Peripheral Blood Mononuclear Cells Of Multiple Sclerosis Patients Research Articles

Altered gene expression of miR-155 in peripheral blood mononuclear cells of Multiple sclerosis patients: Correlation with TH17 frequency, inflammatory cytokine profile and autoimmunity

In the chronic, organ-specific autoimmune disorder known as multiple sclerosis (MS), the myelin sheath is attacked by immune cells, leading to damage to the central nervous system (CNS). It has been discovered that miRNAs are important in the etiology of MS, since deregulation of miRNAs can lead to defects in immune tolerance. In this study, we sought to investigate the involvement of miR-155 in MS disorder through examination of its altered expression in peripheral blood mononuclear cells (PBMCs) of patients with MS in compare with healthy controls. Furthermore, we investigated the frequency of T helper 17 cells (Th17) in MS patients and analyzed not only the expression of inflammatory cytokines including IL-6, IL-17 and IL-21 in patients’ PBMCs, but also their secreted levels in serum of patients suffering from MS. Subsequently, we assessed the correlation between miR-155 expression with Th17 frequency and levels of released cytokines in serum. Upregulated expression of miR-155 was detected in PBMCs of MS patients and the positive correlation between its expression with increased frequency of Th17 cells and their related inflammatory cytokine profile augmented secretion in serum were identified. In conclusion, our study revealed the significant association between Th17 frequency, increased level of cytokines related to Th17 differentiation and function with miR-155 augmented expression in PBMCs. So, our findings suggested that miR-155 and especially its expression in immune cells including effector T cells can be the target of future therapeutic strategies for the management and prevention of MS progression, however, further research is requisite before this approach can be utilized in clinical practice.

Sex-dependent expression levels of VAV1 and P2X7 in PBMC of multiple sclerosis patients.

Multiple sclerosis (MS) is an inflammatory autoimmune disorder of the central nervous system and the leading cause of progressive neurological disability in young adults. It decreases the patient's lifespan by about 10 years and affects women more than men. No medication entirely restricts or reverses neurological degradation. However, early diagnosis and treatment increase the possibility of a better outcome. To identify new MS biomarkers, we tested the expression of six potential markers (P2X4, P2X7, CXCR4, RGS1, RGS16 and VAV1) using qPCR in peripheral blood mononuclear cells (PBMC) of MS patients treated with interferon β (IFNβ), with glatiramer acetate (GA) or untreated. We showed that P2X7 and VAV1 are significantly induced in MS patients. In contrast, the expression of P2X4, CXCR4, RGS1 and RGS16 was not significantly modified by MS in PBMC. P2X7 and VAV1 are essentially induced in female patients, suggesting these markers are connected to sex-specific mechanisms. Strikingly, VAV1 expression is higher in healthy women than healthy men and IFNβ treatment of MS reduced VAV1 expression in female MS patients while it up-regulated VAV1 in male MS patients. Our data point to the differential, sex-dependent value of MS markers and treatment effects. Although rgs16 expression in PBMC was not a valid MS marker in patients, the strong upregulation of P2X4 and P2X7 induced in the spinal cord of WT mice by EAE was abrogated in rgs16KO mice suggesting that rgs16 is required for P2X4 and P2X7 induction by neurological diseases.

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

ABSTRACT 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-β). 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.

The expression pattern of VISTA in the PBMCs of relapsing-remitting multiple sclerosis patients: A single-cell RNA sequencing-based study

