Disturbance in the reconstitution of distinct T-cell subsets and the incidence of GvHD following allo-HSCT in pediatric patients with non-malignant hematological disorders.

BACKGROUND: The reconstitution of different T-cell subsets following allogeneic hematopoietic stem cell transplantation (allo-HSCT) is critical for efficient pathogen protection and the prevention of graft-versus-host disease (GvHD). In particular, studies have highlighted the importance of balanced ratios of regulatory T-cells (Tregs) and distinct functionally T-cells in preventing acute and chronic GvHD. METHODS: We evaluated the regeneration of CD4 and CD8 T-cell subpopulations in nine pediatric patients with non-malignant disorders following allo-HSCT from a fully HLA-identical donor. RESULTS: CD4 and CD8 T-cells were higher 12 months after the transplant but still lower than in healthy controls and pre-transplant. However, we found after allo-HSCT, central memory and effector memory cell subsets were the predominant phenotypes in the CD4 and CD8 T-cell populations, respectively. In patients who had developed acute GvHD, there was an increase in the frequency of TEMRA (effector memory T cells that re-express CD45RA) cells within the CD4 T-cell population. Meanwhile, in patients with chronic GvHD, we observed a decrease in Th1 cells in CD4 T-cells and effector memory cells within the CD8 T-cell population. In addition, we found decreased TEMRA cell subsets in CD4 and CD8 T-cell populations in chronic GvHD. CONCLUSION: Our findings suggest a possible relationship between the influence of acute GvHD and its prevention on delayed CD4 T-cell reconstitution and, reciprocally, unbalanced regeneration of CD4 and CD8 T-cell subsets in the development of chronic GvHD.

T helper 17 and regulatory T-cell profile and graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in pediatric patients with beta-thalassemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment option for hereditary hemoglobin disorders, such as beta-thalassemia; However, this procedure is not without constraints, mainly engendering complications such as acute graft-versus-host disease (aGvHD), chronic GvHD (cGvHD), and susceptibility to infections. The clinical outcomes of allo-HSCT are highly dependant on the quality and quantity of T-cell subsets reconstitution. Following the allo-HSCT of six pediatric patients afflicted with beta-thalassemia, their mononuclear cells were isolated, and then cultured with a combination of phorbol myristate acetate (PMA)/ionomycin and Brefeldin A. The content of CD4 T-cell subsets, including T helper 17 (Th17) cells and regulatory T cells (Tregs), were determined by specific conjugated-monoclonal antibodies three and six months post-HSCT. An increased frequency of total CD4 T-cells, Tregs and Th17 cells was observed at day 90 and 180 after allo-HSCT, albeit the numbers were still lower than that of our healthy controls. In patients who developed cGvHD, a lower Th17/Treg ratio was observed, owing it to a decreased proportion of Th17 cells. In conclusion, creating balance between Th17 and Treg subsets may prevent acute and chronic GvHD in patients after allo-HSCT.

Efficacy of ß-D-Mannuronic Acid [M2000] on the Pro-Apoptotic Process and Inflammatory-Related Molecules NFκB, IL-8 and Cd49d using Healthy Donor PBMC.

OBJECTIVE: This investigation evaluates the pro-apoptotic and anti-inflammatory effects of ß-D-mannuronic acid [M2000] compared to diclofenac, based on gene expression involved in apoptosis and inflammation process [including Bcl2, NFκB, IL-8 and Cd49d] in Peripheral Blood Mononuclear Cells [PBMCs] of healthy donors under exvivo conditions. MATERIALS: The venous blood samples of twelve healthy volunteers with aged 25-60 years were collected in heparinized tubes. The healthy volunteers were selected from no smoking group and without using illicit drugs and suffering from diabetes. The PBMCs were separated and divided into untreated and treated groups. METHODS: The PBMCs of each sample were cultured in 5 wells of culture plate, so that the first well consisted of 2×106 cells exposed by LPS-EB [1µg/ml] to stimulate PBMCs and absence of M2000 [untreated well]. The second, third, fourth and fifth wells containing 2×106 cells/well and LPS-EB, after 4 hours incubation at 37ºC, received 5, 25 and 50 µg/well of M2000 and 5 µg/well of diclofenac, respectively as treated group. RESULTS: The PBMCs were separated and RNAs were then extracted and cDNAs synthesized and gene expression levels were assessed by qRT-PCR. Furthermore, we studied whether M2000 is able to facilitate apoptosis in PBMCs. Our findings represent that the high dose of M2000 could significantly decrease the expression level of NFκB gene compared to untreated group (p < 0.0002). On the other hand, no significant change was observed in treated cells with diclofenac. All doses of M2000 could significantly augment apoptosis compared to untreated group [p < 0.0001]. Additionally, we observed the same apoptotic effects between the medium dose of M2000 and diclofenac. Besides, no significant reduction was shown in expression levels of IL8, Bcl2 and Cd49d genes in all doses of M2000 and diclofenac compared to untreated group. This experiment demonstrates M2000 as a new effective NSAID with immunosuppressive characteristics capable of stimulating apoptosis through lowering expression levels of NFκB gene, which might be probably considered as an appropriate drug for reducing the risk of developing inflammatory diseases and cancer.

