progression in patients with rheumatoid arthritis.

We examined whether polymorphisms upstream of the TNF-alpha gene (TNFA) were associated with the radiological progression of rheumatoid arthritis (RA). One hundred and twenty-three patients with early RA (disease duration <1 year) were enrolled in a prospective follow-up study. The laboratory findings (ESR, CRP, and RF) were evaluated every 2 months for 2 years. Radiological progression in hands/wrists and feet was evaluated every 6 months for 2 years using Larsen's score. HLA-DRB1 genotype was determined by PCR-RFLP method. The genotypes for -1031, -863, and -857 single-nucleotide polymorphisms in the upstream 5'-flanking region of TNFA were determined by a PCR-preferential homoduplex formation assay in patients with RA and 265 healthy controls. Four TNFA alleles (U01, U02, U03, and U04) were identified. The frequency of individuals with U02 was significantly higher in patients than in controls (P = 0.0025). Radiographs of hands/wrists/feet were available for 72 patients after 1 year and for 73 patients after 2 years. When the HLA-DRB1 genotype was analyzed simultaneously, patients possessing U02 without an HLA-DRB1 shared epitope (SE) (U02+SE-) showed the lowest progression of Larsen's score (12 months). There was no difference in the level of ESR, CRP, or RF at the first visit among U02+SE+, U02+SE-, U02-SE+, and U02-SE- groups. The combination of the polymorphism of the TNFA upstream promoter region and HLA-DRB1 allele was associated with radiological progression in the early stage of RA.

Combining effects of polymorphism of tumor necrosis factor α 5'-flanking region and HLA-DRB1 on radiological progression in patients with rheumatoid arthritis.

We examined whether polymorphisms upstream of the TNF-α gene (TNFA) were associated with the radiological progression of rheumatoid arthritis (RA). One hundred and twenty-three patients with early RA (disease duration <1 year) were enrolled in a prospective follow-up study. The laboratory findings (ESR, CRP, and RF) were evaluated every 2 months for 2 years. Radiological progression in hands/wrists and feet was evaluated every 6 months for 2 years using Larsen's score. HLA-DRB1 genotype was determined by PCR-RFLP method. The genotypes for -1031, -863, and -857 single-nucleotide polymorphisms in the upstream 5'-flanking region of TNFA were determined by a PCR-preferential homoduplex formation assay in patients with RA and 265 healthy controls. Four TNFA alleles (U01, U02, U03, and U04) were identified. The frequency of individuals with U02 was significantly higher in patients than in controls (P = 0.0025). Radiographs of hands/wrists/feet were available for 72 patients after 1 year and for 73 patients after 2 years. When the HLA-DRB1 genotype was analyzed simultaneously, patients possessing U02 without an HLA-DRB1 shared epitope (SE) (U02+SE-) showed the lowest progression of Larsen's score (12 months). There was no difference in the level of ESR, CRP, or RF at the first visit among U02+SE+, U02+SE-, U02-SE+, and U02-SE- groups. The combination of the polymorphism of the TNFA upstream promoter region and HLA-DRB1 allele was associated with radiological progression in the early stage of RA.

Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways.

Wnts are secreted glycoproteins that control diverse biological processes, such as proliferation, differentiation, and apoptosis. We here found that Wnt5a inhibited apoptosis induced by serum deprivation in primary-cultured human dermal fibroblasts. Anti-apoptotic activity of Wnt5a was not inhibited by a dickkopf-1 (DKK), which blocks the canonical Wnt pathway. On the other hand, loss of function of protein kinase A (PKA), induced by treatment with PKA inhibitors, siRNA-mediated knocking down of endogenous PKA catalytic subunits, or enforced expression of dominant-negative PKA inhibited the Wnt5a anti-apoptotic activity, indicating the involvement of PKA in the Wnt5a anti-apoptotic activity. In agreement, phosphorylation levels of a cAMP response element binding protein (CREB), a representative downstream effector of PKA, the activation of which is known to lead to the pro-survival effects, was elevated by Wnt5a. In addition, Wnt5a increased the nuclear beta-catenin level and treatment with imatinib or ionomycin, either of which blocks the beta-catenin pathway, reduced the anti-apoptotic activity of Wnt5a, together suggesting the simultaneous involvement of the beta-catenin-mediated pathway in the Wnt5a anti-apoptotic activity. Based on another finding indicating that Wnt5a upregulated PKA-mediated phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at serine 9 that caused inactivation of GSK-3beta and subsequently resulted in activation of the beta-catenin pathway, we have speculated that the Wnt5a anti-apoptotic activity may be partially mediated by PKA-mediated phosphorylation of GSK-3beta and subsequent activation of the beta-catenin pathway.

Antidepressant-like effect of Cordyceps sinensis in the mouse tail suspension test.

Cordyceps sinensis (CS) has been known as a component of traditional medicines that elicit various biological effects such as anti-fatigue, immunomodulatory, and hypoglycemic actions. Since it has been well-established that fatigue is closely related to depression, we used the tail suspension test (TST) in mice to examine the antidepressant-like effects of hot water extract (HWCS) and supercritical fluid extract (SCCS) of CS. Immobility time in the TST was reduced by administration of SCCS (2.5-10 ml/kg, p.o.) dose-dependently though it was not reduced by treatment with HWCS (500-2000 mg/kg, p.o.). Neither HWCS nor SCCS altered locomotor activity in the open field test, excluding the possibility that the effect of SCCS is due to activation of locomotion. Pretreatment with prazosin (an adrenoreceptor antagonist) or sulpiride (a dopamine D2 receptor antagonist) reduced the effect of SCCS on the immobility time. In contrast, pretreatment with p-chlorophenylalanine (p-CPA, a serotonin synthesis inhibitor) did not alter the anti-immobility effect of SCCS. The last finding is consistent with an additional observation that SCCS had no effect on head twitch response induced by 5-hydroxy-L-tryptophan in mice. Taken altogether, these results suggest that SCCS may elicit an antidepressant-like effect by affecting the adrenergic and dopaminergic systems, but not by affecting the serotonergic system.

