Evaluation of cell adhesion molecules (LFA-1 and L-selectin) in ankylosing spondylitis patients after treatment with ß-D-mannuronic acid (M2000).

Background & objectives: To examine ß-D-mannuronic acid (M2000) effects on L-selectin shedding and leucocyte function-associated antigen-1 (LFA-1) expression as mechanisms of action of this drug in patients with ankylosing spondylitis (AS). Methods: To investigate the molecular consequences of ß-D-mannuronic acid on L-selectin shedding, flow cytometry method was used. Furthermore, the effect of it on LFA-1 gene expression was analyzed by using quantitative real time (qRT)-PCR technique. Results: The LFA-1 expression in patients with AS was higher than controls (P=0.046). The LFA-1 expression after 12 wk therapy with ß-D-mannuronic acid was meaningfully decreased (P=0.01). After 12 wk treatment with ß-D-mannuronic acid, the frequency of CD62L-expressing CD4+ T cells in patients with AS, was not considerably altered, compared to the patients before therapy (P=0.5). Furthermore, after 12 wk therapy with ß-D-mannuronic acid, L-selectin expression levels on CD4+ T-cells in patients with AS, were not remarkably changed, compared to the expression levels of these in patients before treatment (P=0.2). Interpretation & conclusions: The results of this study for the first time showed that ß-D-mannuronic acid can affect events of adhesion cascade in patients with AS. Moreover, ß-D-mannuronic acid presented as an acceptable benefit to AS patients and could aid in the process of disease management.

Immunophenotypic and functional analysis of lymphocyte subsets in common variable immunodeficiency patients without monogenic defects.

Common variable immunodeficiency (CVID) is accompanied by various lymphocyte abnormalities believed to be mostly responsible for disease features in patients with no diagnosed monogenic defects. Here, we evaluated the association of B and T lymphocyte abnormalities with the incidence of CVID. Twenty-six genetically unsolved CVID patients were examined for B and T lymphocyte subsets by flow cytometry and CD4+ T-cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) test. We detected a reduction in total, naive, memory B cells and plasmablasts, and also total, naive, central memory and regulatory CD4+ T cells, besides naive CD8+ T cells. There was an increase in CD21low and transitional B cells, effector memory (EM) and terminally differentiated effector memory (TEMRA ) CD4+ T-cell subsets as well as total, EM, TEMRA , activated and cytotoxic CD8+ T cells among non-monogenic CVID patients. CD4+ T-cell proliferation response was reduced regarding both division index and percent divided. In conclusion, regarding the similarity of lymphocyte abnormalities between patients without genetic defects and those with monogenic defects, genetic mutations are not responsible for these specific lymphocyte changes. However, the novel correlations observed between lymphocyte alterations among genetically unsolved CVID patients may serve as a guide to predict the potential of future CVID development for hypogammaglobulinemia children.

Evaluation of G2013 (α-L-guluronic acid) efficacy on PC-3 cells through inhibiting the expression of inflammatory factors.

Given multiple treatment strategies for prostate cancer, its mortality rate is still high; therefore, novel treatment strategies seem necessary. G2013 or α-L-guluronic acid is a new patented drug with immunomodulatory and anti-inflammatory properties. This study aimed to evaluate the property of G2013 on inflammatory molecules involved in tumorigenesis of prostate cancer. MTT assay was used to assess the effect of the drug on the proliferation of PC-3 cells. Expression of interleukin 8 (IL-8), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), myeloid differentiation factor 88 (MYD-88), cyclooxygenase 2 (COX-2), matrix metalloproteinase-2 (MMP-2), and MMP-9 genes were studied in the PC-3 cells treated with 25 (low dose) or 50 (high dose) µg/mL of G2013 for 24 h using quantitative real-time polymerase chain reaction (qRT-PCR) technique. Protein expression of NF-κB and protein activities of MMP-2 and MMP-9 were assayed using flow cytometry and gelatin zymography, respectively. The expression of COX-2 (p = 0.007 at low dose), MMP-2 (p = 0.023 at low dose, p = 0.002 at high dose), NF-κB (p = 0.004 at low dose) and IL-8 (p < 0.0001 in both doses) genes, NF-κB protein (p < 0.0001 in both doses), and MMP-2 activity (p < 0.0001 in both doses) were significantly reduced in the presence of G2013 as compared to the control group. Cancer cell proliferation was also inhibited under 10-500 µg/mL G2013 treatment. Our results revealed that G2013 has the potential to inhibit PC-3 cell proliferation and reduce the expression of tumour-promoting mediators, COX-2, MMP-2, NF-κB, and IL-8 involved in the progression and metastasis of prostate cancer.