Interleukin-1ß and interleukin-6 in Common Variable Immunodeficiency and their association with subtypes of B cells and response to the Pneumovax-23 vaccine.

INTRODUCTION: Common Variable Immunodeficiency (CVID) is the most common symptomatic form of primary immunodeficiencies. Current research data show altered B cells, TLRs, and cytokine profile in CVID patients. The aim of this study was to determine levels of IL-1ß and IL-6 in CVID patients in response to TLRs stimulation and the association of these cytokines with subtypes of B cells and response to Pneumovax-23 vaccination. METHOD: Peripheral blood mononuclear cells of CIVD patients were stimulated with and without TLR2 and TLR4 agonist and specific inhibitors including lipopolysaccharide (LPS), lipoteichoic (LTA), and OxPAPC. The levels of IL-1ß and IL-6 were assessed by ELISA in different treatment groups. Finally, association of cytokines levels was assessed among different subtypes of B cells and types of response to Pneumovax-23 vaccine. RESULTS: Secretion of IL-6 and IL-1ß was significantly diminished in CVID patients (p = 0.015 and p = 0.019), but ligand engagement of TLR2 and TLR4 leads to significant increase in IL-6 and IL-1ß production. IL-6 was significantly lower in Pneumovax-23 hypo responder patients (p = 0.05) and significant correlations between the concentration of IL-6 and the number of switched memory and CD21low expressing B cells were found. CONCLUSION: Secretion of IL-6 and IL-1ß is abolished in CVID patients. However, TLR2 and TLR4 are hyper responsive to stimulation with their cognate ligands resulting in the secretion of higher levels of proinflammatory cytokines. This characteristic of CVID TLRs leads to an improvement of cytokine secretion compared to baseline levels. Also, our novel findings about the association concentrations of serum IL-6 and the frequency of with switched memory and CD21low expressing B cells as well as the poor response to Pneumovax-23 should be substantiated by the use of a higher sample size in future studies.

Therapeutic effects of pegylated-interferon-α2a in a mouse model of multiple sclerosis.

INTRODUCTION: Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS). EAE is mainly mediated by adaptive and innate immune responses that lead to an inflammatory demyelination and axonal damage. The aim of the present research was to examine the therapeutic efficacy of Peg interferon alpha 2a (Peg-IFN α-2a) as a serine protease inhibitor on EAE model. MATERIAL AND METHODS: EAE induction was performed in female C57BL/6 mice by myelin oligodendrocyte glycoprotein (35-55) (MOG35-55) in Complete Freund's Adjuvant (CFA) emulsion, and Peg-IFN α-2a was used for the treatment of EAE. During the course of the study, clinical evaluation was assessed, and on day 21 post-immunisation blood samples were taken from the heart of mice for evaluation of IL-6, and enzymatic and non-enzymatic antioxidants. The mice were sacrificed and the brains and cerebellums were removed for histological analysis. RESULTS: Our findings indicated that Peg-IFN α-2a had beneficial effects on EAE by attenuation of the severity and a delay in the onset of disease. Histological analysis showed that treatment with Peg-IFN α-2a can reduce inflammation criteria. Moreover, in Peg-IFN α-2a-treated mice the serum level of IL-6 was significantly less than in controls, and total antioxidant capacity was significantly more than in the control animals. CONCLUSIONS: These data indicate that Peg-IFN α-2a as an anti-serine protease with immunomodulatory properties may be useful for the treatment of MS.

Immunomodulatory Effect of G2013 (α-L-Guluronic Acid) on the TLR2 and TLR4 in Human Mononuclear Cells.

BACKGROUND: Inhibition of Toll-like receptors (TLRs) signaling has been established as a new method for the development of anti-inflammatory drugs instead of NSAIDs (non-steroid anti-inflammatory drugs). Since the immunomodulatory role of G2013 (α-L-Guluronic acid) was reported in some recent experiments, we decided to assess the effects of G2013 on the protein expression of TLR2 and TLR4, their downstream signaling cascade, and the secretion of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs). METHODS: After blood sampling from 16 healthy donors, PBMCs were isolated and treated with/without lipopolysaccharide (LPS), lipopolyteichoic acid (LTA), and G2013. Flow cytometry was done for detecting the protein expression of TLR2 and TLR4. MyD88, IκB, Tollip, and NF-κB mRNA expression were assessed by realtime PCR. ELISA was performed for assessing the concentration of IL-1ß and IL-6. RESULTS: G2013 at a concentration of 25 µg/mL (high dose) significantly downregulated NF-κB, IκB and MyD88 mRNA expression and suppressed the secretion of IL-1ß by PBMCs. The findings indicate that G2013 may exert its regulatory effect under normal condition via Tollip in a dose dependence pathway. Our results demonstrated that G2013 had no profound impact on the protein expression of TLR2 and TLR4. CONCLUSION: In conclusion, our findings point to the immunomodulatory effect of G2013 on the TLR2 and TLR4 signaling cascade and cytokine production by PBMCs. These findings could lead to an establishment of new safe anti-inflammatory drugs in the future.

