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.

B cells and T cells abnormalities in patients with selective IgA deficiency.

BACKGROUND: Selective IgA deficiency (SIgAD) is the most prevalent inborn errors of immunity with almost unknown etiology. This study aimed to investigate the clinical diagnostic and prognostic values of lymphocyte subsets and function in symptomatic SIgAD patients. METHODS: A total of 30 available SIgAD patients from the Iranian registry and 30 age-sex-matched healthy controls were included in the present study. We analyzed B and T cell peripheral subsets and T cell proliferation assay by flow cytometry in SIgAD patients with mild and severe clinical phenotypes. RESULTS: Our results indicated a significant increase in naïve and transitional B cells and a strong decrease in marginal zone-like and switched memory B-cells in SIgAD patients. We found that naïve and central memory CD4+ T cell subsets, as well as Th1, Th2 and regulatory T cells, have significantly decreased. On the other hand, there was a significant reduction in central and effector memory CD8+ T cell subsets, whereas proportions of both (CD4+ and CD8+) terminally differentiated effector memory T cells (TEMRA) were significantly elevated in our patients. Although some T cell subsets in severe SIgAD were similar, a decrease in marginal-zone and switched memory B cells and an increase in CD21low B cell of severe SIgAD patients were slightly prominent. Moreover, the proliferation activity of CD4+ T cells was strongly impaired in SIgAD patients with a severe phenotype. CONCLUSION: SIgAD patients have varied cellular and humoral deficiencies. Therefore, T cell and B cell assessment might help in better understanding the heterogeneous pathogenesis and prognosis estimation of the disease.

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.

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.

The Anti-tumoral Effect of ß-D-Mannuronic Acid (M2000) as a Novel NSAID on Treg Cells Frequency and MMP-2, MMP-9, CCL22 and TGFß1 Gene Expression in Pre-surgical Breast Cancer Patients.

With respect to the role of chronic inflammation in the induction and progression of breast cancer (BC). The relationship between tumor and tumor microenvironment may be a hopeful strategy for BC therapy. According to the effect of ß-D-Mannuronic acid (M2000) as a novel non-steroidal anti-inflammatory drug (NSAID) on BC murine model and 4T1 cell line, we started to study that was a phase II, randomized, controlled clinical trial. 24 women with BC were included in this study and were followed by fixed oral doses of M2000, 500 mg two times a day (6-8 weeks). Blood samples were collected at baseline and weeks 6-8. To compare the patterns of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), C-C motif chemokine ligand 22 (CCL22) and The transforming growth factor-beta 1 (TGFß1) gene expression and T regulatory cells (Tregs) frequency of healthy women normal controls with BC patients, a set of 10 blood samples of  women healthy volunteers was collected. The gene expression was evaluated by quantitative Real-time PCR (qRT-PCR) and the frequency of Tregs was assessed by flow cytometry. Our results showed, reduction in MMP-2 (p=0.08), MMP-9 (p=0.03), CCL22 (p=0.003) and TGFß1 (p=0.1) gene expression and Tregs frequency (p=0.01) which play a main role in the development of chronic inflammation, angiogenesis, tumorigenesis and metastasis. Our findings demonstrated that M2000 therapy as a novel designed NSAID had valuable therapeutic effects on BC. No adverse effects were observed following the use of M2000 after 6-8 weeks.

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.

Efficacy and Safety of G2013 as a Novel Immunosuppressive Agent on Differentiation, Maturation and Function of Human Dendritic Cells.

BACKGROUND: The expanse of dendritic cells (DC) differentiation plays an important role in determining immune response. DC-based immunosuppressive drugs have notable side effects in increasing the risk of infectious diseases and cancers. G2013, as a novel anti-inflammatory and immunosuppressive agent, has been tested in experimental model of multiple sclerosis. The aim of this study was to conduct the safety property of G2013 on dendritic cells biology. METHODS: The effect of G2013 on differentiation, maturation, and function of dendritic cells was examined at Tehran University in 2014. To investigate how G2013 affects human dendritic cells (DC) in a defined inflammatory environment, human peripheral blood mononuclear cells (PBMC) were isolated from healthy blood. Monocytes were then purified using anti-CD14 microbeads. Monocytes were treated with G2013 in two different doses (6 and 12 µg/well) along with adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 for inducing monocytes to immature DC and adding lipopolysaccharide for running DC maturation. Differentiation, maturation, and function of dendritic cells were examined with flow cytometry and ELISA. RESULTS: G2013 therapy had no significant effect on CD83, CD86 and DR expression, as well as IL-10 and IL-12 cytokine levels and it, has no remarkable side on differentiation, maturation and function of dendritic cells in immature DC and mature DC process in vitro. CONCLUSION: G2013 is a safe agent with no adverse effect on differentiation, maturation, and function of dendritic cells. It may be recommended as a novel immunosuppressive agent with no or little side effect in increasing the risk of infectious diseases and cancers.