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Dysregulated immune responses have been implicated in MS development. Growing evidence has indicated that inhibitory immune checkpoint molecules can substantially regulate immune responses and maintain immune tolerance. V-domain Ig suppressor of T cell activation (VISTA) is a novel inhibitory immune checkpoint molecule that can suppress immune responses; however, its expression pattern in the peripheral blood mononuclear cells (PBMCs) of relapsing-remitting multiple sclerosis (RRMS) has not thoroughly been studied. Herein, we evaluated Vsir expression in PBMCs of RRMS patients and characterized the expression pattern of the Vsir in the PBMCs of MS patients. Besides, we investigated the effect of fingolimod, IFNβ-1α, glatiramer acetate (GA), and dimethyl fumarate (DMF) on Vsir expression in PBMCs of RRMS patients. Our results have shown that Vsir expression is significantly downregulated in the PBMCs of patients with RRMS. Besides, the single-cell RNA sequencing results have demonstrated that Vsir expression is downregulated in classical monocyte, intermediate monocytes, non-classical monocytes, myeloid DCs (mDC), Plasmacytoid dendritic cells (pDCs), and naive B-cells of PBMCs of MS patients compared to the control. In addition, DMF, IFNβ-1α, and GA have significantly upregulated Vsir expression in the PBMCs of RRMS patients. Collectively, the current study has shed light on Vsir expression in the PBMCs of MS patients; however, further studies are needed to elucidate the significance of VISTA in the mentioned immune cells.

In Vitro Effects of Olive Vegetation Water on IFN-γ and IL-10 Secretion in Multiple Sclerosis Patients

Background: Multiple Sclerosis (MS) is an autoimmune and inflammatory disorder of the central nervous system (CNS), which is associated with the imbalance of pro- and anti-inflammatory cytokines. Evidence indicates that nutritional interventions have some immunomodulatory impacts. Objectives: In this study, we investigated the effect of olive vegetation water (OVW) on IFN-γ and IL-10 secretion by peripheral blood mononuclear cells (PBMCs) of MS patients. Methods: In this study, PBMCs of MS patients were separated by Ficoll-Hypaque centrifugation. The cytotoxicity of OVW was assessed by the MTT assay. The treatments were performed for 48 and 72 hours, and IFN-γ and IL-10 were measured by ELISA. Results: No cytotoxicity was observed for OVW. Besides, OVW showed a significant inhibitory effect on IFN-γ secretion but augmenting effect on IL-10 secretion by PBMCs dose-dependently. Conclusions: This study indicated that OVW could have immunoregulatory effects on inflammatory reactions in MS patients.

PBMC of Multiple Sclerosis Patients Show Deregulation of OPA1 Processing Associated with Increased ROS and PHB2 Protein Levels.

Multiple sclerosis (MS) is an autoimmune disease in which activated lymphocytes affect the central nervous system. Increase of reactive oxygen species (ROS), impairment of mitochondria-mediated apoptosis and mitochondrial alterations have been reported in peripheral lymphocytes of MS patients. Mitochondria-mediated apoptosis is regulated by several mechanisms and proteins. Among others, optic atrophy 1 (OPA1) protein plays a key role in the regulating mitochondrial dynamics, cristae architecture and release of pro-apoptotic factors. Very interesting, mutations in OPA1 gene, have been associated with multiple sclerosis-like disorder. We have analyzed OPA1 and some factors involved in its regulation. Fifteen patients with MS and fifteen healthy control subjects (HC) were enrolled into the study and peripheral blood mononuclear cells (PBMCs) were isolated. H2O2 level was measured spectrofluorimetrically, OPA1, PHB2, SIRT3, and OMA1 were analyzed by western blotting. Statistical analysis was performed using Student’s t-test. The results showed that PBMC of MS patients were characterized by a deregulation of OPA1 processing associated with increased H2O2 production, inactivation of OMA1 and increase of PHB2 protein level. The presented data suggest that the alteration of PHB2, OMA1, and OPA1 processing could be involved in resistance towards apoptosis. These molecular parameters could also be useful to assess disease activity.

