Association of Killer Cell Immunoglobulin-like Receptor (KIR) Genes and their HLA Ligands with Susceptibility to Takayasu Arteritis in the Iranian Population.

Takayasu arteritis (TA) is a chronic inflammatory disorder characterized by vascular damage and fibrosis in the intima that commonly occurs in the aorta. In many damaged sites in TA patients, natural killer (NK) cells have been shown to be hyperactivated and produce inflammatory cytokines and toxic components. Killer cell immunoglobulin-like receptors (KIRs) are found on NK cells and interact with human leukocyte antigen (HLA) class I ligands to activate or suppress NK cells. The present study assessed the possible role of KIR and their HLA ligand genes in susceptibility to TA in Iranian patients. This case-control study included 50 TA patients and 50 healthy subjects. DNA was extracted from whole peripheral blood samples, and polymerase chain reaction with sequence-specific primers (PCR-SSP) was performed to recognize the presence or absence of polymorphism in 17 KIR genes and 5 HLA class I ligands in each participant. Among the KIR and HLA genes, a significant decrease was detected in the frequency of 2DS4 (full allele) in TA patients (38%) compared with healthy controls (82%) (OR=0.13, 95% CI=0.05-0.34). However, none of the KIR and HLA genotypes or the interactions between these genes were associated with susceptibility to TA. The KIR2DS4 gene might be involved in the regulation of activation as well as the production of cytotoxic mediators of NK cells in patients with TA.

A2A adenosine receptor agonist reduced MMP8 expression in healthy M2-like macrophages but not in macrophages from ankylosing spondylitis patients.

BACKGROUND: Ankylosing spondylitis (AS) is an inflammatory autoimmune disease that mostly affects different joints of the body. Macrophages are the predominant cells that mediate disease progression by secreting several pro-inflammatory mediators. Different receptors are involved in macrophages' function including the adenosine receptors (AR). Our main objective in this study was to assess the effect of applying A2A adenosine receptor agonist (CGS-21,680) on the gene expression of inflammatory mediators including bone morphogenetic proteins (BMP)-2, 4 and matrix metalloproteinases (MMP)-3, 8, 9, and 13 on the macrophages from AS patients compared to healthy macrophages. METHODS: Monocytes were isolated from the whole blood of 28 individuals (AS patients and healthy controls in a 1:1 ratio). Macrophages were differentiated using macrophage colony-stimulating factor (M-CSF), and flow cytometry was performed to confirm surface markers. CGS-21,680 was used to treat cells that had been differentiated. Using SYBR green real-time PCR, relative gene expression was determined. RESULTS: Activating A2AAR diminished MMP8 expression in healthy macrophages while it cannot reduce MMP8 expression in patients' macrophages. The effect of A2AAR activation on the expression of BMP2 and MMP9 reached statistical significance neither in healthy macrophages nor in the patients' group. We also discovered a significant positive connection between MMP8 expression and patient scores on the Bath ankylosing spondylitis functional index (BASFI). CONCLUSION: Due to the disability of A2AAR activation in the reduction of MMP8 expression in patients' macrophages and the correlation of MMP8 expression with BASFI index in patients, these results represent defects and dysregulations in the related signaling pathway in patients' macrophages.

Efficacy and safety of the biosimilar denosumab candidate (Arylia) compared to the reference product (Prolia®) in postmenopausal osteoporosis: a phase III, randomized, two-armed, double-blind, parallel, active-controlled, and noninferiority clinical trial.

