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.

The Open, Randomized, Positive Control Clinical Trial of Guluronic Acid (G2013) on SARS-CoV-2 Patients.

INTRODUCTION: Recently, the coronavirus disease 2019 (COVID-19) infection, with a vast spectrum of clinical and paraclinical symptoms has been a major health concern worldwide. Therapeutical management of COVID-19 includes antiviral and anti-inflammatory drugs. NSAIDs, as the second-line therapy, are often prescribed to relieve the symptoms of COVID-19. The α-L-guluronic acid (G2013) is a non-steroidal patented (PCT/EP2017/067920) agent with immunomodulatory properties. This study investigated the effect of G2013 on the outcome of COVID-19 in moderate to severe patients. METHODS: The disease's symptoms were followed up during hospitalization and for 4 weeks postdischarge in G2013 and control groups. Paraclinical indices were tested at the time of admission and discharge. Statistical analysis was performed on clinical and paraclinical parameters and ICU admission and death rate. RESULTS: The primary and secondary outcomes indicated the efficiency of G2013 on COVID-19 patients' management. There were significant differences in the duration of improvement of fever, coughing, fatigue/malaise. Also, a comparison of paraclinical indices at the time of admission and discharge showed significant change in prothrombin, D-dimer, and platelet. As the main findings of this study, G2013 significantly decreased the percentage of ICU admission (control:17 patients, G2013:1 patient) and death (control: 7 cases, G2013:0). CONCLUSION: These results conclude that G2013 has sufficient potential to be considered for moderate to severe COVID-19 patients, can significantly reduce the clinical and physical complications of this disease, has a positive effect on modulating the coagulopathy process, and aids in saving lives.

The immunomodulatory effects of all-trans retinoic acid and docosahexaenoic acid combination treatment on the expression of IL-2, IL-4, T-bet, and GATA3 genes in PBMCs of multiple sclerosis patients.

OBJECTIVES: Multiple sclerosis (MS) is a potentially disabling autoimmune disease of the central nervous system. Neither the pathogenesis nor the effectiveness of treatment of MS has been fully understood. This in vitro trial evaluated the beneficial immunomodulatory effects of single and combined treatments of all-trans retinoic acid (ATRA) and docosahexaenoic acid (DHA) on the peripheral blood mononuclear cells (PBMCs) of relapsing-remitting MS (RRMS) patients who were receiving interferon beta (IFN-ß). METHODS: The PBMCs of 15 RRMS patients were isolated, cultured, and treated with single and combined treatments of ATRA and DHA. The expressions of IL-2, IL-4, T-bet, and GATA3 genes were evaluated using real-time PCR. RESULTS: The results showed that a single treatment of ATRA could significantly suppress the gene expression of the pro-inflammatory cytokine, IL-2 (P < 0.05), and related transcription factor, T-bet (P < 0.001). The gene expression level of the anti-inflammatory cytokine, IL-4, and its transcription factor, GATA3, were not significantly changed. The expression of IL-2 and T-bet genes was significantly decreased in combination treatments of ATRA and DHA (P < 0.001). Significant suppression of IL-2 and T-bet (P < 0.001) was observed in ATRA and DHA combination therapy with half doses of their single treatment, which suggested a synergistic effect of these components. DISCUSSION: Co-administration of vitamin A and DHA, an omega-3 fatty acid derivative, may exert a synergistic effect in modulating the immune system in MS patients; however, more studies are needed to evaluate the exact effects and mechanism of their actions on the immune cells.

Digging for the discovery of SARS-CoV-2 nsp12 inhibitors: a pharmacophore-based and molecular dynamics simulation study.

Aim: COVID-19 is a global health threat. Therapeutics are urgently needed to cure patients severely infected with COVID-19. Objective: to investigate potential candidates of nsp12 inhibitors by searching for druggable cavity pockets within the viral protein and drug discovery. Methods: A virtual screening of ZINC natural products on SARS-CoV-2 nsp12's druggable cavity was performed. A lead compound with the highest affinity to nsp12 was simulated dynamically for 10 ns. Results: ZINC03977803 was nominated as the lead compound. The results showed stable interaction between ZINC03977803 and nsp12 during 10 ns. Discussion: ZINC03977803 showed stable interaction with the catalytic subunit of SARS-CoV-2, nsp12. It could inhibit the SARS-CoV-2 life cycle by direct interaction with nsp12 and inhibit RdRp complex formation.

