Intravenous injection of autologous bone marrow-derived mesenchymal stem cells on the gene expression and plasma level of CCL5 in refractory rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is the most prevalent autoimmune disease, in which CCL2 and CCL5 are critically involved. The objective was to evaluate the therapeutic effects of bone marrow-derived mesenchymal stem cells (MSCs) on the foregoing chemokines in RA patients. MATERIALS AND METHODS: Thirteen RA patients were evaluated in terms of clinical manifestations, paraclinical factors, gene expression, and plasma levels of CCL2 and CCL5 prior to treatment and 1 and 6 months after intervention. Real-time-polymerase chain reaction and enzyme-linked immunosorbent assay were employed to assess the gene expression and plasma levels of CCL2 and CCL5 at different time points after MSC therapy. Statistical analysis was performed by SPSS 16 and Prism 7. RESULTS: The CCL2 gene expression had statistically significantly increased (P = 0.034), and its plasma level had insignificantly reduced after 1 month. Furthermore, the gene expression and plasma level of CCL5 had statistically significantly decreased (P = 0.032, P < 0.001). The CCL5 gene expression had statistically significantly increased after 6 months (P = 0.001) and its plasma level had insignificantly reduced. CONCLUSION: The most significant inhibitory effects of MSC therapy on the gene expression and plasma level of CCL5 were observed at the end of 1 month. The differences between the gene expression and protein levels during the treatment might be related to microRNA effects or the insufficient number of MSC injection.

Gene expression pattern of CCL2, CCL3, and CXCL8 in patients with bipolar disorder.

BACKGROUND: Bipolar disorder (BD) is one of the most important psychiatric disorders in the world. There is evidence suggesting the role of inflammatory mediators such as chemokines in the etiology of BD. The objective of the current study was to evaluate the gene expression of CCL2, CCL3, and CXCL8 in patients with BD and compare them to healthy controls. MATERIALS AND METHODS: A total of 48 patients with confirmed BD and 48 healthy controls enrolled in this study. All patients were recruited from April to August 2016 at Ibn-Sina Psychiatric Hospital, Mashhad University of Medical Sciences, Mashhad, Iran. RNA was extracted from the whole blood samples and then cDNA was synthesized. Gene expression of CCL2, CCL3, and CXCL8 was measured using SYBR® Green real-time polymerase chain reaction. The difference of delta-CT values between patients and healthy controls was compared with the independent samples t-tests. RESULTS: CCL2 and CXCL8 genes expressed at higher levels in patients with BD as compared to healthy controls, but not significant. On the contrary, we found lower expression levels for CCL3 gene in our patients compared to healthy controls, but the difference was not statistically significant. CONCLUSION: Our findings do not show an association between the gene expression of CCL2, CCL3 and CXCL8 and BD. Increasing the sample size and evaluation on the gene expression of other chemokines in depression and mania phases of BD might be helpful to get a better conclusion.

The Effects of Mesenchymal Stem Cells on the Gene Expression of TGF-beta and IFN-gamma in Patients with Rheumatoid Arthritis.

The therapeutic and immunomodulatory potential of mesenchymal stem cells (MSCs) in rheumatoid arthritis (RA) has attracted considerable scientific attention in recent decades. This study aimed to evaluate the expression of genes encoding interleukin (IL)4 and IL10, as well as interferon-gamma (IFNG) and transforming growth factor beta (TGFB1) in refractory RA patients following intravenous injection of autologous bone marrow-derived MSCs (BM-MSCs). This study was registered in Iranian Registry of Clinical Trials (IRCT) (2015102824760N1) and ClinicalTrials.gov (identifier: NCT03333681). Blood samples were taken from 13 patients before and 1 and 6 months after the MSC injection to evaluate the clinical manifestations, paraclinical factors, and expression of IL4, IL10, IFNG, and TGFB1 genes employing the SYBR Green real-time reverse-transcriptase polymerase chain reaction (RT-PCR) technique. There was a significant increase in the expression of TGFB1 at 1 and 6 months after the MSC injection compared to that in the baseline, while the expression of IL4 and IL10 did not change significantly. On the other hand, the expression of IFNG increased significantly after 1 month but decreased significantly at 6 months compared to 1 month after the intervention. Nevertheless, it showed no significant decrease compared to the baseline. A significant decrease was observed for the expression of IFNG 6 months after the injection compared to that after 1 month, which was in concordance with the rise in the expression of the TGFB1 gene. A significant change in the gene expression of TGFB1 and IFNG in our study was consistent with the amelioration of clinical manifestations, suggesting a mechanism of action for MSCs in the treatment of RA.