BACKGROUND/OBJECTIVE: Osteoporosis is a global health concern with an increasing prevalence worldwide. Denosumab is an antiresoptive agent that has been demonstrated to be effective and safe in osteoporotic patients. This study aimed to compare the efficacy and safety of the biosimilar denosumab candidate (Arylia) to the originator product (Prolia®) in postmenopausal osteoporotic patients. METHODS: In this randomized, double-blind, active-controlled, noninferiority trial, postmenopausal osteoporotic patients received 60 mg of subcutaneous Arylia or Prolia® at months 0, 6, and 12 and were followed up for 18 months. The primary endpoint was the noninferiority of the biosimilar product to the reference product in the percentage change of bone mineral density (BMD) in 18 months at the lumbar spine (L1-L4), total hip, and femoral neck. The secondary endpoints were safety assessment, the incidence of new vertebral fractures, and the trend of bone turnover markers (BTMs). RESULTS: A total of 190 patients were randomized to receive either biosimilar (n = 95) or reference (n = 95) denosumab. In the per-protocol (PP) analysis, the lower limits of the 95% two-sided confidence intervals of the difference between Arylia and Prolia® in increasing BMD were greater than the predetermined noninferiority margin of - 1.78 at the lumbar spine, total hip, and femoral neck sites (mean differences [95% CIs] of 0.39 [- 1.34 to 2.11], 0.04 [- 1.61 to 1.69], and 0.41 [- 1.58 to 2.40], respectively). The two products were also comparable in terms of safety, new vertebral fractures, and trend of BTMs. CONCLUSION: The efficacy of the biosimilar denosumab was shown to be noninferior to that of the reference denosumab, with a comparable safety profile at 18 months. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03293108 ; Registration date: 2017-09-19.

Prototypic P2X7 Receptor Agonist, BzATP, Induced the Expression of Unfolded Protein Response Genes in Human M1 Macrophages.

Purinergic receptors stimulation by adenosine triphosphate (ATP) contributes significantly to macrophage activation, and also macrophage cell death. Upon the macrophage activation, the protein load of the endoplasmic reticulum is increased which is resulted in the activation of unfolded protein response (UPR). In the current study, we aimed to evaluate the connection between prototypic P2X7 receptor agonist, extracellular 2'(3')-O-(4-Benzoylbenzoyl)-ATP (BzATP), and the UPR pathway in macrophages. The monocyte-derived macrophages from blood samples of 14 healthy volunteers were skewed toward M1 macrophages after incubation with LPS and IFN-γ. M1 macrophages were treated with 200 µM BzATP. The expression levels of UPR genes, including CHOP, HERP, GADD34, XBP1, and ATF6 in macrophages before and after treatment were measured using real-time polymerase chain reaction. The results demonstrated that the expression of CHOP, HERP, and ATF6 is significantly decreased and the expression level of GADD34 and XBP1 is significantly increased after M1 polarization. BzATP not only significantly increased the expression levels of CHOP, GADD34, ATF6, and HERP but also significantly decreases the XBP1 expression level in M1 macrophages. The present study showed that BzATP induces cellular stress in M1 macrophages by elevating the expression levels of UPR genes including CHOP, GADD34, ATF6, and reducing cell viability.

Upregulation of Unfolded Protein Response and ER Stress-Related IL-23 Production in M1 Macrophages from Ankylosing Spondylitis Patients.

The inflammatory interleukin (IL)-23/IL-17 axis plays an important role in the pathogenesis of ankylosing spondylitis (AS), but with an unknown regulatory mechanism. This study aimed to investigate the role of endoplasmic reticulum (ER) stress and autophagy pathway in the expression of IL-23 in peripheral blood-derived macrophages in AS patients. Peripheral blood samples were obtained from 15 AS and 15 healthy control subjects. MACS was used to isolate monocytes from PBMCs. Then, M-CSF was used to differentiate monocytes to M2 macrophages. IFN-γ and/or LPS were used to activate macrophages and M2 polarization towards M1 macrophages. Thapsigargin was used to induce ER stress and 3-MA to inhibit autophagy. The purity of extracted monocytes and macrophage markers was evaluated by flow cytometry. mRNA expression of HLA-B and-B27, ER stress-related genes, autophagy-related genes, and IL-23p19 was performed using RT-qPCR. Soluble levels of IL-23p19 were measured using ELISA. Significant increase in mRNA expression of HLA-B, HLA-B27, BiP, XBP1, CHOP, and PERK mRNAs was observed in macrophages of AS patients before and after stimulation with IFN-γ and LPS. No significant change in autophagy gene expression was detected. mRNA and soluble levels of IL-23p19 demonstrated a significant increase in macrophages of AS patients compared to healthy subjects. ER stress induction led to a significant increase in IL-23p19 in macrophages. Inhibition of autophagy did not affect IL-23 expression. ER stress, unlike autophagy, is associated with increased IL-23 levels in macrophages of AS patients.Key Messages ER stress in macrophages from AS patients plays a role in the increased production of IL-23. The autophagy pathway is not involved in the modulation of IL-23 production by AS macrophages.

