Modulation of Natural HLA-B*27:05 Ligandome by Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 2 (ERAP2).

The HLA-B*27:05 allele and the endoplasmic reticulum-resident aminopeptidases are strongly associated with AS, a chronic inflammatory spondyloarthropathy. This study examined the effect of ERAP2 in the generation of the natural HLA-B*27:05 ligandome in live cells. Complexes of HLA-B*27:05-bound peptide pools were isolated from human ERAP2-edited cell clones, and the peptides were identified using high-throughput mass spectrometry analyses. The relative abundance of a thousand ligands was established by quantitative tandem mass spectrometry and bioinformatics analysis. The residue frequencies at different peptide position, identified in the presence or absence of ERAP2, determined structural features of ligands and their interactions with specific pockets of the antigen-binding site of the HLA-B*27:05 molecule. Sequence alignment of ligands identified with species of bacteria associated with HLA-B*27-dependent reactive arthritis was performed. In the absence of ERAP2, peptides with N-terminal basic residues and minority canonical P2 residues are enriched in the natural ligandome. Further, alterations of residue frequencies and hydrophobicity profile at P3, P7, and PΩ positions were detected. In addition, several ERAP2-dependent cellular peptides were highly similar to protein sequences of arthritogenic bacteria, including one human HLA-B*27:05 ligand fully conserved in a protein from Campylobacter jejuni These findings highlight the pathogenic role of this aminopeptidase in the triggering of AS autoimmune disease.

Association of rs27044 and rs30187 polymorphisms in endoplasmic reticulum aminopeptidase 1 gene and ankylosing spondylitis susceptibility: A meta-analysis.

BACKGROUND: The findings regarding association of endoplasmic reticulum aminopeptidase 1 (ERAP1) gene polymorphisms and ankylosing spondylitis (AS) susceptibility are inconsistent. Our aim is to appraise and merge the existing evidence on the relationship of rs27044 and rs30187 polymorphisms in ERAP1 gene and susceptibility to AS. METHODS: Electronic databases of PubMed, Web of Science, EMBASE, Cochrane Library, Wanfang, and CNKI were retrieved for relevant publications. The search was conducted from inception to 10 June, 2019. Studies on the association of rs27044 and rs30187 polymorphisms and risk of AS were included. Quality evaluation was carried out using Newcastle-Ottawa Scale (NOS). Summary odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to appraise the associations under allelic and genotypic models. RESULTS: In total, 26 case-control studies with 31 cohorts containing 17 223 AS patients and 36 915 controls were eligible for this meta-analysis. According to NOS, each study received ≥ 5 scores. The pooled data indicated that rs27044 and rs30187 polymorphisms were significantly associated with AS susceptibility in the overall population: rs27044, G versus C, OR = 1.24, 95% CI 1.16-1.33, P<.001; rs30187, T versus C, OR = 1.24, 95% CI 1.17-1.33, P<.001. When stratified by ethnicity, rs27044 appeared to be significantly correlated with AS in both Asians and Caucasians. For rs30187, despite positive association being observed under the allelic model in both Asians and Caucasians, the findings of genotypic comparisons supported the association only existed in Caucasians but not Asians. CONCLUSION: This study suggests that rs27044 and rs30187 polymorphisms are significantly associated with increased risk of AS, especially in Caucasians.