The Sufficient Immunoregulatory Effect of Autologous Bone Marrow-Derived Mesenchymal Stem Cell Transplantation on Regulatory T Cells in Patients with Refractory Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an advanced autoimmune disease described by joint involvement. The special properties of mesenchymal stem cells (MSCs) introduced them as a potential therapeutic candidate for RA. In this study, a single dose of autologous MSCs isolated from bone marrow (autologous BM-MSCs, 1 × 106 per kg) was injected intravenously into 13 patients suffering from refractory RA who were followed up within 12 months after the intervention to evaluate immunological elements. Our results showed that the gene expression of forkhead box P3 (FOXP3) in peripheral blood mononuclear cells (PBMCs) considerably increased at month 12. We found a substantial increasing trend in the culture supernatant levels of IL-10 and transforming growth factor-beta 1 (TGF-ß1) in PBMCs from the beginning of the intervention up to the end. Our data may reflect the sufficient immunoregulatory effect of autologous BM-MSCs on regulatory T cells in patients suffering from refractory RA.

Immunoregulatory Effects of Krocina™, a Herbal Medicine Made of Crocin, on Osteoarthritis Patients: A Successful Clinical Trial in Iran.

Osteoarthritis (OA) is the major cause of joint pain and disability. This research was planned to examine the effects of Krocina™, aherbal medicine made of crocin, an ingredient of saffron, in patients with OA. Forty patients suffering from OA were enrolled in our study and randomly divided into two groups, receiving Krocina™ and placebo, and the clinical trial continued for four months.Peripheral blood was taken from all patients and the percentage ofvarious subsets of T cells in addition to the levels of forkhead box protein P3 (FOXP3) and interleukin (IL)-17 were measured by flow cytometry technique. The visualan alog scale (VAS) index analysis decreased significantly in both groups (krocinaTM and placebo) (p<0.05). Assessment of the C-reactive protein (CRP) level in serum showed a significant decrease in the krocinaTM group (p<0.05). Moreover, we found a meaningful increase in the percentage of regulatory T cells (Tregs)cellin samples gathered from Krocina™ group (P=0.02) patients. The mean percentages of T helper (Th) 17 cellsinthe Krocina™ group and CD8+ T cellsin the placebo group patients were also meaningfully reduced (p<0.05). The geometric mean fluorescence intensity (GMFI) for IL-17 showed a significant decrease and increase in Krocina™ and placebo groups, respectively (p<0.05). No noticeable difference was observed in the percentages of Th cells and GMFI-FOXP3 in either group. Treg/Th17 ratio was shifted towards Tregscell in Krocina™ group at the end of the intervention. It is concluded that Krocina™ has immunoregulatory effects on patients with OA, ameliorating the disease.

A significant decrease of BAFF, APRIL, and BAFF receptors following mesenchymal stem cell transplantation in patients with refractory rheumatoid arthritis.

In the present study, we aimed to evaluate effects of autologous mesenchymal stem cells (MSCs) intravenous administration on the response of B cells, BAFF, APRIL, and their receptors on the surface of B cells at 1, 6, and 12 month follow-up periods in refractory rheumatoid arthritis (RA) patients. Thirteen patients with refractory RA received autologous MSCs. Plasma levels of BAFF and APRIL were measured employing ELISA method, followed by estimating B cell population and BAFFRs evaluation by flow cytometry technique. Gene expression of BAFF, APRIL, and their receptors on B cell surface in PBMCs was evaluated by SYBR Green real-time PCR technique. Plasma concentration of BAFF significantly decreased 1 and 6 months after the MSCT (MSCs Transplantation). Plasma concentration of APRIL significantly decreased 1 month after the MSCT. Percentages of CD19 + B cells in the PBMC population significantly decreased 12 months after the MSCT. Percentages of BR3 + CD19 + B cells and BCMA + CD19 + B cells significantly decreased at the 12th month after the MSCT. The gene expression of BAFF in the PBMC population significantly decreased during 6, and 12 months after the MSCT. The gene expression of APRIL significantly decreased on month 6 after the MSCT. The gene expression of BR3 significantly decreased during 1, 6, and 12 months after the MSCT. The MSCT seems to decrease B cells response because of the reduced production of BAFF and APRIL cytokines and decrease the expression of their receptors on the surface of B cells.