Monocyte-derived and M1 macrophages from ankylosing spondylitis patients released higher TNF-α and expressed more IL1B in response to BzATP than macrophages from healthy subjects.

Macrophages participate in the pathogenesis of ankylosing spondylitis (AS) by producing inflammatory cytokines. Extracellular adenosine triphosphate (eATP), released during cell stress, acts through purinergic receptors (P2XR and P2YR) and induces inflammatory responses. We investigated the effect of 2'(3')-O-(4-benzoyl benzoyl) ATP (BzATP) (a prototypic agonist of P2X7R) on the production of inflammatory cytokines in both monocyte-generated (M2-like) and M1 macrophages from patients and controls. Macrophages were differentiated from isolated periphery-monocytes (n = 14 in each group) by macrophage colony-stimulating factor (M-CSF). Using LPS and IFN-γ, macrophages were skewed toward M1 type and were treated with BzATP. Gene expression and protein release of IL-1ß, IL-23, and TNF-α were evaluated by real-time PCR and ELISA methods respectively before and after treatment. BzATP significantly increased the protein release of TNF-α and the expression of TNFA and IL1B in monocyte-generated macrophages. Besides, BzATP treatment significantly upregulated IL1B expression, reduced TNFA and IL23A expression, and TNF-α release in M1 macrophages from both groups. Monocyte-generated and M1 macrophages from AS patients released higher TNF-α and expressed more IL1B in response to the same concentration of BzATP treatment respectively. Based on our results, AS macrophages were more sensitive to BzATP treatment and responded more intensively. Besides, the diverse effects of BzATP on monocyte-derived and M1 macrophages in our study may represent the differed inflammatory properties of these two groups of macrophages in response to eATP in the body.

Efficacy and safety of Romosozumab in treatment for low bone mineral density: a systematic review and meta-analysis.

Osteoporosis is a chronic skeletal disease with an increasing prevalence. Romosozumab, as a monoclonal anti-sclerostin antibody with a dual function, has been produced. In this meta-analysis, we aimed to examine the efficacy of Romosozumab in patients with low bone mineral density. A systematic search was conducted in the most important electronic search engines like Cochrane Library, PubMed, Web of Science, Scopus, Google Scholar, and ClinicalTrials.gov at the end of July 2019 to retrieve randomized controlled trials (RCTs), which evaluated the effect of Romosozumab in patients with osteoporosis and/or low bone mineral density. After evaluating the quality of articles with the Cochrane checklist, data related to the outcomes of bone mineral density (BMD) of lumbar spine, femoral neck, and total hip, risk of clinical, vertebral and non-vertebral fractures, and risk of adverse events were extracted. Quality of evidence was assessed according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. Heterogeneity between studies was evaluated by I2 and Q statistics. The meta-analysis was performed using CMA v.2.0 software. Of all the 671 initially retrieved articles, seven articles were entered into the meta-analysis after removing duplicates and reviewing papers with inclusion and exclusion criteria. The results of the meta-analysis showed that Romosozumab 210, 140, and 70 mg compared with Alendronate, Teriparatide, and placebo can increase the bone mineral density in the lumbar spine, femoral neck, and total hip. The risk of adverse events like adjudicated cardiovascular serious adverse events and adjudicated cardiovascular death was more in Romosozumab 210 mg in comparison with placebo. However, this difference was not statistically significant. Treatment with anti-sclerostin antibodies can be a proper therapeutic option in patients with osteoporosis and low bone mineral density. Based on the results of this meta-analysis, it seems that Romosozumab, with its dual function, has a positive role in the treatment of osteoporosis and low bone mineral density.

Identification of RELN variant p.(Ser2486Gly) in an Iranian family with ankylosing spondylitis; the first association of RELN and AS.

Ankylosing spondylitis (AS) is a common complex inflammatory disease; however, up to now distinct genes with monogenic pattern have not been reported for this disease. In the present study, we report a large Iranian family with several affected members with AS. DNAs of the three affected and two healthy cases were chosen for performing whole-exome sequencing (WES). After several filtering steps, candidate variants in the following genes were detected: RELN, DNMT1, TAF4ß, MUC16, DLG2, and FAM208. However, segregation analysis confirmed the association of only one variant, c.7456A>G; p.(Ser2486Gly) in the RELN gene with AS in this family. In addition, in silico predictions supported the probable pathogenicity of this variant. In this study, for the first time, we report a novel variant in the RELN gene, c.7456A>G; p.(Ser2486Gly), which completely co-segregates with AS. This association suggests potential insights into the pathophysiological bases of AS and it could broaden horizons toward new therapeutic strategies.