Evaluation of ERAP1 gene single nucleotide polymorphisms in immunomodulation of pro-inflammatory and anti-inflammatory cytokines profile in ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a prototype of chronic inflammatory arthritis termed seronegative spondyloarthropathies that typically affects the joints. Among the non-Human leukocyte antigen (HLA) loci, the strongest association has been observed with Endoplasmic reticulum aminopeptidase 1 (ERAP1) gene single nucleotide polymorphisms (SNPs). Moreover, the effect of ERAP1 gene SNPs on the pro-inflammatory and anti-inflammatory cytokines in AS disease has still been poorly elucidated. In this study, we aimed to determine the association of ERAP1 gene SNPs (rs30187 and rs2287987) with AS risk as well as their effect on the mRNA expression of pro-inflammatory and anti-inflammatory cytokines, with emphasis on the immunoregulation of the IL-17/IL-23 pathway, in an Iranian population. METHODS: We performed Single specific primer (SSP)-PCR for genotyping of 160 AS patients and 160 healthy controls. After isolation of peripheral blood mononuclear cells (PBMCs), total RNA of PBMCs was isolated, complementary DNA (cDNA) was synthesized, and quantitative analyses of mRNA expression of cytokines were performed by Real-time PCR for 40 HLA-B27 positive AS patients and 40 healthy individuals as controls. RESULTS: It was seen that T allele of rs30187 (OR = 1.54, 95% CI = 1.07-2.22, P =  0.017) and C allele of rs2287987 (OR 1.50, 95% CI 1.05-2.14, P = 0.024) were associated with the risk of AS. Both of these alleles were associated more strongly in the HLA-B27 positive AS patients. There was a significant overexpression of mRNAs of pro-inflammatory (IL-17A, IL-17F, IL-23, TNF-α and IFN-γ), while downregulation of anti-inflammatory cytokines (IL-10 and TGF-ß) in PBMCs from 40 HLA-B27 positive AS patients in comparison to controls. AS patients with rs30187 SNP TT genotype expressed mRNA of IL-17A, IL-17F, and IL-23 significantly higher than patents with CT and CC genotypes for this SNP. CONCLUSIONS: This study represented the association of ERAP1 gene rs30187 and rs2287987 polymorphism with the risk of AS. Additionally, it appears that rs30187 polymorphism may be involved in the immunomodulation of the IL-17/IL-23 pathway in the AS disease.

Regulation of osteoblasts by alkaline phosphatase in ankylosing spondylitis.

AIM: Ankylosing spondylitis (AS) is characterized by excessive spinal ankylosis and bone formation. Alkaline phosphatase (ALP) activity is reported to be high in AS, but little is known about the molecular relationship between ALP and AS. The aims of this study were to investigate the relevance of ALP to AS and the role of ALP in the regulation of osteoblast differentiation in AS. METHODS: High-throughput data with accession numbers GSE73754 and GSE41038 were downloaded from the Gene Expression Omnibus. We retrospectively collected and compared the ALP levels of male patients with AS to those of sex- and age-matched healthy controls (HC) and rheumatoid arthritis (RA) patients. Total serum ALP and ALP activity were measured in the AS and RA groups. ALP gene expression and intracellular ALP activity were analyzed in microarray data from primary diseases control (Ct) and AS-bone-derived cells (BdCs) and in vitro experiments. Furthermore, the effect of ALP inhibitor was examined in both primary Ct- and AS-BdCs under osteoblast differentiation. Regulation of runt-related transcription factor 2 (RUNX2) by ALP was also analyzed. RESULTS: Alkaline phosphatase level was higher in AS compared with RA and HC and was associated with radiograph progression. ALP expression was also enriched in the bone tissue of AS patients. Furthermore, AS-BdCs exhibited increasing ALP activity, leading to accelerated osteoblastic activity and differentiation. Intriguingly, inhibition of ALP reduced RUNX2 expression, a master transcriptional factor in osteoblasts, and differentiation status of both primary Ct- and AS-BdCs. Treatment of ALP activator or inhibitor modulated RUNX2 protein level and RUNX2 regulated ALP promoter activity, indicating a reciprocal ALP-RUNX2 positive feedback to regulate osteoblast differentiation. CONCLUSION: Alkaline phosphatase was highly expressed in AS patients, may be involved in the ankylosis of AS, and represents a possible therapeutic target for ankylosis.

MiR-124 regulates osteoblast differentiation through GSK-3ß in ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is a spastic and spinal joint disease with the characteristic of pathological ossification. Bioinformatics analysis demonstrated that there is a complementary binding site between microRNA-124 (miR-124) and the 3'-UTR of glycogen synthase kinase-3ß (GSK-3ß) mRNA. We aimed to investigate the role of miR-124 in regulating GSK-3ß expression, Wnt/ß-catenin pathway activity, and osteoblast differentiation of spinal ligament fibroblasts. PATIENTS AND METHODS: The ligament tissues of AS and the femoral neck fracture patients were collected. MiR-124 and GSK-3ß mRNA expressions were detected by using quantitative Real-time PCR (qRT-PCR). GSK-3ß and ß-catenin protein expressions were detected by using Western blot. Ligament fibroblasts were isolated and induced to differentiate into osteoblasts. Alizarin red S staining (ARS) was used to identify osteoblast differentiation. Expressions of miR-124, GSK-3ß, ß-catenin, Osterix, and runt-related transcription factor 2 (RUNX2) were detected during differentiation. The cells were divided into two groups as agomiR-normal control (NC) transfection group and agomir miR-124 transfection group. Alkaline phosphatase (ALP) activity and Alizarin Red S staining were detected. RESULTS: MiR-124 and ß-catenin expressions in the ligament of AS patients increased, while GSK-3ß level reduced compared with control. MiR-124, ß-catenin, Osterix, and RUNX2 expressions gradually elevated, whereas GSK-3ß level gradually declined following increased osteoblasts differentiation. Antagomir miR-124 transfection significantly up-regulated the expression of GSK-3ß in osteoblast differentiation, significantly decreased the expression of ß-catenin, Osterix, and RUNX2, and significantly inhibited osteoblast differentiation. CONCLUSIONS: MiR-124 decreased and GSK-3ß elevated in AS ligament tissue. Down-regulation of miR-124 expression enhanced GSK-3ß expression, weakened Wnt/ß-catenin pathway activity, and inhibited the differentiation of ligament fibroblasts into osteoblasts.