Evaluation of the acute and 28-day sub-acute intravenous toxicity of α-l-guluronic acid (ALG; G2013) in mice.

α-l-Guluronic acid (ALG; G2013) has been previously introduced as a new anti-inflammatory agent with promising therapeutic effects. Thus, in the present study, we aimed to evaluate the acute and sub-acute toxicity of ALG through intravenous (i.v.) administration in Balb/C mice. ALG was administrated i.v. to the mice with doses of 300, 600, and 1000 mg/kg of body weight to investigate acute toxicity (single dose) and with doses of 25, 50, and 100 mg/kg of body weight to sub-acute toxicity study (daily injections for a period of 28 days). The mortality rate, food and water intake, behavior, body weight, gross necropsy, hematological and biochemical parameters as well as histopathological presentations of the vital organs (kidneys, liver, lungs, spleen, and heart) were examined in treated groups and compared to the healthy controls. The results of both acute and sub-acute studies showed that i.v. administrations of ALG did not affect the investigated parameters in both sexes, indicating that the LD50 of ALG was higher than 1000 mg/kg of body weight. As no difference was observed in toxicity profiles of investigated doses, no-observed-adverse-effect-level for i.v. administration of ALG in the sub-acute study was greater than 100 mg/kg body weight in both female and male mice. According to the finding, i.v. administration of ALG did not lead to any clinical sign in abovementioned doses, suggesting that ALG was well tolerated up to 1000 mg/kg. These pre-clinical findings support the application of ALG in the future clinical trials.

A controlled, randomized phase II clinical trial for efficacy and safety evaluation of mannuronic acid in secondary progressive form of multiple sclerosis.

BACKGROUND: The ß-D-Mannuronic acid (M2000) as a novel immunosuppressive drug, patented (PCT/EP2017/067920), has shown positive effects in experimental model of multiple sclerosis (MS). In this study, our aim was to assess efficacy and safety outcomes in MS treated patients with mannuronic acid compared to the conventional drug. METHODS: In a 6-month, randomized controlled, phase II trial, we enrolled patients who had secondary progressive multiple sclerosis (SPMS), were 21-54 years of age, with a score of 1-7 on the Expanded Disability Status Scale (EDSS), and who had at least one relapse in the previous 6 months. Patients were administered orally 1000 mg/day (two 500 mg/capsule daily) of M2000. Endpoints included changes in brain magnetic resonance imaging (MRI) measures and the EDSS score, as compared to the conventional drug (interferon beta-1a, interferon beta-1b). RESULTS: A total of 25 (92.5%) of the M2000 treated patients and 25 conventionally treated patients completed the study. M2000 had better performance compared to the conventional drug regarding to MRI-related measurements, however, the differences between groups were not statistically significant. M2000 decreased the disability progression over the 6-month period. The EDSS score was decreased in the M2000 treated group in the sixth month versus the conventional drug (p < 0.009). Furthermore, we did not observe any short-term side effects. CONCLUSIONS: As compared with the conventional drug, mannuronic acid (M2000) improved the rate of disability progression. This clinical trial demonstrated the efficacy and safety of mannuronic acid in patients with SPMS. (Registered Clinical Trials number, IRCT2016111313739N6).