Activation of adenosine A2A receptor induced interleukin-23 mRNA expression in macrophages of ankylosing spondylitis patients.

BACKGROUND: Ankylosing spondylitis (AS) is an auto-inflammatory debilitating disorder with a complex pathogenesis. The adenosinergic pathway is an immunologic regulating pathway with a potential role in AS pathophysiology. In the present study, we have aimed to investigate the influence of A2A adenosine receptor (A2AAR) activation on tumor necrosis factor-α (TNF-α) and interleukin-23 (IL-23) expression and secretion by monocyte-generated macrophages of AS patients. METHODS: Whole-blood separated monocytes were extracted from 14 AS patients and 14 healthy controls. Macrophages were differentiated by macrophage colony-stimulating factor (M-CSF), and surface markers were confirmed by flow cytometer. Cells were treated with CGS-21680 as a known agonist of A2AAR. Analysis of ADORA2A, TNFA, and IL23A gene expression was performed by SYBR green real-time PCR. The concentration of secreted cytokines was also measured by ELISA kits. RESULTS: Based on our analysis, CGS-21680 significantly decreased TNF-α secretion by monocyte-derived macrophages of AS patients. Moreover, A2AAR agonist increased the IL23A mRNA expression level in monocyte-derived macrophages of AS patients considerably. Whereas, CGS-21680 did not have any influence on macrophages of healthy individuals. CONCLUSION: According to our results, it appears that A2AAR activation can increase IL-23 secretion by monocyte-derived macrophages of AS patients. Although the TNF-α reducing effect of A2AAR agonists can be a potential target in AS treatment, robust increasing of IL-23 should be considered as the undesirable effect of these agents.

P2 receptors mRNA expression profiles in macrophages from ankylosing spondylitis patients and healthy individuals.

BACKGROUND: Ankylosing spondylitis (AS) is a multifactorial rheumatic disease which mainly involves the axial skeleton. Macrophages and extracellular nucleotides have been shown to contribute to the inflammation process in autoimmune diseases. Membrane-bound purinergic P2 receptors might be involved in the modulation of immune cells in AS. Therefore, we aimed to analyze the messenger RNA (mRNA) expression of P2 receptors in the macrophages of AS patients and healthy controls. METHODS: Twenty-three AS patients and 23 age- and sex-matched healthy individuals were included in our study. Whole blood-separated monocytes of study participants were stimulated by macrophage colony-stimulating factor for 7 days and differentiated to macrophages. Monocyte and macrophage markers were analyzed by flow cytometry. SYBR green real-time polymerase chain reaction was used to measure the relative expression levels of P2RX1 , P2RX2 , P2RX3 , P2RX4 , P2RX5 , P2RX6 , P2RX7 , P2RY1 , P2RY2 , P2RY4 , P2RY6 , P2RY11 , P2RY12 , P2RY13 , P2RY14 , and PANX1 genes. RESULTS: P2RY13 and P2RY6 genes had the highest expression levels in macrophages among P2RY genes. P2RY1 mRNA expression was significantly down-regulated (-1.75 fold) and P2RY14 was up-regulated (2.6 fold) in macrophages of AS patients compared to healthy individuals. P2RX4 gene had the highest expression in monocyte-derived macrophages, followed by P2RX7 and P2RX1  genes. There was no significant difference in P2X receptor mRNA expression level between macrophages of AS patients and healthy individuals. CONCLUSIONS: Our results indicate that AS patients show altered expression levels of P2 receptor genes. Moreover, these changes might be associated with disease activity and patients' status.

Genome-wide association study in Turkish and Iranian populations identify rare familial Mediterranean fever gene (MEFV) polymorphisms associated with ankylosing spondylitis.