Inhibiting ex-vivo Th17 responses in Ankylosing Spondylitis by targeting Janus kinases.

Treatment options for Ankylosing Spondylitis (AS) are still limited. The T helper cell 17 (Th17) pathway has emerged as a major driver of disease pathogenesis and a good treatment target. Janus kinases (JAK) are key transducers of cytokine signals in Th17 cells and therefore promising targets for the treatment of AS. Here we investigate the therapeutic potential of four different JAK inhibitors on cells derived from AS patients and healthy controls, cultured in-vitro under Th17-promoting conditions. Levels of IL-17A, IL-17F, IL-22, GM-CSF and IFNγ were assessed by ELISA and inhibitory effects were investigated with Phosphoflow. JAK1/2/3 and TYK2 were silenced in CD4+ T cells with siRNA and effects analyzed by ELISA (IL-17A, IL-17F and IL-22), Western Blot, qPCR and Phosphoflow. In-vitro inhibition of CD4+ T lymphocyte production of multiple Th17 cytokines (IL-17A, IL-17F and IL-22) was achieved with JAK inhibitors of differing specificity, as well as by silencing of JAK1-3 and Tyk2, without impacting on cell viability or proliferation. Our preclinical data suggest JAK inhibitors as promising candidates for therapeutic trials in AS, since they can inhibit multiple Th17 cytokines simultaneously. Improved targeting of TYK2 or other JAK isoforms may confer tailored effects on Th17 responses in AS.

Influence of CYP2C9 and COX-2 Genetic Polymorphisms on Clinical Efficacy of Non-Steroidal Anti-Inflammatory Drugs in Treatment of Ankylosing Spondylitis.

BACKGROUND The aim of this study was to evaluate the relationships of CYP2C9 and COX-2 genetic polymorphisms with therapeutic efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in treatment of ankylosing spondylitis (AS). MATERIAL AND METHODS We enrolled 130 AS inpatients and outpatients in the Arthritis and Rheumatism Department of Peking University First Hospital and 106 healthy people getting routine check-ups between September 2013 and July 2014. CYP2C9 and COX-2 genetic polymorphisms were detected by PCR-RFLP. All AS patients underwent medical treatment and 12-week follow-up treatment. Score differences of BASDAI, ASAS20, ASAS50, and ASAS70 for AS patients with different genotypes before and after treatment were compared. RESULTS In terms of COX-2-1290A/G and -1195G/A gene polymorphism genotype and allele frequency, the case group and control group were obviously different (all P<0.05), but CYP2C9*3 polymorphism genotype and allele frequency were not statistically different between the 2 groups (P>0.05). AS patients had improved BASDAI, ASAS20, ASAS50, and ASAS70 scores after they received NSAID treatment (all P<0.05). Furthermore, the efficacy of NSAID in treatment of AS and COX-2 gene -1290A/G and -1195G/A polymorphism were associated (all P<0.05), but it is not associated with CYP2C9 *3 polymorphism (all P>0.05). CONCLUSIONS COX-2-1290A/G and -1195G/A polymorphism may increase AS risk and they both can be considered as biological indicators for prediction of efficacy of NSAIDs in treatment of AS.

ERAP1 association with ankylosing spondylitis is attributable to common genotypes rather than rare haplotype combinations.