The effects of guluronic acid (G2013), a new emerging treatment, on inflammatory factors in nonalcoholic steatohepatitis patients under in vitro conditions.

BACKGROUND: Nonalcoholic Steatohepatitis (NASH) results from the accumulation of fatty acids in the liver. The elevated production of pro-inflammatory factors is the reason for the hyper inflammation in NASH. The α-L-Guluronic acid (G2013), a new member of NSAID family, is a plant-originated agent with immunomodulatory properties. The current study investigated the effects of G2013 on inflammatory factors in PBMCs of NASH patients. METHODS: PBMCs of 14 NASH patients and 14 healthy controls were isolated and cultured. The patient's cells were treated with low (5 µg/mL) and moderate (25 µg/mL) doses of G2013 alongside the diclofenac optimum dose (3 µg/mL). The expression and secretion levels of variables were assessed by real-time PCR and ELISA, respectively. RESULTS: Findings indicated that the expression levels of TLR4 and NF-κB, as well as the secretion levels of TNF-α and IL-6 cytokines, were significantly elevated in NASH patients compared to healthy individuals. The expression levels of TLR4 and NF-κB were strikingly downregulated in treated cells of patients in both low and moderate doses of G2013. A considerable reduction was obtained in the secretion level of IL-6 using both low and moderate doses of G2013 and in the secretion level of TNF-α using the moderate dose of G2013. CONCLUSION: The results indicated that G2013 could meaningfully decrease the expression and secretion levels of evaluated factors (TLR4, NF-κB, TNF-α, and IL-6) in PMBCs of NASH cases. Since there is no effective treatment for NASH patients, we hope that G2013 would be a promising immunomodulatory agent in reducing inflammation and improvement of patients.

Anti-tumor effect of M2000 (ß-d-mannuronic acid) on the expression of inflammatory molecules in the prostate cancer cell.

Aim: The importance of chronic inflammation during the progression of prostate cancer (PCa) is well-known. M2000 (ß-d-mannuronic acid) is a novel anti-inflammatory drug. According to its potential capacity for the inhibition of molecules involved in creating conditions of inflammation, it is reasonable to assess the anti-inflammatory role of M2000 in PCa cells.Methods: MTT assay was performed to determine the cytotoxicity of M2000 in PC3 cells. Correspondingly, these cells were cultured and then treated with low (25 µg/ml) and high (50 µg/ml) doses of M2000 as optimal doses. Thereafter, real-time RT-PCR, flow cytometry analysis, and zymography were performed to evaluate the expressions of MYD-88, NF-kB, IL-8, COX-2, MMP-2, and MMP-9 molecules. Results: Of note, the M2000 at the concentration of ≤200 µg/ml had no cytotoxicity effect on the cells. MYD-88 gene expression was significantly down-regulated at both low and high doses in the M2000-treated cells compared to the control (p = .017 and p = .001, respectively). The expression of the NF-kB was also reduced at both the gene and protein levels (all p values were <.001). The expression of IL-8 and COX-2 genes was also down-regulated in the high dose of M2000 (p<.001, p = .001, respectively). The decreased expression of the MMP-9 gene was observed at both doses (both p values were <.001).Conclusion: Inhibitory effects of M2000 on the activity of MMPs in the LPS/M2000-treated cells were evident, but not in the M2000-treated cells. M2000 as a new anti-inflammatory drug appears to constitute a potential agent for down-regulation of inflammatory molecules in the PCa cells.

Evaluation of the oral administration of α-l-guluronic acid on COX-1 and COX-2 gene expression profile in ankylosing spondylitis patients.