Ankylosing spondylitis (AS) is a highly heritable immune-mediated arthritis common in Turkish and Iranian populations. Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disease most common in people of Mediterranean origin. MEFV, an FMF-associated gene, is also a candidate gene for AS. We aimed to identify AS susceptibility loci and also examine the association between MEFV and AS in Turkish and Iranian cohorts. We performed genome-wide association studies in 1001 Turkish AS patients and 1011 Turkish controls, and 479 Iranian AS patients and 830 Iranian controls. Serum IL-1ß, IL-17 and IL-23 cytokine levels were quantified in Turkish samples. An association of major effect was observed with a novel rare coding variant in MEFV in the Turkish cohort (rs61752717, M694V, OR = 5.3, P = 7.63×10(-12)), Iranian cohort (OR = 2.9, P = 0.042), and combined dataset (OR = 5.1, P = 1.65×10(-13)). 99.6% of Turkish AS cases, and 96% of those carrying MEFV rs61752717 variants, did not have FMF. In Turkish subjects, the association of rs61752717 was particularly strong in HLA-B27-negative cases (OR = 7.8, P = 8.93×10(-15)), but also positive in HLA-B27-positive cases (OR = 4.3, P = 7.69×10(-8)). Serum IL-1ß, IL-17 and IL-23 levels were higher in AS cases than controls. Among AS cases, serum IL-1ß and IL-23 levels were increased in MEFV 694V carriers compared with non-carriers. Our data suggest that FMF and AS have overlapping aetiopathogenic mechanisms. Functionally important MEFV mutations, such as M694V, lead to dysregulated inflammasome function and excessive IL-1ß function. As IL-1 inhibition is effective in FMF, AS cases carrying FMF-associated MEFV variants may benefit from such therapy.

The safety and efficacy of Guluronic acid (G2013) in ankylosing spondylitis: A randomized controlled parallel clinical trial.

BACKGROUND: To assess the therapeutic efficacy, safety and tolerability of Guluronic acid (G2013) in patients with ankylosing spondylitis (AS) patients. METHODS: This investigation was a 12-week randomized, placebo-controlled, phase I/II clinical trial involving 75 AS patients that were randomly divided into 3 groups: 25 as placebo, 25 Guluronic acid and 25 naproxen groups. Patients who had AS with active disease at baseline according to the modified New York criteria were considered for this trial. The primary consequence measure was the Appraisement of Spondyloarthritis International Society (ASAS) 20 response-rate at week 12. RESULTS: There were no statistically significant differences between groups at the entry. ASAS20 response at week 12 was achieved (60.8%) in patients receiving Guluronic acid compared with - (68.4% of) - patients in the naproxen group (p > 0.05) and (21.0%) of patients in the placebo group. In comparison with the placebo group from the baseline to week 12, patients who received Guluronic acid and naproxen showed significantly greater improvement in all secondary endpoints. Moreover, Guluronic acid decreased some inflammatory parameters more dramatically than naproxen and placebo group. Patients in the naproxen group had more incidence of gastrointestinal and others adverse events in comparison with Guluronic acid and placebo groups. CONCLUSION: The present research indicated that Guluronic acid and naproxen are similar in terms of efficacy. However, Guluronic acid had more notable safety characteristics identifying information than naproxen. Accordingly, it is proposed that Guluronic acid could be appropriate for management of AS. Clinical trial identifier; IRCT2016091813739N4.

microRNA involvement in the regulation of survivin in peripheral blood mononuclear cells from rheumatoid arthritis patients.

AIM: Impaired regulation of immune tolerance results in autoimmune diseases, such as rheumatoid arthritis (RA). Survivin is an anti-apoptotic protein and can induce cellular mitosis. In the current study, we assessed the transcript level of total survivin (survivin-TS) and its three major variants and evaluated the expression level of important micro RNAs (miRNAs) involved in survivin expression regulation in RA patients. METHOD: Peripheral blood mononuclear cells (PBMCs) were isolated from 50 healthy controls and 50 RA-active patients. RNA extraction was performed and then single-strand complementary DNA was synthesized. Quantitative real-time polymerase chain reaction was used to assess the expression level of survivin-TS and its variants with effective miRNAs in PBMCs. RESULTS: Overexpression of survivin-2B (fold change = 1.57, P = 0.005), survivn-ΔEx3 (fold change = 1.93, P = 0.009) and downregulation of survivin-WT (fold change = 0.64, P = 0.0002) were found in PBMCs of patients, while messenger RNA (mRNA) expression of survivin-TS had no significant difference between RA patients and controls. Expression levels of miR-335-5p, miR-485-5p, miR-16-5p, miR-150-5p, miR-34a-5p, and miR-203a-3p were significantly increased in PBMCs from patients compared with healthy controls. In a correlation study, dysregulation of these miRNAs were not correlated with mRNA expression level of survivin. CONCLUSION: While survivin-TS was not differently expressed in RA patients, its variants had altered expression. Although miRNAs were aberrantly expressed in PBMCs from RA subjects, they did not regulate survivin-TS. miRNAs might be involved in RA pathogenesis, but not through controlling survivin.