We investigated the proposal that ankylosing spondylitis (AS) is associated with unusual ERAP1 genotypes. ERAP1 haplotypes were constructed for 213 AS cases and 46 rheumatoid arthritis controls using family data. Haplotypes were generated from five common ERAP1 single nucleotide polymorphisms (SNPs)-rs2287987 (M349V), rs30187 (K528R), rs10050860 (D575N), rs17482078 (R725Q), and rs27044 (Q730E). Haplotype frequencies were compared using Fisher's exact test. ERAP1 haplotypes imputed from the International Genetics of AS Consortium (IGAS) Immunochip study were also studied. In the family study, we identified only four common ERAP1 haplotypes ("VRNQE," "MKDRQ," "MRDRE," and "MKDRE") in both AS cases and controls apart from two rare (<0.5%) previously unreported haplotypes. There were no examples of the unusual ERAP1 haplotype combination ("*001/*005") previously reported by others in 53% of AS cases. As expected, K528-bearing haplotypes were increased in the AS family study (AS 43% vs. control 35%), due particularly to an increase in the MKDRQ haplotype (AS 35% vs. control 25%, P = 0.01). This trend was replicated in the imputed Immunochip data for the two K528-bearing haplotypes MKDRQ (AS 33% vs. controls 27%, P = 1.2 × 10-24) and MKDRE (AS 8% vs. controls 7%, P = 0.004). The ERAP1 association with AS is therefore predominantly attributable to common ERAP1 haplotypes and haplotype combinations.

[Correlation of DNase I in serum and synovial fluid with inflammatory activity in patients with rheumatoid arthritis].

OBJECTIVE: To investigate the potential role of deoxyribonuclease I (DNase I) in the pathogenesis of rheumatoid arthritis (RA). METHODS: DNase I activity was measured by radial enzyme-diffusion method in serum samples from 83 RA patients and 60 healthy volunteers and in the synovial fluid (SF) from 27 RA patients and 38 patients with other inflammatory arthritis. SF cfDNA level was measured with Pico Green Kit, and the correlation among DNase I activity, cfDNA level and clinical parameters of RA patients was analyzed. RESULTS: Serum DNase I activity was significantly lower in RA patients than in the healthy control subjects (0.3065∓0.1436 vs 0.4289∓0.1976 U/mL, P<0.001), and was negatively correlated with ESR (r=-0.2862, P=0.0122), CRP (r=-0.2790, P=0.0184) and neutrophil cell counts (r=-0.287, P=0.011). SF DNase I activity was almost negative in patients with RA, ankylosing spondylitis (AS) and gouty arthritis (GA). SF cfDNA level in RA patients was significantly higher than that in patients with osteoarthritis (100.81∓142.98 vs 18.98∓31.40 µg/mL, P=0.002), but similar to that in patients with AS (45.85∓47.67 µg/mL, P=0.428) and GA (162.95∓97.49 µg/mL, P=0.132). In patients with inflammatory arthritis, SF cfDNA level was positively correlated with ESR (r=0.4106, P=0.0116) and CRP (r=0.5747, P=0.0002). CONCLUSION: Impairment of DNase I activity may be responsible for the enhanced NETs generation and plays a role in the pathogenesis of RA.

The serum angiotensin converting enzyme and lysozyme levels in patients with ocular involvement of autoimmune and infectious diseases.

BACKGROUND: Increased serum levels of angiotensin converting enzyme and lysozyme are considered as inflammatory markers for diagnosis of sarcoidosis which is an autoimmune inflammatory disease. The purpose of this study is to evaluate the significance of differences in serum angiotensin converting enzyme and lysozyme levels of patients with ocular involvement of other autoimmune inflammatory and infectious diseases. METHODS: This is a prospective study involving patients with ankylosing spondylitis, behcet's disease, presumed sarcoidosis, presumed latent tuberculosis, presumed latent syphilis, and control group. The serum levels of angiotensin converting enzyme and lysozyme were analyzed by enzyme-linked immunosorbent assay. Bonnferoni analysis was used to assess pairwise comparisons between the groups. RESULTS: There was a significant increase in serum angiotensin converting enzyme level in patients with presumed sarcoidosis compared to ankylosing spondylitis (p = 0.0001), behcet's disease (p = 0.0001), presumed latent tuberculosis (p = 0.0001), presumed latent syphilis (p = 0.0001), and control group (p = 0.0001). The increase in serum lysozyme level was significant for patients with presumed sarcoidosis with respect to ankylosing spondylitis (p = 0.0001), behcet's disease, (p = 0.0001) presumed latent tuberculosis (p = 0.001), presumed latent syphilis (p = 0.033), and control group (p = 0.0001). CONCLUSION: Elevated serum angiotensin converting enzyme levels are significant for patients with presumed sarcoidosis compared to ocular involvement of other autoimmune diseases such as behcet's disease and ankylosing spondylitis, and ocular involvement of infectious diseases such as presumed latent tuberculosis and presumed latent syphilis. However, elevated serum lysozyme level might be also detected in ocular involvement of infectious diseases such as presumed latent tuberculosis and presumed latent syphilis. TRIAL REGISTRATION NUMBER: NCT02627209. Date of registration: 12/09/2015.