Possible Anti-inflammatory Effects of Mesenchymal Stem Cells Transplantation via Changes in CXCL8 Levels in Patients with Refractory Rheumatoid Arthritis.

The synovial- lining cells have been involved with rheumatoid arthritis (RA) through the secretion of various cytokines and chemokines. Increased levels of these cytokines and chemokines are seen first in the synovial and subsequently in the bloodstream of RA patients. The synovial and circulating levels of CXCL8, CXCL12, and CXCL13 are higher in the RA patients than in the healthy subjects, causing migration of immune cells to the joints, which is associated with increased joint destruction. We aimed to evaluate the effects of autologous mesenchymal stem cells intravenous administration on plasma levels of CXCL8, CXCL12 and CXCL13 at 1, 6, and 12 month follow-up periods in refractory RA patients. 13 patients with refractory RA received autologous mesenchymal stem cells (MSCs). The ELISA technique was used to evaluate the plasma level of these chemokines. CXCL8 levels were significantly decreased at month 6 after MSCs transplantation in comparison with pre-injection level, and the concentration of this chemokine was significantly increased at month 12 in comparison with the month 6 after injection (P <0.05). The levels of CXCL12 and CXCL13 were insignificantly decreased at months 1 and 6 after the MSCs transplantation. The interaction of MSCs after migration to the inflamed joints with CXCL8-producing cells could be one but not the only possible mechanism that reduces its production in the joints and subsequently in the plasma of RA patients. CXCL8 reduction as a consequence of MSCs application returned to pre-injection levels after 12 months. Therefore, increasing the dose of MSCs and replication of injections may maintain the potential anti-inflammatory effects of MSCs on the production of CXCL8 as an inflammatory mediator in patients with refractory RA.

Amelioration of clinical symptoms of patients with refractory rheumatoid arthritis following treatment with autologous bone marrow-derived mesenchymal stem cells: A successful clinical trial in Iran.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune arthropathy characterized by synovial hyperplasia leading to functional impairment. Although the exact cause of RA is unknown, there is evidence suggesting the role of T cell subtypes in the pathogenesis of RA. Conventional therapy in some RA patients is associated with mild or severe side effects, and resistance of some patients has been reported to these types of therapy. The therapeutic potential of mesenchymal stem cells (MSCs) introduced them as a novel therapeutic choice for the treatment of rheumatic diseases. The aim of our study was to evaluate the effects of intravenous administration of autologous bone marrow-derived MSCs on the immunological, clinical and para-clinical factors such as regulatory T cells, Th17 cells, CD8+ T cells, CD4+ T cells, disease activity score 28-erythrocyte sedimentation rate (DAS28-ESR), visual analogue scale (VAS), ESR, C-reactive protein (CRP), rheumatoid factor (RF), and anti-cyclic citrullinated peptide (anti-CCP) antibodies in patients with refractory RA. Nine refractory RA patients with no other rheumatologic disorders were included in this study. All patients received a single intravenous dose of 1 × 106 autologous bone marrow-derived MSCs/kg, and were followed up at 1, 6 and 12 months after injection of MSCs. We found a significant decreasing trend in Th17 percentage and geometric mean fluorescence intensity for IL-17A following injection of MSCs at 12 months compared to the time point zero. Furthermore, a significant increase in regulatory T cells percentage was observed at the end of the first month after the intervention. DAS28-ESR decreased significantly at 1 and 12 months after MSC therapy. VAS score showed a significant decreasing trend during the follow-up periods. No significant difference was found for serum CRP and anti-CCP levels after the intervention. In conclusion, our data indicated that clinical symptoms were significantly ameliorated following the intravenous injection of autologous bone marrow-derived MSCs to the patients with refractory RA.

Influence of vitamin D on cell cycle, apoptosis, and some apoptosis related molecules in systemic lupus erythematosus.