Association study of copy number variation in BMP8A gene with the risk of ankylosing spondylitis in Iranian population.

BACKGROUND: Copy number variation (CNV) of DNA segments has been considered as an important component of genetic variation, affecting the quality and quantity of gene expression. Bone morphogenic protein 8A (BMP8A) has been reported to function in bone formation. With respect to the bone and joint complications in ankylosing spondylitis (AS), this investigation aimed to study the role of BMP8A gene CNV in impressing the gene expression as well as the disease risk. METHODS: A total of 900 individuals, including 450 patients with AS and 450 healthy controls were enrolled. The copy numbers of BMP8A gene were detected by TaqMan real-time polymerase chain reaction (PCR) method. BMP8A messenger RNA (mRNA) transcript level in peripheral blood mononuclear cells (PBMCs) was also measured by SYBR Green real-time gene expression PCR method. RESULTS: No significant association of BMP8A copy number was detected with the risk of AS. BMP8A mRNA expression level was significantly downregulated in patients compared with controls. mRNA expression level of BMP8A in both AS patients with and without syndesmophyte was significantly lower than the healthy control group. There was no correlation between the mRNA expression level of BMP8A and both demographic and clinical data of the patients. CONCLUSIONS: Although BMP8A gene expression was downregulated in patients with AS, its copy number could not affect the transcript level of BMP8A gene in PBMCs and was not associated with susceptibility to AS in Iranian population. BMP8a may take into account as an indicator of bone formation process in AS, but it seems that mechanisms other than CNV may regulate this protein.

Increased inflammatory responsiveness of peripheral blood mononuclear cells (PBMCs) to in vitro NOD2 ligand stimulation in patients with ankylosing spondylitis.

Background: Ankylosing spondylitis (AS) is a common debilitating rheumatic disease in which the innate immune components especially the Interleukin (IL)-23/IL-17 axis related genes play important role in its pathogenesis. Nucleotide binding oligomerization domain-containing protein (NOD)2, as an innate receptor, is critical for IL-23 production in cells. Therefore, we aimed to stimulate NOD2 signaling and study its effects on cytokine production in peripheral blood mononuclear cells (PBMC) of these patients. Methods: PBMCs from 18 patients with active AS and 18 healthy individuals were separated by Ficoll-Hypaque density gradient centrifugation and cultured in the presence of muramyl dipeptide (MDP), as NOD2 ligand. Quantitative expression analysis of NOD1, NOD2, RIPK2, SLC15A4, NLRP1, NLRP3, IL23A, IL17A, IL1B, and TNFA genes was performed using Real-time polymerase chain reaction (PCR). Finally, protein changes of IL23A and IL17A expression were validated using enzyme linked immunosorbent assay (ELISA). Results: Apart from NOD1 that tend to be downregulated in the controls, all the selected genes showed overexpression in response to MDP in cells from the studied groups. Except RIPK2, all the genes had higher expression changes upon MDP stimulation in the AS population. Overexpression of IL23A and IL17A were confirmed at protein levels using ELISA. The strong positive correlation between NLRP3 and NOD2 was decreased after stimulation but new correlations between NLRP3 and IL1B, RIPK2 and SLC15A4 were observed after treatment. Conclusions: This study indicated that AS PBMCs were hyper-responsive to MDP stimulation. This observation implies an important role of NOD2 in the pathogenesis of inflammatory diseases including AS.

Association between rs6759298 and Ankylosing Spondylitis in Iranian Population.