Histopathological changes in supraspinous ligaments, ligamentum flava and paraspinal muscle tissues of patients with ankylosing spondylitis.

OBJECTIVE: To examine the histopathological changes in spinal tissues of ankylosing spondylitis (AS) patients. METHODS: Tissue samples from 10 AS patients and 10 control subjects were obtained. Hematoxylin and eosin, picrosirius, Masson and van Gieson stainings were utilized to determine the pathological changes in tissues. Ultrastructural alterations were examined by electronic microscopy. Proteoglycan levels were assessed by enzyme-linked immunosorbent assays (ELISA). Matrix metalloproteinase-3 (MMP-3), transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α) levels were evaluated by immunohistochemistry. RESULTS: Our results demonstrate that the density of collagen fibrils was reduced in the supraspinous ligaments of AS tissue and fibrils were loosely and irregularly organized as compared to a regular distribution of collagen fibrils in controls. In ligamentum flava from AS patients, activated fibroblasts with enlarged nuclei were detected, while the number of elastic fibers was greatly decreased. Paraspinal muscle tissues of AS patients exhibited increased collagen fibril accumulation and atrophy. Significantly decreased proteoglycan and elevated MMP-3 levels were found in supraspinous ligament samples from AS patients (P < 0.01). Additionally, the levels of TGF-ß1 in ligamentum flava and paraspinal muscle tissues of AS patients were increased (P < 0.01). The expression of TNF-α was also upregulated in the ligamentum flavum (P < 0.01), with no significant difference in the paraspinal muscle between control and AS patients (P > 0.05). CONCLUSIONS: Our findings reveal histopathological changes that occur in certain spinal tissues of AS patients and suggest that increased levels of MMP-3 and TGF-ß1 may contribute to the pathogenesis of AS.

ERAP1 Gene Expression Is Influenced by Nonsynonymous Polymorphisms Associated With Predisposition to Spondyloarthritis.

OBJECTIVE: Several polymorphisms in ERAP1 are strongly associated with susceptibility to spondyloarthritis (SpA). The combination of rs17482078, rs10050860, and rs30187 results in the construction of 3 major haplotypes that are associated with SpA (the "protective" haplotype T/T/C, the "neutral" haplotype C/C/C, and the "susceptibility" haplotype C/C/T). The aim of the present study was to determine whether such haplotypes might affect endoplasmic reticulum aminopeptidase 1 (ERAP-1) messenger RNA (mRNA) expression, protein level, and/or enzymatic activity in antigen-presenting cells, a type of cell that is potentially relevant to disease pathogenesis. METHODS: Monocyte-derived dendritic cells (DCs) were generated in 2 cohorts (a discovery cohort and a replication cohort) comprising a total of 23 SpA patients and 44 healthy controls. Lymphoblastoid B cell lines were established from individuals who were homozygous for the risk, the neutral, or the protective ERAP1 haplotype, respectively. In those samples, we investigated the relationship between ERAP1 haplotypes and mRNA expression level. We also used Western blot analysis to measure the relative protein expression of ERAP-1 and a fluorogenic assay to measure its enzymatic activity. RESULTS: In monocyte-derived DCs, there was a strong association between ERAP1 haplotypes and the ERAP-1 mRNA expression level, with higher levels in subjects harboring the susceptibility haplotype (P = 0.001 and P = 5.6 × 10(-7) in the discovery and replication cohorts, respectively). In lymphoblastoid B cell lines, we observed a significant correlation between haplotype risk score and ERAP1 transcript or protein level (P = 0.003, ρ = 0.92 for both). Enzymatic activity followed a similar trend both in monocyte-derived DCs and in lymphoblastoid B cell lines. CONCLUSION: These data provide strong evidence that SpA-associated ERAP1 polymorphisms affect the level of gene expression in antigen-presenting cells. How increased production/activity of ERAP-1 may influence susceptibility to SpA remains to be determined.