Ankylosing spondylitis (AS) is a chronic autoimmune arthritis disease with a genetic background, affecting the skeletal axis, sacroiliac, and peripheral joints. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first-line treatment for AS to alleviate the inflammation and pain. Despite the beneficial effect, their use is accompanied by a wide variety of possible side effects in the gastrointestinal and kidneys. The α-l-guluronic acid (G2013) is a new nonsteroidal anti-inflammatory patented (PCT/EP2017/067920) drug, which has shown its anti-inflammatory properties in the previous investigations. The present study revealed the oral administration effect of G2013 on COX-1 and COX-2 gene expression in AS patients. The blood samples of twelve 18-45 years old patients suffering AS and BASDAI >4, and BASFI >4, before and after 12 weeks of treatment with G2013 and 12 blood samples of healthy volunteers were collected and the effect of G2013 on the gene expression of COX-1 and COX-2 enzymes were assessed by Real-Time PCR. The results indicate that G2013 is able to reduce the gene expression level of COX-1 and COX-2 enzymes in treated AS patients compared to healthy control. Statistically significant differences were not observed between the treatment and the healthy control groups. According to the findings, G2013 might be categorized and introduced as a novel NSAID for the treatment of AS.

Effects of guluronic acid, as a new NSAID with immunomodulatory properties on IL-17, RORγt, IL-4 and GATA-3 gene expression in rheumatoid arthritis patients.

Aim: Rheumatoid arthritis (RA) is a prevalent inflammatory, autoimmune diseases characterized by inflammation and destruction of joints. Disease-modifying anti-rheumatic drugs (DMARDs) and biological drugs can have modulatory interference in disease process. In this study, the effect of Guluronic Acid (G2013) as a novel non-steroidal anti-inflammatory drug (NSAID) with immunomodulatory effects was evaluated on IL-17, RORγt, IL-4 and GATA-3 gene expression in RA patients.Methods: Fourteen patients with RA who had an inadequate response to conventional treatments were included in this clinical trial. During this trial, patients were permitted to continue the conventional therapy excluding NSAIDs. G2013 was administered orally at dose of 500 mg twice daily for 12 weeks. The peripheral blood mononuclear cells (PBMCs) were collected before and after treatment to evaluate the gene expression levels of IL-4, GATA-3, IL-17 and RORγt.Results: Primary and secondary efficacy endpoints and Disease Activity Score (DAS) 28 showed an improvement after 12 weeks of treatment. G2013 has a potent efficacy on gene expression of these molecules, so that it could decrease IL-17 and RORγt levels and increase IL-4 and GATA-3 levels after 12 weeks of treatment. Reduction of IL-17 was statistically non-significant whereas for its transcription factor (RORγt) was statistically significant. Moreover, the gene expression results were in accordance with the clinical and preclinical assessments.Conclusion: G2013 as a natural novel drug showed a significant increase on IL-4 and GATA-3 and a significant decrease on RORγt gene expression after 12 weeks oral administration of this drug in RA patients. (Clinical trial identifier: IRCT2016092813739N5).

The role of ß-d-mannuronic acid, as a new non-steroidal anti-inflammatory drug on expression of miR-146a, IRAK1, TRAF6, NF-κB and pro-inflammatory cytokines following a clinical trial in rheumatoid arthritis patients.

Context: miR-146a, its targets (IRAK1, TRAF6) and NF-κB transcription factor play a fundamental role in rheumatoid arthritis (RA). Positive effects of drug ß-d-mannuronic acid (M2000) were proven on their expression in the HEK-Blue hTLR2 cell line, and results of its phase III clinical trial on RA patients were encouraging.Objective: This research aimed to investigate the effects of M2000 on expression of these genes and serum levels of IL-6 and TNF-α as pro-inflammatory cytokines in RA patients.Material and methods: In this study (Trial Registration Number: IRCT2017100213739N10), 12 RA patients (according to the American College of Rheumatology criteria) and 12 healthy subjects (as control group) were selected. The gene expression of miR-146a, IRAK1, TRAF6, and NF-κB were measured at the baseline and after 12 weeks M2000 therapy, using quantitative real-time PCR method. Moreover, the serum levels of IL-6 and TNF-α were evaluated at the similar times by ELISA method.Results: Our findings showed that the gene expression of miR-146a, IRAK1, TRAF6, and NF-κB significantly decreased after 12 weeks M2000 therapy in RA patients (0.81-, 0.68-, 0.79-, 0.82-fold, with p < .05, p < .01, p < .01, p < .05, respectively). Furthermore, the serum levels of IL-6 and TNF-α significantly reduced in these patients after 12 weeks M2000 therapy (both with p < .05).Conclusions: The present research results determined the part of molecular mechanisms of drug M2000 in RA treatment, based on the expression and function modification of miR-146a, IRAK1, TRAF6, NF-κB, IL-6 and TNF-α.