Wnt5a promotes adhesion of human dermal fibroblasts by triggering a phosphatidylinositol-3 kinase/Akt signal.

Frizzled-3 (Fzd3), highly expressed in both the central nervous system (CNS) and skin, plays essential roles in axonal growth and guidance during the CNS development and may be involved in maintenance of skin integrity, although its ligand remains undetermined. In this study, we demonstrate that Wnt5a specifically binds to Fzd3 in vitro and triggers phosphorylation of Akt mediated by phosphatidylinositol-3 kinase (PI3K), but not that of ERK or protein kinase C, in human primary-cultured dermal fibroblasts. We have further found that such Wnt5a/Fzd3-triggered activation of the PI3K/Akt signal promotes integrin-mediated adhesion of human dermal fibroblasts to collagen I-coated dishes. Based on another finding that Wnt5a/Fzd3-triggered activation of the PI3K/Akt signal was blocked by an excess amount of a recombinant Fzd3-cysteine-rich domain (CRD), but not by that of a recombinant Fzd6-CRD, it is concluded that Wnt5a is a natural ligand of Fzd3 that triggers the PI3K/Akt signal and promotes adhesion of human dermal fibroblasts.

Immunogenetic features in 120 Japanese patients with idiopathic inflammatory myopathy.

OBJECTIVE: To examine the role of HLA-DRB1 and tumor necrosis factor (TNF) promoter genotypes in the development and the autoantibody profiles of idiopathic inflammatory myopathy (IIM) in Japanese patients. METHODS: HLA-DRB1 and TNF promoter genotypes were determined, and serum antinuclear autoantibodies were identified in 120 adult Japanese patients with IIM [72 with dermatomyositis (DM), 30 with polymyositis (PM), 18 with myositis overlapping with other collagen vascular diseases], as well as in 265 controls. RESULTS: Forty-two patients (35%) were positive for myositis-specific autoantibodies (MSA), including 37 (31%) for anti-aminoacyl-tRNA synthetase (ARS) autoantibodies. Allele carrier frequency of HLA-DRB1*0803 was increased in the patients with IIM [p = 0.02, corrected p (pc) NS, 23% vs 14%, odds ratio (OR) = 1.9 (95% confidence interval, CI = 1.1-3.2)], with PM [p = 0.006, pc NS, 33%, OR 3.1 (95% CI 1.3-7.1)], and with anti-ARS autoantibodies [27%, p = 0.04, OR 2.3 (95% CI 1.0-5.1)] compared with controls. DRB1*0405 was increased in patients with anti-ARS autoantibodies compared with controls [41% vs 25%, p = 0.04, pc NS, OR 2.1 (95% CI 1.0-4.3)]. TNF promoter genotype was associated with the presence of interstitial lung disease (ILD). The carriage of a TNF-a haplotype formed by -1031C, -863A, and -857C was increased in the patients with ILD versus those without ILD [33% vs 18%, p = 0.05, pc NS, OR 2.3 (95% CI 0.94-5.5)]. CONCLUSION: HLA-DRB1 alleles were associated with development of IIM and MSA in a Japanese population.

Genetic background of Japanese patients with antineutrophil cytoplasmic antibody-associated vasculitis: association of HLA-DRB1*0901 with microscopic polyangiitis.

OBJECTIVE: To examine association of 8 candidate genes with susceptibility to antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in Japanese patients. Little is known on the genetic background of AAV in Japanese patients mainly because of the difficulty in collecting a sufficient number of samples for the genetics study. METHODS: Sixty-nine patients, including 50 with microscopic polyangiitis (MPA), were recruited in a multicenter study. Among them, 64 patients were positive for myeloperoxidase (MPO)-ANCA. Associations of HLA-DRB1, tumor necrosis factor-alpha promoter (TNF), TNF receptor 2 (TNFR2), Fcgamma receptor IIa (FCGR2A), IIb (FCGR2B), IIIa (FCGR3A), IIIb (FCGR3B), and CTLA-4 (CTLA4) polymorphisms were examined in a case-control analysis. RESULTS: A significant association of HLA-DRB1*0901 with MPA (p = 0.0037, OR 2.44, 95% CI 1.33-4.46), as well as with MPO-ANCA positivity (p = 0.0014, OR 2.44, 95% CI 1.41-4.22), was detected. There was no difference in the TNF promoter haplotype frequencies between patients with MPA and controls, excluding the possibility that the association of DRB1*0901 was secondarily caused by linkage disequilibrium with TNF. No association was observed for TNFR2, FCGR, or CTLA4 with MPA, nor with the presence of MPO-ANCA, although the combined genotype FCGR2A-131H/H and 3A-176F/F was increased in patients with MPA (p = 0.025). CONCLUSION: There was an association of HLA-DRB1*0901 with MPA and MPO-ANCA positive vasculitis in Japanese patients.