A functional variant of PTPN22 confers risk for Vogt-Koyanagi-Harada syndrome but not for ankylosing spondylitis.

BACKGROUND: Protein tyrosine phosphatase non-receptor 22 (PTPN22) is a key negative regulator of T lymphocytes and has emerged as an important candidate susceptibility factor for a number of immune-related diseases. This study aimed to examine the predisposition of PTPN22 SNPs to Vogt-Koyanagi-Harada (VKH) syndrome and acute anterior uveitis (AAU) associated with ankylosing spondylitis (AS). METHODS: A total of 1005 VKH syndrome, 302 AAU+AS+ patients and 2010 normal controls among the Chinese Han population were enrolled in the study. Genotyping, PTPN22 expression, cell proliferation, cytokine production and cell activation were examined by PCR-RFLP, Real-time PCR, CCK8, ELISA and Flow cytometry. RESULTS: The results showed significantly increased frequencies of the rs2488457 CC genotype and C allele but a decreased frequency of the GG genotype in VKH syndrome patients (PBonferroni correction (Pc) = 3.47×10(-7), OR = 1.54; Pc = 3.83×10(-8), OR = 1.40; Pc = 6.35×10(-4), OR = 0.62; respectively). No significant association of the tested SNPs with AAU+AS+ patients was observed. Functional studies showed a decreased PTPN22 expression, impaired cell proliferation and lower production of IL-10 in rs2488457 CC cases compared to GG cases (Pc = 0.009, Pc = 0.015 and Pc = 0.048 respectively). No significant association was observed concerning T cell activation and rs2488457 genotype. CONCLUSIONS: The study showed that a functional variant of PTPN22 confers risk for VKH syndrome but not for AAU+AS+ in a Chinese Han population, which may be due to a modulation of the PTPN22 expression, PBMC proliferation and IL-10 production.

The active form of MMP-3 is a marker of synovial inflammation and cartilage turnover in inflammatory joint diseases.

BACKGROUND: Matrix metalloproteinase-3 (MMP-3) plays an important role in the pathology of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Measurement of active MMP-3 in clinical samples could provide information about progression of rheumatoid diseases, and potentially response to treatment. Hence, we aimed to develop a sensitive assay specifically measuring the active form of MMP-3 (act-MMP-3) both in ex vivo models and in human sera. METHODS: A monoclonal antibody against the first 6 amino acids of act-MMP-3 was developed, and the specificity was carefully tested by comparing total and active MMP-3. A technically robust act-MMP-3 ELISA was produced. For biological validation, human synovial membrane and human cartilage explant (HEX) culture models were measured and compared by ELISA and immunoblots. For clinical relevance, the serum levels of act-MMP-3 in AS and RA patients before and after anti-TNF-α treatment were evaluated. RESULTS: A highly specific and technically robust ELISA detecting act-MMP-3 in serum was developed. The lower limit of detection was 33.7 pg/mL. The dilution and spiking recovery of human serum was within 100 ± 20%. The average intra- and inter-assay variations were 3.1% and 13.5% respectively.High levels of act-MMP-3 expression were observed in human synovial membrane culture and oncostatin M and TNF-α stimulated human cartilage. In a cross-sectional study of both AS and RA patients, serum act-MMP-3 level was correlated with C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). In addition, in patients receiving anti-TNF-α treatment, the serum level of act-MMP-3 was significantly reduced compared to baseline level reflecting the anti-inflammatory effects of the treatment. CONCLUSION: We have successfully developed an assay measuring act-MMP-3 in human serum showing correlation to inflammatory markers. Further studies are required to clarify, whether act-MMP-3 can serve as a predictive marker for outcome in chronic rheumatoid disorders.

Is there a relationship between endothelial nitric oxide synthase gene polymorphisms and ankylosing spondylitis?