Immunopharmacological effect of ß-d-mannuronic acid (M2000), as a new immunosuppressive drug, on gene expression of miR-155 and its target molecules (SOCS1, SHIP1) in a clinical trial on rheumatoid arthritis patients.

The positive impacts of ß-d-mannuronic acid (M2000) on the gene expression of miR-155, its target molecules (SOCS1 and SHIP1), and NF-κB transcription factor were demonstrated in a study using the HEK293-TLR2 cell line. This new drug has been approved as a safe and effective medication by a randomized, multinational, phase III clinical trial on RA patients. The present study aimed to evaluate the oral administration effect of M2000 on the expression levels of the mentioned genes in RA patients. This research was conducted on 12 RA patients and 12 healthy individuals. After extraction of total RNA from PBMCs of patients and synthesis of cDNA, the expression levels of miR-155, SOCS1, SHIP1, and NF-κB genes were measured through quantitative Real-time PCR at baseline and after 12 weeks of M2000 therapy. Our findings showed that the miR-155 gene expression level significantly decreased in the M2000-treated patients compared with the baseline (0.76-fold, with p < .05). The expression levels of SOCS1 and SHIP1 genes significantly increased in the patients treated with M2000 compared with the before treatment (1.46-, 1.54-fold, with p < .01, p < .05, respectively). In addition, it was found that the gene expression level of the NF-κB transcription factor significantly reduced in M2000-treated patients compared with the baseline (0.81-fold, with p < .05). This study showed that the oral administration of M2000 was able to reduce the expression of the miR-155, increase the expression of SOCS1 and SHIP1, and decrease the NF-κB gene expression (Trial Registration Number: IRCT2017100213739N10).

An in vitro assessment for evaluating the efficiency of ß-d-mannuronic acid (M2000) in myelodysplastic syndrome.

ß-d-Mannuronic acid (M2000), a novel non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive properties, has been previously shown to exhibit potential therapeutic effects on autoimmune diseases. Immunosuppression therapy has been a standard approach for myelodysplastic syndrome (MDS) for many years. We evaluated the effect of M2000 on isolated peripheral blood mononuclear cells (PBMCs) from patients with MDS. The PBMCs were isolated from 13 patients with MDS and 13 normal donors. The cells were then treated with low, moderate, and high doses of M2000 and diclofenac as a control group. The level of interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), IL-3, granulocyte colony-stimulating factor (G-CSF) gene expression and the serum level of IL-6 and TNF-α production were evaluated by real-time polymerase chain reaction and enzyme-linked immunosorbent assay methods, respectively. Our findings indicated a significant reduction in the production of IL-6 and TNF-α as inflammatory cytokines. Furthermore, the level of G-CSF gene expression was significantly increased. In conclusion, M2000, a newly designed NSAID, has a remarkable effect on isolated PBMC in patients with MDS, which might bring a potential hope for its oral administrations in these patients.

Vitamin D downregulates key genes of diabetes complications in cardiomyocyte.