Evaluating Mannuronic Acid Effect on Gene Expression Profile of Inflammatory Mediators in Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is a multisystem disorder. Various studies have shown the important role of inflammatory factors tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-22, MYD88, and toll-like receptor 2 (TLR2) in this disease. In this study, we investigated the anti-inflammatory effects of B-D-Mannuronic acid (M2000), as a new immunosuppressive drug, on the expression of these inflammatory markers in peripheral blood mononuclear cells (PBMCs) of RA patients. The blood samples of active RA patients and healthy volunteers were used for PBMCsl separation. The cells were cultured with LPS (1 µg/mL), low (5 µg/mL), moderate (25 µg/mL), and high (50 µg/mL) doses of M2000 and a single dose of diclofenac (1 µg/mL) to evaluate TNF-α, IL-6, IL-22, MYD88, and TLR2 genes expression by quantitative real-time (qRT-PCR). Cell surface expression and MFI of TLR2 were assessed; using flow cytometry. Our findings exhibited a significant reduction of TNF-α, IL-6, and MYD88 gene expressions after treatment with three doses of M2000 and an optimum dose of diclofenac. TLR2 gene expression was significantly diminished by moderate and high doses of M2000 and a single dose of diclofenac. Moreoversurface expression of TLR2 was significantly downregulated by moderate and high doses of M2000, while MFI of this receptor was significantly reduced by three doses of M2000. The results of this research showed that M2000 was able to significantly reduce the gene expression of inflammatory molecules  TNF-α, IL-6, MYD88, and TLR2 in patients PBMCs. factor-alpha; Rheumatoid arthritis. These data revealed a part of the molecular mechanisms of M2000 in the treatment process.

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.

ß-D-mannuronic Acid (M2000) and Inflammatory Cytokines in COVID-19; An In vitro Study.

coronavirus disease of 2019 (COVID-19) can be complicated by acute respiratory distress syndrome (ARDS) and may be associated with cytokine storm and multiorgan failure. Anti-inflammatory agents, such as systemic corticosteroids, monoclonal antibodies, and nonsteroidal anti-inflammatory drugs (NSAIDs) can be used for this purpose. In this study, we evaluated the immunomodulatory effect of mannuronic acid (M2000), which is a novel NSAID, on COVID-19-related cytokine storms. This study was conducted in vitro on blood samples of 30 COVID-19 patients who presented with ARDS to a referral center. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples and incubated with phorbol myristate acetate for 24 hours. M2000 was administered with the dosages of 25 µg/well and 50 µg/well after 4 hours of incubation at 37°C. The quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess mRNA gene expression. Enzyme-linked immunosorbent assay (ELISA) was performed to evaluate the supernatant PBMC levels of interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ. Both mRNA expression and the supernatant PBMC levels of IL-17, TNF-α, IL­6, and IFN­Î³ were decreased in PBMCs of COVID-19 patients treated with M2000 compared with the control  group. For the first time, it was observed that M2000 could be effective in alleviating the inflammatory cascade of COVID-19 patients based on an in vitro model. After further studies in vitro and in animal models, M2000 could be considered a novel NSAID drug in COVID-19 patients.