OBJECTIVE: Nitric oxide is produced by endothelial nitric oxide synthase, and its production can be influenced by polymorphisms of the endothelial nitric oxide synthase gene. Because candidate genes responsible for susceptibility to ankylosing spondylitis are mostly unknown and available data suggest that there may be problems related to the nitric oxide pathway, such as endothelial dysfunction and increased asymmetric dimethylarginine, this study aimed to assess the association of common endothelial nitric oxide synthase gene polymorphisms with ankylosing spondylitis. METHODS: One hundred ninety-four unrelated Turkish ankylosing spondylitis patients and 113 healthy without apparent cardiovascular disease, hypertension or diabetes mellitus were included. All individuals were genotyped by PCR-RFLP for two single-nucleotide polymorphisms, namely 786T>C (rs2070744, promoter region) and 786 Glu298Asp (rs1799983, exon 7). Variable numbers of tandem repeat polymorphisms in intron 4 were also studied and investigated by direct electrophoresis on agarose gel following polymerase chain reaction analysis. The Bath ankylosing spondylitis metrology index of the patients was calculated, and human leukocyte antigen B27 was studied. RESULTS: All studied polymorphisms satisfied Hardy-Weinberg equilibrium. Sex distributions were similar between the patient and control groups. No significant differences were found in the distributions of allele and genotype frequencies of the studied endothelial nitric oxide synthase polymorphisms between patients and controls. There were no correlations between endothelial nitric oxide synthase polymorphisms, disease duration, Bath ankylosing spondylitis metrology index or human leukocyte antigen B27. CONCLUSION: The results presented in this study do not support a major role of common endothelial nitric oxide synthase polymorphisms in Turkish ankylosing spondylitis patients.

Association of mineralization-related genes TNAP and ANKH polymorphisms with ankylosing spondylitis in the Chinese Han population.

The aim of this study was to investigate two mineralization-related genes TNAP and ANKH polymorphisms associated with ankylosing spondylitis (AS) in the North Chinese Han population. We carried out a case-control study in Chinese AS cohorts involving 278 AS patients and 286 unrelated healthy controls. Five TNAP SNPs (rs3200254, rs1256348, rs1472563, rs1780329, rs3767155) and four ANKH SNPs (rs25957, rs26307, rs27356, rs28006) were genotyped by the Multiplex Snapshot method. There were significant differences in genotype (permutated p = 0.00481) and allele (permutated p = 0.0126) frequencies of the rs26307 ANKH SNP between AS patients and controls. Logistic regression analysis suggested an association of AS with the polymorphism in an additive model (OR = 0.640, 95%CI = 0.480-0.853, p = 0.0023, permutation 10,000 corrected p = 0.0158) and a dominant model (OR = 0.599, 95%CI = 0.423-0.846, p = 0.0037, permutation 10,000 corrected p = 0.022). Haplotype analysis identified the ANKH haplotype rs26307(C)/rs27356 (T) as a predisposing factor for AS (OR = 1.53, 95%CI = 1.165-2.071, p = 0.0026, permutation 10,000 corrected p = 0.0103). This study provides evidence that variation in the ANKH gene influences susceptibility to AS in the Northern Han Chinese population.

The development and characterization of an ELISA specifically detecting the active form of cathepsin K.

OBJECTIVE: Cathepsin K plays essential roles in bone resorption and is intensely investigated as a therapeutic target for the treatment of osteoporosis. Hence an assessment of the active form of cathepsin K may provide important biological information in metabolic bone diseases, such as osteoporosis or ankylosing spondylitis. METHODS: Presently there are no robust assays for the assessment of active cathepsin K in serum, and therefore an ELISA specifically detecting the N-terminal of the active form of cathepsin K was developed. RESULTS: The assay was technically robust, with a lowest limit of detection (LOD) of 0.085 ng/mL. The average intra- and inter-assay CV% were 6.60% and 8.56% respectively. The dilution recovery and spike recovery tests in human serum were within 100±20% within the range of the assay. A comparison of latent and active cathepsin K confirmed specificity towards the active form. Quantification of the levels of active cathepsin K in supernatants of purified human osteoclasts compared to corresponding macrophages showed a 30-fold induction (p<0.001). In contrast, in serum samples from osteoporotic women on estrogen or bisphosphonate therapy and from ankylosing spondylitis patients no clinically relevant differences were observed. CONCLUSION: In summary, we have developed a robust and sensitive assay specifically detecting the active form of cathepsin K; however, while it monitors osteoclasts with high specificity in vitro, it appears that circulating levels of active cathepsin K do not reflect bone changes under these circumstances.

Evaluation of paraoxonase and arylesterase activities in Egyptian patients with ankylosing spondylitis.