OBJECTIVE: Vitamin D deficiency has been reported to be associated with the incidence of type 1 and type 2 diabetes and worsening of diabetes complications. This study was designed to investigate the effect of vitamin D treatment on the expression of five key genes involved in the development of diabetic cardiomyopathy. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into three groups. The first group served as control and the other two groups received an intraperitoneal injection of 45 mg/kg streptozotocin (STZ) to develop diabetes. Then groups were treated for 4 weeks either with placebo or vitamin D (two injections of 20,000 IU/kg). Serum levels of glucose, insulin, HbA1c, and advanced glycation end products (AGEs), as well as the gene expression of AGE cellular receptor (RAGE), glyoxalase, aldose reductase, O-GlcNAc transferase (OGT), and glutamine-fructose-6-phosphate aminotransferase (GFAT) and nuclear factor-kB (NF-kB) activity of nuclear extracts were assessed at the end of experiment. RESULTS: Increment in serum cholecalciferol could improve hyperglycaemia and hypoinsulinemia in diabetic rats. In addition, a significant reduction was observed in RAGE, OGT, and GFAT gene expression and NF-kB activity in cardiac myocytes. CONCLUSIONS: Vitamin D might contribute in reducing diabetic cardiomyopathy not only by improving blood glucose and insulin levels but also via downregulating AGE and hexosamine pathways and decreasing NF-kB activity in heart tissue.

A randomized, controlled, phase II clinical trial of ß-D-mannuronic acid (M2000) in pre-surgical breast cancer patients at early stage (T1-T2).

Following the potent efficacy of ß-D-Mannuronic acid in a breast cancer murine model, we evaluated the efficacy of this novel non-steroidal anti-inflammatory drug in breast cancer patients in the present clinical trial. The study was an 8-week randomized, controlled, phase II clinical trial (IRCT: 2017012213739N7 (in 48 pre-surgical breast cancer patients. Patients who had breast cancer at early stage, with invasive ductal carcinoma, were placed on a waiting-list for surgery and were allocated to the study. ß-D-Mannuronic was administrated at a dose of two capsules (1000 mg/d) orally during a period of 8 weeks. The end point of this study was when the patients were admitted for surgery. Moreover, the patients' well-being status was followed up on for safety. There were no statistically significant differences between treatment and non-treatment groups at baseline. ß-D-Mannuronic acid therapy, from 20 patients, showed that in one patient (5%) tumour size was decreased; in five patients (25%) tumour growth was stopped; and in 14 patients (70%) the growth rate in the treatment group did not show significant change, compared to the non-treatment group. Evaluation of two tumour markers (carcinoembryonic antigen and cancer antigen 15-3) showed that there was no significant difference between before and after treatment. Although the use of some non-steroidal anti-inflammatory drugs in a long time period has shown a prophylactic effect in breast cancer, their therapeutic efficacy in a short time period is unknown, whereas treatment with ß-D-Mannuronic acid during 8 weeks could show 30% therapeutic effects in pre-surgical breast cancer patients.

The role of T helper 17 and regulatory T cells in tumor microenvironment.

T helper 17 (Th17) cells were first described as a novel T helper cell lineage independent from Th1 and Th2 subsets. Th17 cells play vital roles in inflammation and tumor immunity. It causes the dissipation of antitumor immunity and contribution to the survival of tumor cells, worsening tumor growth and metastasis. Tumor-infiltrating Th17 cells were seen innumerous cancers in mice and humans. There has been an association between intratumoral Th17 cell infiltration and both good and bad prognoses. Besides the protumoral roles defined for IL-17 andTh17 cells, several reports have shown that Th17 cells also drive antitumoral immunity. Various mechanisms by which Th17 cells control tumor growth are as following: recruitment of several immune cells including DCs, CD4+ T cells, and CD8+ T cells within tumors, activation of CD8+ T cells, and probably plasticity toward Th1 phenotype, related to IFN-γ and TNF-α production. Regulatory T cells (Tregs) have been exhibited to infiltrate human tumors and are believed to restrict antitumor immunity. The effect of Treg cells has been more controversial. Whereas some studies have proposed that a high density of Treg cells within the tumor associated with a poor clinical prognosis, other studies have presented a positive clinical prognosis, underlining the importance of elucidating the clinical significance of Treg cells further. Treg and Th17 cells play both positive and negative roles in regulating antitumor immune responses. In spite of the presence of these cells, yet some tumors develop and grow. These T cells by themselves are not adequate to efficiently mount antitumor immune responses.