The Effects of Mannuronic Acid on IL-1ß, IL-17A, STAT1, and STAT3 Gene Expressions and TLR2 and TLR4 Molecules in Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic neurologic disease defined by inflammation and demyelination of the central nervous system that comes with variable degrees of axonal and neuronal damage. The efficacy of ß-D-mannuronic acid (M2000) as a novel drug with immunosuppressive properties (patented: PCT/EP2017/067920), has been shown in an experimental model of MS. In this study, the effects of M2000 on interleukin (IL)-1ß, IL-17A, signal transducer and activator of transcription (STAT) 1, and STAT3 gene expressions and Toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) molecules in patients with secondary progressive MS were evaluated. In this study, 14 patients with secondary progressive MS and 14 healthy subjects (as control group) were entered from the phase 2 clinical trial (Clinical Trial identifier, IRCT2016111313739N6). The gene expressions of IL-1ß, IL-17A, STAT1, and STAT3 were assessed at the baseline and then measured after 6 months of therapy with M2000 by using the quantitative real-time polymerase chain reaction method. Moreover, the expressions of TLR2 and TLR4 molecules on peripheral blood mononuclear cells were evaluated by the flow cytometry method. The gene expressions of IL-17A, STAT1, and STAT3 in patients with MS decreased after 6 months of therapy with M2000 comparing before treatment. Also, the gene expression of IL-1ß decreased numerically after 6 months. Furthermore, the expressions of TLR2 and TLR4 on PBMCs of the patients declined when compared to baseline. The results of this investigation revealed that M2000 could downregulate IL-17, STAT1, and STAT3 genes in patients with secondary progressive MS and also reduce the expressions of TLR2 and TLR4 on PBMCs. Moreover, M2000 declined numerically IL-ß gene expression.

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.

Tau and amyloid beta differentially affect the innate immune genes expression in Drosophila models of Alzheimer's disease and ß- D Mannuronic acid (M2000) modulates the dysregulation.

Alzheimer's disease (AD) is the most common cause of dementia and neuroinflammation is considered as one of the main culprits. The aim of this study was to evaluate the independent role of Aß42 and tau on the inflammatory pathway in the Drosophila models of AD and investigating the potential modulating effect of M2000 as a novel NSAIDs in those flies. The expression levels of relish, orthologs of NF-κB, antimicrobial peptide (AMP) including attacin A, diptericin B and a dual oxidase (Duox) as a ROS mediator, were evaluated in both M2000 treated and untreated groups followed by brain histology analysis to assess the extent of neurodegeneration. The potential inhibitory role of M2000 (ß-D Mannuronic acid) on the aggregation of tau protein was also investigated in vitro. According to the result, there was a significant induction of Duox, AMPs and its transcription factor expression in both aged and Drosophila models of AD which was in accordance with the increase in the number of vacuoles in the brain section of Drosophila models of AD. Interestingly M2000 treatment revealed a significant reduction in all neurodegeneration indexes; in vivo and anti-aggregating property; in vitro. Findings suggest that M2000 has potential to be an AD therapeutic agent.

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.

The Evaluation of Safety Property and Apoptotic Efficacy of α-L-Guluronic Acid (G2013), as a Novel NSAID, Under In Vitro Examination on L929 and Hepatocellular Carcinoma Cell Lines.

BACKGROUND: Many investigations have expanded this concept that liver chronic inflammation has an essential role in persistent cell damages along with altering the liver microenvironment leading to fibrosis, cirrhosis, and finally, hepatocellular carcinoma (HCC). To reduce inflammation and relieve symptoms, Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) are commonly used; however, their long-term usage can lead to severe adverse events on vital organs like the liver. Interestingly, the α-L-Guluronic Acid (G2013), as a novel NSAID with immunomodulatory properties, has shown the inhibitory effects on inflammation and metastasis in experimental models. OBJECTIVE: This study was conducted to determine the effects of G2013 on cytotoxicity and induction of apoptosis, as a new therapeutic target for cancer therapy, in the HepG2 cell line and the mouse fibroblast cell line L929, as a control. METHODS: MTT assay and flow cytometry method were carried out using the different concentrations of G2013 (5, 15, 25, 50, 100, 200 and 400 µg/ml) in 3 distinct incubation times. RESULTS: Our data showed that treatment of HepG2 cells with high concentration (400µg/mL) of G2013 could effectively cause a decrease in cell viability, so that they were statistically different after 72 hours compared to other concentrations (5 to 200 µg/ml) (p<0.05 and p<0.01, respectively). Moreover, the proportion of apoptosis of HepG2 cells at the dose of 200µg/mL considerably increased, suggesting that the induction of apoptosis by G2013 in HepG2 cells is dose- and time-dependent, which could promote its anticancer properties. CONCLUSION: The present study revealed that G2013 could induce apoptosis in the liver cancer model. Therefore, based on these findings, G2013 might be considered as a therapeutic option in cancer therapy.