The role of paraoxonase (PON) and arylesterase (ARE) in the pathogenesis of inflammatory arthritis has been investigated, and their serum levels have been evaluated, but clinical study concerning PON1 and ARE and ankylosing spondylitis (AS) is little reported in literature. The aim of this study was to investigate PON1 and ARE activities in AS in comparison with healthy persons and their relation with the disease activity parameters. 35 AS patients and 35 healthy controls (matched for age and sex) participated. Disease activity of AS patients was assessed clinically according to the Bath AS disease activity index, and AS functional impairment was assessed using the Bath AS Functional Index. Serum samples were collected from all subjects to evaluate serum PON1, ARE activities, and lipid profile. The mean serum triglycerides, total cholesterol, and low density lipoprotein (LDL) were significantly higher in the AS patients than in controls, while the high-density lipoprotein (HDL) is significantly lower in the AS patients than controls. Serum PON1 and ARE activities were significantly lower in AS patients than in controls, while malondialdehyde (MDA) was significantly higher. AS patients with active disease had significantly higher serum triglycerides, total cholesterol, LDL and MDA while lower HDL, PON1 and ARE than those with no active AS. Decrease in the PON1/ARE activity leading to generation of oxidative stress may play an important role in the pathogenesis of AS. Moreover, it seems that activity of PON1/ARE in patients with AS is strictly correlated with the activity of the inflammatory process.

Is there a relationship between endothelial nitric oxide synthase gene polymorphisms and ankylosing spondylitis?

OBJECTIVE: Nitric oxide is produced by endothelial nitric oxide synthase, and its production can be influenced by polymorphisms of the endothelial nitric oxide synthase gene. Because candidate genes responsible for susceptibility to ankylosing spondylitis are mostly unknown and available data suggest that there may be problems related to the nitric oxide pathway, such as endothelial dysfunction and increased asymmetric dimethylarginine, this study aimed to assess the association of common endothelial nitric oxide synthase gene polymorphisms with ankylosing spondylitis. METHODS: One hundred ninety-four unrelated Turkish ankylosing spondylitis patients and 113 healthy without apparent cardiovascular disease, hypertension or diabetes mellitus were included. All individuals were genotyped by PCR-RFLP for two single-nucleotide polymorphisms, namely 786T>C (rs2070744, promoter region) and 786 Glu298Asp (rs1799983, exon 7). Variable numbers of tandem repeat polymorphisms in intron 4 were also studied and investigated by direct electrophoresis on agarose gel following polymerase chain reaction analysis. The Bath ankylosing spondylitis metrology index of the patients was calculated, and human leukocyte antigen B27 was studied. RESULTS: All studied polymorphisms satisfied Hardy-Weinberg equilibrium. Sex distributions were similar between the patient and control groups. No significant differences were found in the distributions of allele and genotype frequencies of the studied endothelial nitric oxide synthase polymorphisms between patients and controls. There were no correlations between endothelial nitric oxide synthase polymorphisms, disease duration, Bath ankylosing spondylitis metrology index or human leukocyte antigen B27. CONCLUSION: The results presented in this study do not support a major role of common endothelial nitric oxide synthase polymorphisms in Turkish ankylosing spondylitis patients.

Association between endoplasmic reticulum aminopeptidase-1 (ERAP-1) and susceptibility to ankylosing spondylitis in Iran.

Ankylosing spondylitis (AS) is an inflammatory arthritis, which affects mainly spine and sacroiliac joints. According to recent studies, ERAP1 is the second most common candidate gene for AS susceptibility after HLA-B27. The aim of this study was to determine the association of ERAP1 gene polymorphisms with AS in Iranian population.The study group comprised 387 Iranian AS patients and 316 healthy controls from Iran. Using Real Time PCR allelic discrimination method, we genotyped four SNPs (rs30187, rs469876, rs13167972 and rs27434) of ERAP1. We found that rs30187 and rs27434 were significantly associated with AS in Iranian population (P=6×10-5, P=7×10-3, respectively). The rs30187 T/T genotype was associated with AS compared with C/C genotype (P=1.5×10-5). The rs27434 G/G genotype was inversely associated with AS (P=5×10-3). Two specific haplotypes including: rs30187/ rs469876/ rs13167972/ rs27434 TAAA and CAGG were associated with increased and decreased risk of AS in Iranian population, respectively. These results indicated that ERAP1 SNPs and haplotypes were associated with AS in Iranian population.