BACKGROUND: Ankylosing Spondylitis (AS) is a chronic autoinflammatory Spondyloarthropathy (SpA) which is characterized by sacroiliitis, which progresses to the axial skeleton. It seems that non-Human Leukocyte Antigen (HLA) and also HLA-B27 are associated with the susceptibility and pathogenesis of the disease. The recent Ge-nome-Wide Association Studies (GWASs) have reported intergenic rs6759298 to be associated with AS etiology. The aim of this study was investigation of the rs6759298 polymorphism in Iranian AS patients. In addition, probable correlations with clinical indices and manifestations were considered. METHODS: This study included 403 patients with AS. The control group consisted of 506 healthy individuals who were matched for sex, age, and ethnicity with AS group. Genotyping of rs6759298 was determined using the Amplification-Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR). RESULTS: The GG genotype and G allele were found to be significantly more prevalent in the patient group in comparison to the control group [(p=2×10-6 and 7.44×10-9; OR (95% CI) =2.16 (1.56-2.98) and 1.73 (1.43-2.08)], respectively. CONCLUSION: No associations were found between patients with three genotypes and any disease manifestations or clinical indices. This investigation confirmed a highly significant association of rs6759298 with disease susceptibility, with no effect on disease progress or clinical presentations. Since rs6759298 belongs to the 2p15 gene desert, further studies would elucidate the exact role of this polymorphism in the pathogenesis of AS.

A phase I/II randomized, controlled, clinical trial for assessment of the efficacy and safety of ß-D-mannuronic acid in rheumatoid arthritis patients.

BACKGROUND: Following the potent efficacy of ß-D-mannuronic acid (M2000) in phase I/II trial in ankylosing spondylitis patients, the present clinical trial was conducted to evaluate the efficacy, safety, and tolerability of this novel drug in rheumatoid arthritis (RA) patients who had inadequate response to conventional therapy. METHOD: The study was a 12-week randomized, controlled, phase I/II clinical trial with two treatment arms: M2000 and conventional treatment. Patients who had RA according to the modified American College of Rheumatology (ACR) criteria, with active disease at baseline also inadequate response to conventional therapy, were enrolled in this study. M2000 was administrated at a dose of two capsules (500 mg) per day orally during a period of 12 weeks. The primary endpoint was the proportion of patients fulfilling the ACR 20% improvement criteria after 12 weeks of M2000 therapy. Moreover, the patients were also followed up for safety. RESULTS: There were no statistically significant differences between treatment and conventional groups at baseline characteristics. The ACR20 response rate was significantly higher among M2000-treated patients than conventional-treated control, so that 74% of patients in treatment group showed an ACR20 response after 12 weeks of M2000 therapy (74 versus 16%; P = 0.011). 10% of M2000-treated patients and 57.1% of conventional-treated patient's adverse events occurred during this study. CONCLUSION: Treatment with M2000 in combination with conventional therapy showed a significantly superior efficacy along with a high safety profile compared to conventional-treated patients. Thereby, M2000 might be suggested as a suitable option in the treatment of RA.

Ankylosing spondylitis monocyte-derived macrophages express increased level of A2A adenosine receptor and decreased level of ectonucleoside triphosphate diphosphohydrolase-1 (CD39), A1 and A2B adenosine receptors.

Macrophages play an important role in the ankylosing spondylitis (AS) auto-inflammatory responses and fibrocartilage destruction. Adenosine is a key modulator of inflammatory conditions. The various effects of adenosine are mediated by its interaction with adenosine receptors (AR). In this study, we investigated the mRNA expression of A1, A2A, A2B, and A3 adenosine receptors, ectonucleoside triphosphate diphosphohydrolase-1 (CD39), and ecto-5'-nucleotidase (CD73) in the monocyte-derived macrophages from AS patients in comparison to healthy controls. We also explored the correlation between analyzed gene expression and patients' clinical manifestations. Whole blood-separated monocytes from 23 healthy controls and 23 active AS patients were stimulated by macrophage colony-stimulating factor (M-CSF) for 7 days and differentiated to macrophages. Monocyte and macrophage markers were analyzed by flow cytometry. Analysis of adenosine receptors (ADORA1، ADORA2A، ADORA2B، ADORA3), CD39 and CD73 gene expression was performed by SYBR green real-time PCR. Our results demonstrated monocyte-derived macrophages from AS patients expressed increased level of A2AAR and reduced level of A1, A2BAR, and CD39 mRNA compared to healthy controls. We found an inverse correlation between A2AAR mRNA expression and Bath Ankylosing Spondylitis Functional Index (BASFI) score in AS patients. According to our results, altered expression level of adenosine-relying system would be involved in AS macrophage dysfunction and inflammation and correlated with functional status in AS patients.