Preclinical and pharmacotoxicology evaluation of α-l-guluronic acid (G2013) as a non-steroidal anti-inflammatory drug with immunomodulatory property.

CONTEXT: Therapeutic effects of α-l-guluronic acid with the greatest tolerability and efficacy (G2013) have been shown in experimental model of multiple sclerosis and other in vitro and in vivo examinations regarding α-l-guluronic acid; there are no toxicological researches on its safety although the pharmacological impacts have been recorded. OBJECTIVE: This study was designed to determine the acute and sub chronic toxicity of α-l-guluronic acid in healthy male and female BALB/c mice. MATERIALS AND METHODS: For the acute toxicity study, the animals orally received five different single doses of α-l-guluronic acid and were kept under observation for 14 d. In the sub-chronic study, 24 male and female BALB/c mice were divided into four groups and treated daily with test substance preparation at dose levels of 0, 50, 250, and 1250 mg/kg body weight for at least 90 consecutive days. The mortality, body weight changes, clinical signs, hematological and biochemical parameters, gross findings, histopathological, and organs weight determinants were monitored during this study. RESULTS: The results of acute toxicity indicated that the LD50 of α-l-guluronic acid is 4.8 g/kg. We found no mortality or abnormality in clinical signs, body weight, relative organs weight, or necropsy in any of the animals in the subchronic study. Additionally, the results showed no significant difference in hematological, biochemical, and histopathological parameters in rats. CONCLUSIONS: Our results suggest that α-l-guluronic acid has high safety when administered orally in animals.

Preclinical assessment of ß-d-mannuronic acid (M2000) as a non-steroidal anti-inflammatory drug.

CONTEXT: ß-d-Mannuronic acid (M2000) has shown its therapeutic effects with the greatest tolerability and efficacy in various experimental models such as experimental autoimmune encephalomyelitis (EAE), adjuvant induced arthritis (AIA), nephrotic syndrome, and acute glomerulonephritis. Despite pharmacological effects of ß-D-mannuronic acid, there have been no systematic toxicological studies on its safety so far. OBJECTIVE: The study was designed to determine the acute and subchronic toxicity of ß-D-mannuronic acid, an anti-inflammatory agent, in healthy male NMRI mice and Wistar rats, respectively. MATERIALS AND METHODS: For the acute toxicity study, the animals received orally five different single doses of ß-D-mannuronic acid and were kept under observation for 14 d. In the subchronic study, 24 Wistar male rats were divided into four groups and were treated orally (gavage) once daily with test substance preparation at dose levels of 0, 50, 250, and 1250 mg/kg body weight for at least 63 consecutive days (9 weeks). Mortality, clinical signs, body weight changes, hematological and biochemical parameters, gross findings, organ weights, and histopathological determinations were monitored during the study. RESULTS: The results of acute toxicity indicated that the LD50 of ß-D-mannuronic acid is 4.6 g/kg. We found no mortality and no abnormality in clinical signs, body weight, relative organ weights, or necropsy in any of the animals in the subchronic study. Additionally, the results showed no significant difference in hematological, biochemical, and histopathological parameters in rats. CONCLUSIONS: Our results suggest that ß-D-mannuronic acid is relatively safe when administered orally in animals.

Effects of imatinib mesylate in mouse models of multiple sclerosis and in vitro determinants.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS), This autoimmune disease is mainly mediated by adaptive and innate immune responses that lead to an inflammatory demyelination and axonal damage. Imatinib mesylate is a selective protein tyrosine kinase inhibitor with immunomodulatory properties that abrogates multiple signal transduction pathways in immune cells. In the present research, our aim was to test the therapeutic efficacy of imatinib in experimental model of MS. We performed EAE induction in 23 female C57 mice by myelin oligodendrocyte glycoprotein-35-55 (MOG35-55) in Complete Freund's Adjuvant (CFA) emulsion and used imatinib for treatment of EAE. The clinical evaluation and histopathology were assessed. Also for in vitro analysis, we used U-87 MG, C6 and WEHI-164 cell lines to evaluate the inhibitory effects of imatinib in cell proliferation, as well as pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) and matrix metalloproteinase (MMP) secretion. Our findings demonstrated that this drug had beneficial effects on EAE by attenuation in the severity and a delay in the onset of disease. In vitro, imatinib inhibited cell proliferation, MMP-2 expression and activity and also attenuated the production of proinflammatory cytokines. Imatinib with its potential therapeutic effects and immunomodulatory properties may be considered, after additional necessary tests and trials, for treatment of MS.