OBJECTIVES: Genetic and environmental factors are involved in the pathogenesis of systemic lupus erythematosus (SLE). Autoreactive lymphocytes are cleared through apoptosis and any disturbance in the apoptosis or clearance of apoptotic cells may disturb tolerance and lead to autoimmunity. Vitamin D has anti-proliferative effects and controls cell cycle progression. In this study we investigated the effects of vitamin D on cell cycle and apoptosis induction in lupus patients. MATERIALS AND METHODS: Isolated peripheral blood mononuclear cells (PBMCs) from 25 SLE patients were cultured in the presence of 50 nM of 1,25(OH)2D3; then one part of the cells were stained with FITC labeled Annexin V and PI and were analyzed for apoptosis determination. For gene expression assessment of FasL, Bcl-2 and Bax, RNA was extracted from one another part of the cells, cDNA was synthesized and gene expression analysis was performed using Real time PCR. An additional part of the cells were treated with PI and the cell cycle was analyzed using flowcytometer. RESULTS: The mean number of early apoptotic cells in vitamin D treated cells decreased significantly (18.48±7.9%) compared to untreated cells (22.02±9.4%) (P=0.008). Cell cycle analysis showed a significant increase in G1 phase in vitamin D treated cells (67.33±5.2%) compared to non treated ones (60.77±5.7%) (P =0.02). Vitamin D up-regulated the expression levels of Bcl-2 by (18.87 fold increase), and down-regulated expression of Bax (23%) and FasL (25%). CONCLUSION: Vitamin D has regulatory effects on cell cycle progression, apoptosis and apoptosis related molecules in lupus patients.

Association between lower frequency of R381Q variant (rs11209026) in IL-23 receptor gene and increased risk of recurrent spontaneous abortion (RSA).

Recurrent spontaneous abortion (RSA) is defined as three or more consecutive spontaneous abortions before the 20th week of gestation. The purpose of the present study was to investigate the association between a functional single nucleotide polymorphism (SNP) in the interleukin (IL)-23 receptor gene (IL-23R; rs11209026, 1142 G wild type → A reduced function, Arg381Gln, R381Q) and RSA. For the study, 200 RSA patients (confirmed using established diagnostic criteria) and 200 normal individuals in fertility and infertility centers in the cities of Yazd and Isfahan were recruited during a period from 2012-2013. Using PCR-RFLP, the R381Q variant was screened for in the IL-23R gene of the patients and controls. The results indicated there were significant differences in the frequency of this genetic variant in the patients versus the healthy controls, i.e. 2% and 7.5%, respectively (p value = 0.01; odds ratio = 0.25; CI = 95%). No significant difference was found for the G allelic frequency in patients with RSA and in the control group (p = 0.60). The A allelic frequency was significantly different between the two groups (p = 0.01). Based on these findings, it is concluded that the frequency of single nucleotide polymorphism in the IL-23 receptor (R381Q) in patients with recurrent spontaneous abortion (RSA) is less than that found in normal control women.

Influence of 1 Alpha, 25-Dihydroxyvitamin D3 on T Helper 17 Cells and Related Cytokines in Systemic Lupus Erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Emerging data suggests that T helper 17 (Th17) cells play a pathogenic role in SLE and the increased number of these cells correlates with disease activity. In recent years, 1α, 25-dihydroxyvitamin D3 (1,25VitD3) has been considered as an immunomodulatory factor. OBJECTIVE: To investigate the effect of 1,25VitD3 on Th17 cells and on the expression of related cytokines in SLE patients. METHOD: Thirty SLE patients (newly diagnosed or in remission) were sampled for 10 ml whole blood to isolate peripheral blood mononuclear cells (PBMCs) using Ficoll-Hypaque density gradient centrifugation. Isolated cells were cultured in the presence and absence of 50 nM 1,25VitD3. After incubation, cells were harvested and stimulated for 4-5 hours with phorbol myristate acetate (PMA) and ionomycin in the presence of brefeldin A. IL-17 secreting cells were analyzed by flowcytometry. RNA was extracted from cultured cells, cDNA was synthesized, and the expression levels of IL-6, IL-17, IL-23 and TGF-ß genes were assessed by real-time PCR. RESULTS: The percentage of Th17 cells (CD3+CD8- IL-17+ T cells) decreased significantly in 1,25VitD3-treated cells (3.67 ± 2.43%) compared to untreated cells (4.65 ± 2.75%)( p=0.003). The expression of TGF-ß up regulated (1.38-fold) and the expression of IL-6 (50%), IL-17 (27%) and IL-23 (64%) down regulated after 1,25VitD3 treatment. CONCLUSION: This study showed that 1,25VitD3 modulates Th17 related pathways in SLE patients and revealed the immunomodulatory effect of 1,25VitD3 on the Th17 mediated autoimmunity.