Anti-aging effects of M2000 (ß-D-mannuronic acid) as a novel immunosuppressive drug on the enzymatic and non-enzymatic oxidative stress parameters in an experimental model.

BACKGROUND: The anti-aging property of ß-D-mannuronic acid (M2000) as a novel non-steroidal anti-inflammatory and immunosuppressive agent was investigated on several determinants relative to the oxidative stress in an animal model. METHODS: Sprague-Dawley rats were used for evaluating the safety and efficacy properties of M2000 on some oxidative stress enzymes, including the following: mitochondrial superoxide dismutase (SOD2), catalase (CAT), glutathione peroxidase (GPX1), glutathione S-transferase (GST), myeloperoxidase (MPO), and inducible nitric oxide synthase (iNOS) gene expression by real-time PCR. Malondialdehyde (MDA), carbonyl protein (PCO) (the lipid and protein oxidation marker, respectively), and total antioxidant capacity (TAC) were tested in serum by biochemical analysis. In addition, cortisol as a steroid hormone was surveyed by chemiluminescence immunoassay after 12 weeks of M2000 consumption. The rats were sacrificed 3 months after daily oral administration of M2000. RESULTS: Our findings revealed the favorable effects of M2000 on several antioxidant enzyme and gene expression, including SOD2, CAT, GPX1, and GST; however, our results were not statistically significant. Moreover, there was no significant difference in MDA and PCO as lipid and protein oxidation markers, TAC, and cortisol compared with the control group following M2000 consumption. A slight weight increase in the M2000-treated group was also observed. CONCLUSIONS: Our data showed the anti-aging property of M2000 as a novel designed non-steroidal anti-inflammatory drug (NSAID) with immunosuppressive property on various oxidative stress determinants.

The Biology of ß-D-mannuronic acid (M2000) on Human Dendritic Cell Based on MicroRNA-155 and MicroRNA-221.

BACKGROUND: The aim of this study was to evaluate the effect of ß-Dmannuronic acid (M2000) on related miRNAs to dendritic cells (DCs) differentiation. DC-based immunosuppressive drugs can suppress the progression of autoimmune diseases, however, their notable side effects in increasing the risk of infectious diseases and cancers should be considered. The ß-D-mannuronic acid, as a novel non-steroidal anti-inflammatory agent, has been tested in various experimental models. METHOD: The effect of M2000 on expression of miRNA-155 and miRNA-221 was examined. To investigate how M2000 affects differentiation of human dendritic DCs in a defined inflammatory environment, human peripheral blood mononuclear cells were isolated from healthy blood and the monocytes were purified using anti-CD14 microbeads. The so isolated monocytes were subsequently incubated in the presence of M2000 in two different doses (3 and 6 mMol/well) adding granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 for inducing monocytes to immature DC and lipopolysaccharide for running DC differentiation. The expression of miRNA-155 and miRNA-221 were examined with Real Time PCR. RESULTS: The results demonstrate that M2000 has no significant side effect on expression of miR-155 and miR-221 in both immature DC and mature DC process in vitro. CONCLUSION: Our findings show that ß-D-mannuronic acid is a safe agent which has no adverse effect on regulatory miRNA-155 and miRNA-221 in dendritic cells.

Anti-aging property of G2013 molecule as a novel immunosuppressive agent on enzymatic and non-enzymatic oxidative stress determinants in Rat model.

BACKGROUND: G2013 molecule is a novel non-steroidal anti-inflammatory agent with immunosuppressive property, which was investigated on determinants relative to the oxidative stress in animal model. MATERIALS AND METHODS: The Sprague-Dawley rats were used for evaluating properties of G2013 on some oxidative stress enzymes including: Myeloperoxidase (MPO), Glutathione peroxidase (GPX1), mitochondrial Superoxide dismutase (SOD2), Catalase (CAT), Glutathione S-Transferase (GST), and inducible nitric oxide synthase (iNOS) genes expression by Real Time PCR. The rats were sacrificed 3 months after daily oral administration of G2013. Moreover, Malondialdehyde (MDA), Carbonyl protein (PCO), the lipid and protein oxidation markers respectively and total anti-oxidant capacity (TAC) were tested in serum by biochemical analysis. Also cortisol as a steroid hormone was evaluated by chemiluminescence immunoassay after 12 weeks consumption of G2013 solution. RESULT: Our findings revealed a significant decrease in MPO in G2013 treated group, indicating its favorable effects but has no significant effects on genes expression of another antioxidant enzymes, including: SOD2, CAT, GPX1, and GST. Also, there were no significant differences in PCO, TAC and cortisol compared to control group following G2013 consumption. While an enhancement in serum MDA level was observed in the treatment group. In addition, G2013 therapy did not show any weight loss. CONCLUSIONS: Our data showed the safety and efficacy of G2013 as a novel designed NSAID on various oxidative stress determinants.