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).

Cytokine storm in the pathophysiology of COVID-19: Possible functional disturbances of miRNAs.

SARS-CoV-2, as the causative agent of COVID-19, is an enveloped positives-sense single-stranded RNA virus that belongs to the Beta-CoVs sub-family. A sophisticated hyper-inflammatory reaction named cytokine storm is occurred in patients with severe/critical COVID-19, following an imbalance in immune-inflammatory processes and inhibition of antiviral responses by SARS-CoV-2, which leads to pulmonary failure, ARDS, and death. The miRNAs are small non-coding RNAs with an average length of 22 nucleotides which play various roles as one of the main modulators of genes expression and maintenance of immune system homeostasis. Recent evidence has shown that Homo sapiens (hsa)-miRNAs have the potential to work in three pivotal areas including targeting the virus genome, regulating the inflammatory signaling pathways, and reinforcing the production/signaling of IFNs-I. However, it seems that several SARS-CoV-2-induced interfering agents such as viral (v)-miRNAs, cytokine content, competing endogenous RNAs (ceRNAs), etc. preclude efficient function of hsa-miRNAs in severe/critical COVID-19. This subsequently leads to increased virus replication, intense inflammatory processes, and secondary complications development. In this review article, we provide an overview of hsa-miRNAs roles in viral genome targeting, inflammatory pathways modulation, and IFNs responses amplification in severe/critical COVID-19 accompanied by probable interventional factors and their function. Identification and monitoring of these interventional elements can help us in designing the miRNAs-based therapy for the reduction of complications/mortality rate in patients with severe/critical forms of the disease.

Efficient roles of miR-146a in cellular and molecular mechanisms of neuroinflammatory disorders: An effectual review in neuroimmunology.

Known as one of the most sophisticated systems of the human body, the nervous system consists of neural cells and controls all parts of the body. It is closely related to the immune system. The effects of inflammation and immune reactions have been observed in the pathogenesis of some neurological disorders. Defined as the gene expression regulators, miRNAs participate in cellular processes. miR-146a is a mediator in the neuroimmune system, leaving substantial effects on the homeostasis of immune and brain cells, neuronal identities acquisition, and immune responses regulation in the nervous system. Its positive efficiency has been proven in modulating inflammatory reactions, hemorrhagic complications, and pain. Moreover, the miR-146a targets play a key role in the pathogenesis of these illnesses. Based on the performance of its targets, miR-146a can have various effects on the disease progress. The abnormal expression/function of miR-146a has been reported in neuroinflammatory disorders. There is research evidence that this molecule qualifies as a desirable biomarker for some disorders and can even be a therapeutic target. This study aims to provide a meticulous review regarding the roles of miR-146a in the pathogenesis and progression of several neuroinflammatory disorders such as multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, temporal lobe epilepsy, ischemic stroke, etc. The study also considers its eligibility for use as an ideal biomarker and therapeutic target in these diseases. The awareness of these mechanisms can facilitate the disease management/treatment, lead to patients' amelioration, improve the quality of life, and mitigate the risk of death.

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.

Lymphocytes subsets in correlation with clinical profile in CVID patients without monogenic defects.

Objectives: Common variable immunodeficiency (CVID) patients experience clinical manifestations rather than recurrent respiratory infections including autoimmunity, enteropathy, and lymphoproliferation. We evaluated the correlation of lymphocyte subpopulations with such manifestations.Methods: Twenty-six genetically unsolved CVID patients were subdivided into four phenotypes: infection only (IO), autoimmunity (AI), chronic enteropathy (CE), and lymphoproliferative disorders (LP) and examined for lymphocyte subsets by flow cytometry and TCD4+ proliferation by Carboxyfluorescein succinimidyl ester (CFSE) test.Results: We detected reduced memory B and increased total, effector memory (EM), cytotoxic, and activated TCD8+ in IO, AI and CE, decreased plasmablasts, total and naive TCD4+, Regulatory TCD4+ (Treg) and naive TCD8+ in IO and CE, elevated CD21low B and terminally differentiated effector memory (TEMRA) TCD8+ in IO and AI, increased helper T (Th2) and Th17 in IO, decreased Th1 in AI and defective total and naive B and central memory (CM) TCD4+ in CE. IO showed reduced TCD4+ proliferation response.Conclusions: In genetically unsolved CVID patients, increased Th2 and Th17 and reduced Treg is associated with IO, increased CD21low B and TEMRA TCD8+ and reduced Th1 is contributed to AI and reduced total and naive B, CM TCD4+ and naive TCD8+ and expanded total TCD8+ is correlated with CE.