Effects of guluronic acid (G2013) on gene expression of TLR2, TLR4, MyD88, TNF-α and CD52 in multiple sclerosis under in vitro conditions.

Context: Multiple sclerosis (MS) is an autoimmune and chronic inflammatory disease of CNS. The α-L-guluronic acid (G2013) as novel NSAID with immunomodulatory effects has shown its positive effects in various investigations.Objective: Present research aimed to study the potency of G2013 on gene expression of TLR2, TLR4, MyD88, TNF-α and CD52 in PBMCs of MS patients under in vitro conditions. Materials and methods: 24 blood samples from MS patients and healthy controls were considered for RT-PCR and flow cytometry techniques under two different doses of G2013.Results: Our research indicated that this drug could significantly decrease the gene expression of TLR2, TLR4 and TNF-α compared to untreated group. Conclusion: Data demonstrated that the guluronic acid is able to modify the expression levels of TLR2, TLR4 and TNF-α genes to less than the pathogenic boarder line level, which it might be recommended for reducing the pathological process in multiple sclerosis.

A randomized clinical trial for the assessment of the efficacy and safety of guluronic acid (G2013) in patients with rheumatoid arthritis.

Objective: To evaluate the safety, efficacy and tolerability of guluronic acid (G2013) in order to treat the rheumatoid arthritis patients who had inadequate response to conventional drugs. Methods: A randomized, 12-week clinical trial with two treatment arms: guluronic acid (G2013) and conventional treatment was performed. The diagnosed RA patients according to the ACR/European League against Rheumatism 2010 classification criteria, with an active disease at baseline that had inadequate response to conventional therapy were considered for the study. G2013 was administered orally twice a day with capsules of 500 mg during a period of 12 weeks and the patients were followed up for the safety and efficacy. Results: Our data showed that, the mean changes in the G2013 and control groups were -7.54 and -2.5 for tender joint count; -7.59 and -3.59 for swollen joint count; -30 and -0.9 for physician global assessment; -23.18 and -1.81 for patient global assessment; -14.45 and -1.45 for erythrocyte sedimentation rate, respectively. Improvements seen with G2013 were significantly greater than those with conventional drugs. In total, in 15.3% of G2013-treated patients and 69.2% of conventional-treated patients adverse events (AEs) occurred in this study. Conclusion: These data from routine rheumatology clinical practice highlight the effectivenessof G2013 in combination with conventional therapy with more desirable safety profile compared to the conventional-treated patients. Therefore, G2013 therapy could be an appropriate choice in order to manage the RA disease. (Clinical trial identifier: IRCT2016092813739N5).

International multicenter randomized, placebo-controlled phase III clinical trial of ß-D-mannuronic acid in rheumatoid arthritis patients.

BACKGROUND: The oral administration of drug ß-D-mannuronic acid (M2000) showed a potent therapeutic effect in phase I/II study in rheumatoid arthritis (RA) patients. Here, our aim is to assess the efficacy and safety of this new drug in RA patients under a multinational, randomized placebo-controlled phase III clinical trial. METHOD: Patients (n = 288) with active disease at baseline and inadequate response to conventional drugs were randomly allocated to three groups; (1) receiving mannuronic acid at a dose of two capsules (500 mg) per day orally for 12 weeks, (2) placebo-controlled, and (3) conventional. The primary endpoints were the America College of Rheumatology 20 response (ACR20), 28-joint disease activity score (DAS28) and Modified Health Assessment Questionnaire-Disability Index (M-HAQ-DI). In addition, the participants were followed-up for safety assessment. RESULTS: In this phase III trial, after 12 weeks of treatment, there was a significant reduction in ACR20 between mannuronic-treated patients compared to placebo and conventional groups. Moreover, there was a similar significant improvement for DAS28 following mannuronic therapy. The statistical analysis showed a significant reduction in the swollen and tender joint count in mannuronic-treated patients compared with the placebo group. On the other side, mannuronic acid showed no-to-very low adverse events in comparison to placebo. CONCLUSION: The results of this multinational, phase III clinical trial provided a potent evidence base for the use of ß-D-mannuronic acid as a new highly safe and efficient drug in the treatment of RA.