The effects of ß-D-mannuronic acid (M2000), as a novel NSAID, on COX1 and COX2 activities and gene expression in ankylosing spondylitis patients and the murine monocyte/macrophage, J774 cell line.

Ankylosing spondylitis (AS) is a debilitating chronic inflammatory disease with genetic predisposition, which is characterized by the involvement of spine and sacroiliac joints. Due to the relatively unsuccessful treatments, we designed ß-D-mannuronic (M2000) with the beneficial effects in various experimental models as a novel non-steroidal anti-inflammatory drug (NSAID). The aims of our present study were: first, to compare the therapeutic effects of M2000, as a novel designed NSAID, with naproxen and placebo in Iranian patients with AS during 12 weeks; second, to evaluate the effect of M2000 on gene expression of cyclooxygenase enzyme (COX-1/COX-2), a key enzyme in the initiation of inflammatory pathways in AS patients; and third, to assess the activity of COX-1 and COX-2 enzymes in the presence/absence of M2000 at the different doses in the murine macrophage, J774 cell line. This was a sub-study of phase II, randomized, placebo-controlled trial with three treatment arms: M2000, naproxen, and placebo. The outcome measures were the mean changes from baseline to week 12. The gene expression was assessed by real-time PCR. The COX-1 and COX-2 activities were evaluated by ELISA in J774 cell line induced by LPS and arachidonic acid (AA). Our findings demonstrated that M2000 had beneficial therapeutic effects on pain, stiffness, and inflammation, whereas no adverse effects were observed following the use of M2000 after 12 weeks. The analysis of gene expression showed that M2000 could effectively reduce the expression levels of COX-1 and COX-2 in comparison with untreated patients. In addition, the enzymatic activities in the presence of M2000 were significantly less than LPS- and AA-treated groups. Our results indicate that M2000, as a novel designed NSAID with immunosuppressive properties, can be considered as one of the therapeutic options for the treatment of inflammatory diseases without adverse events. Clinical trial identifier IRCT2013062213739N1/ http://www.IRCT.ir .

Targeting of circulating Th17 cells by ß-D-mannuronic acid (M2000) as a novel medication in patients with rheumatoid arthritis.

OBJECTIVE: This study aimed at investigating the inhibitory effect of ß-D-mannuronic acid (M2000) on the Th17 circulating levels and IL-17 a related cytokine in rheumatoid arthritis (RA) patients. METHODS: The study included 27 patients with RA who had failed response to treatment. All patients were treated orally by M2000 at a dose of 500 mg twice daily for 12 weeks (Clinical trial identifier: IRCT2014011213739N2). The patients based on anti-tumor necrosis factor alpha (TNFα) blocker treatment were classified into two groups (conventional group and etanercept group). They were then allowed to continue their treatment excluding non-steroidal anti-inflammatory drugs (NSAIDs). The frequency of circulating Th17 cells and IL-17 serum level were determined before and 12 weeks after M2000 therapy and were compared to the healthy controls by using flow cytometry analysis and ELISA method, respectively. RESULTS: At baseline, higher circulating Th17 and IL-17 serum levels were significantly observed in both groups of RA patients than in the healthy controls (all P < 0.001). The frequency of Th17 cells significantly decreased in the conventional group as well as in the etanercept group after M2000 therapy but the level of reduction was higher in the conventional group compared to the etanercept group (P < 0.03 and P < 0.04, respectively). The IL-17 serum level significantly decreased in both groups after M2000 therapy (P < 0.01 and P < 0.02, respectively). Furthermore, the frequency of Th17 cells was positively correlated with Disease Activity Score (DAS28) (r = 0.34, P = 0.02). CONCLUSION: M2000 shows the inhibitory effect on the frequency of circulating Th17 cells as well as in the production of IL-17 in RA patients.