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 Effect of Mannuronic Acid as a Novel NSAID With Immunosuppressive Properties on Expression of SOCS1, SOCS3, SHIP1, and TRAF6 Genes and Serum Levels of IL-6 and TNF-α in Patients With Multiple Sclerosis.

Multiple sclerosis (MS) is described as a chronic inflammatory, demyelinating disease of the central nervous system on an autoimmune basis, which is the most frequent reason for nontraumatic disability in youth. The efficacy and safety of ß-D-nannuronic acid (M2000) as a novel immunosuppressive drug (patented PCT/EP2017/067920) has been shown in an experimental model of MS and also in a phase 2 clinical trial. The effects of M2000 on SOCS1, SOCS3, TRAF6, and SHIP1 gene expression and also serum levels of IL-6 and TNF-α in secondary progressive multiple sclerosis patients have been assessed in this study. In this study, 14 secondary progressive multiple sclerosis patients and 14 healthy subjects (as the control group) were recruited from the phase 2 clinical trial (Clinical Trial identifier, IRCT2016111313739N6). Gene expression of SOCS1, SOCS3, TRAF6, and SHIP1 was measured at baseline and after 6 months of therapy with M2000 using a quantitative real-time polymerase chain reaction method. Furthermore, the serum levels of IL-6 and TNF-α were assessed by the enzyme-linked immunosorbent assay method. Our results showed that the gene expression of SOCS1, SOCS3, and SHIP1 was increased after 6 months of therapy with M2000 in MS patients. Moreover, the serum levels of IL-6 and TNF-α of patients declined compared with baseline, but this was not statistically significant. The results of this study demonstrated that M2000, with immunosuppressive properties, could upregulate SOCS1, SOCS3, and SHIP1 genes in patients with secondary progressive multiple sclerosis.

ß-D-Mannuronic Acid (M2000) as a Landmark in Pharmacology.

OBJECTIVES: The goal of this article is to retrace the studies of ß-D-Mannuronic Acid (M2000) as a new immunosuppressive drug with non-steroidal anti-inflammatory drugs (NSAIDs) property in miscellaneous aspects including in vitro, in vivo examinations, clinical trials and related to clinical trials studies. Our goal is to compare the effect of this drug with other similar drugs through varied researches and to follow tolerability, biocompatibility, potency, safety, and efficacy of this medication in different studies, as well as to evaluate its therapeutic effectiveness in various diseases. MATERIALS AND METHODS: Different methods were applied in the studies of ß-D-Mannuronic Acid under in vitro, in vivo examinations, and clinical trials phase I, II and III and related investigations to these clinical trials using different techniques showing the efficacy of this medication in the treatment of various diseases. RESULTS: The administration of ß -D-Mannuronic Acid showed the greatest tolerability and biocompatibility compared to diclofenac, piroxicam, and dexamethasone without or very low side effects. The drug has shown a punchy effect on many molecules which participate either in physiologic or in pathogenic activities in animal models and human. This new drug not only revealed the anti-inflammatory and immunosuppressive properties but also based on the results of various investigations, ß-D-Mannuronic Acid showed the antidiabetic, cardioprotective and anti-tumoral effects. CONCLUSION: ß-D-Mannuronic Acid (M2000) as a novel immunosuppressive drug with NSAID properties along with antidiabetic, cardioprotective and anti-tumoral efficacy showed great tolerability and safety profile. In addition, it has no or mild adverse events compared with many other medicines, therefore this medicament could be considered as a landmark in pharmacology and represent turn point in the treatment of different diseases based on the experimental and in vitro studies explained and clinical and related studies proved.