The imbalance of circulating T helper subsets and regulatory T cells in patients with LRBA deficiency: Correlation with disease severity.

Patients with lipopolysaccharides responsive beige-like anchor protein (LRBA) deficiency suffer from a variety of immunological abnormalities. In the current study, we investigated the role of T helper (Th) cell subsets and regulatory T (Treg) cells and their related cytokines and transcription factors in the immune dysregulation of LRBA deficiency. The study population comprised of 13 LRBA-deficient patients and 13 age- and sex-matched healthy controls (HCs). Th subsets and Treg were examined by flow cytometry. The expression of determinant cytokines (interferon-γ [IFN-γ], interleukin [IL]-17, IL-22, and IL-10), and cell subset-specific transcription factors were evaluated before and after proliferation and activation stimuli. The frequencies of Th1, Th1-like Th17 and Th22 cells along with the expression of T-box transcription factor (TBET) and runt-related transcription factor 1 (RUNX1) were significantly increased in patients with LRBA. Moreover, IFN-γ and IL-22 production in LRBA-deficient CD4+ T cells were elevated after lymphocyte stimulation, particularly in patients with enteropathy. However, CD4+ CD25+ FoxP3+ CD127- cells were significantly decreased in LRBA-deficient patients compared with those of HCs, particularly in patients with autoimmunity. There was a negative correlation between the frequencies of CD4+ CD25+ FoxP3+ CD127- cells and Th1-like Th17 cells in LRBA-deficient patients, and an overlapping phenotype of autoimmunity and enteropathy were observed in ~70% of patients. The frequency of Th17 cells was lower in patients with enteropathy, while Th1-like Th17 cells were higher than in those without enteropathy. Our findings demonstrated an imbalance in Th subsets, mainly in Th1-like Th17 and Treg cells and their corresponding cytokines in LRBA deficiency, which might be important in the immunopathogenesis of autoimmunity and enteropathy.

The effect of 1, 25(OH)2 D3 (calcitriol) alone and in combination with all-trans retinoic acid on ROR-γt, IL-17, TGF-ß, and FOXP3 gene expression in experimental autoimmune encephalomyelitis.

OBJECTIVES: It has been shown that calcitriol and all-trans retinoic acid (ATRA) have modulatory effects on the immune system. The present study investigates the synergistic effects of combination treatment of calcitriol and ATRA in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). METHODS: The mice were allocated to four preventive groups, each consisting of eight animals, ATRA (250 µg/mouse), calcitriol (100 ng/mouse), combination of ATRA and calcitriol (125  µg/mouse and 50 ng/mouse) and vehicle groups. EAE was induced by MOG35-55 peptide in female C57BL/6 mice. Treatments were initiated at day 1 before immunization and continued every other day throughout the study until the day 21 post-immunization. Splenocytes were isolated from EAE-induced mice and the expression of retinoic acid receptor-related orphan receptor gamma t (ROR-γt), Interleukin-17 (IL-17), transforming growth factor beta (TGF-ß), and forkhead box P3 (FOXP3) genes was measured using real-time polymerase chain reaction. RESULTS: The expression of FOXP3 and TGF-ß genes in the splenocytes of combination-treated and calcitriol alone-treated mice was significantly increased compared to vehicle group (P < 0.05). The expression of ROR-γt and IL-17 genes in the splenocytes of ATRA, calcitriol and combination- treated mice was significantly reduced compared to those of vehicle- treated mice (P < 0.05). The relative expression level of ROR-γt was significantly (P < 0.05) lower in the combination group than in the mice treated by ATRA or calcitriol alone. DISCUSSION: This study demonstrated that treatment with combination of calcitriol and ATRA can be considered as a new strategy for MS prevention and treatment.