A critical role for expression of atypical chemokine receptor 2 in multiple sclerosis: A preliminary project

Background: ACKR2 is an atypical chemokine receptor that promotes acute inflammation by acting as a scavenger receptor for inflammatory chemokines in experimental models of some inflammatory disorders, but its function in multiple sclerosis (MS) is unclear. Therefore we aimed to evaluate the mRNA expression of ACKR2 as a scavenger receptor in patients with MS and also the correlation of this expression with certain cytokines that are important for MS pathogenesis. Methods: The ACKR2 mRNA expression was examined on peripheral blood mononuclear cells (PBMCs) of 40 patients with relapsing-remitting MS (RRMS) and 35 healthy individuals. ACKR2 mRNA expressions were measured using real-time quantitative polymerase chain reaction (qPCR). In addition, circulating cytokine levels (TNF-α, IL-6, IL-33) in all patients and controls were evaluated using enzyme-linked immunosorbent assay. Results: mRNA expression of ACKR2 was decreased on PBMCs compared to healthy subjects (p < 0.001). ACKR2 expression in peripheral blood leucocytes could be regulated by circulating cytokines but there are no correlations with these cytokines (p > 0.05). In addition, the patients’ plasma levels of IL-33 significantly increased (p = 0.039) and no significant difference was found between other cytokine levels in the patients (p > 0.05). Conclusion: Our data clearly show a decreased ACKR2 mRNA expression on PBMCs and increased plasma IL-33 levels of patients with MS. There was no significant relationship between ACKR2 and other cytokine levels. Within our knowledge, this is the first study that evaluates the ACKR2 mRNA expression in the PBMCs of MS patients.

SA50PSYCHIATRIC GENETIC COUNSELLING TRAINING IN THE UK OVER THE LAST THREE YEARS

we recently showed that a hypothesized anti-aging and anti-inflammatory protein, namely Klotho, may contribute to the etiology and/or pathogenesis of multiple sclerosis (MS). In addition, Klotho function and its gene expression are dependent on inflammatory pathways. Accordingly, the aim of this study was to investigate the Klotho gene expression within peripheral blood mononuclear cells (PBMCs) of patients with MS.Altogether, 30 patients with relapsing-remitting MS (RRMS) along with 30 age and sex-matched healthy individuals were enrolled in this study. Blood samples were obtained from all participants and then PBMCs were isolated. The quantitative Real-Time PCR was carried out for Klotho mRNA derived from PBMCs.The results showed that klotho gene expression in the PBMCs of patients with RRMS is nearly 2.5-fold less than healthy individuals (P = 0.0006).This is the first study demonstrating a possible role of Klotho in the PBMCs of MS patients.

Klotho gene expression decreases in peripheral blood mononuclear cells (PBMCs) of patients with relapsing-remitting multiple sclerosis

Backgroundwe recently showed that a hypothesized anti-aging and anti-inflammatory protein, namely Klotho, may contribute to the etiology and/or pathogenesis of multiple sclerosis (MS). In addition, Klotho function and its gene expression are dependent on inflammatory pathways. Accordingly, the aim of this study was to investigate the Klotho gene expression within peripheral blood mononuclear cells (PBMCs) of patients with MS. MethodsAltogether, 30 patients with relapsing-remitting MS (RRMS) along with 30 age and sex-matched healthy individuals were enrolled in this study. Blood samples were obtained from all participants and then PBMCs were isolated. The quantitative Real-Time PCR was carried out for Klotho mRNA derived from PBMCs. ResultsThe results showed that klotho gene expression in the PBMCs of patients with RRMS is nearly 2.5-fold less than healthy individuals (P=0.0006). ConclusionThis is the first study demonstrating a possible role of Klotho in the PBMCs of MS patients.

Decreased Level of sRAGE in the Cerebrospinal Fluid of Multiple Sclerosis Patients at Clinical Onset

Objectives: Receptor for advanced glycation end products (RAGE) ligands/RAGE interactions have been proposed to have a pathogenic role in neuroinflammatory disorders. Our study aimed to assess changes in high-mobility group box (HMGB)1 and its receptor RAGE in peripheral blood (PBL) and cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) at the disease onset compared with control subjects. Methods: PBL and CSF were collected from control subjects (n = 30) and MS patients (n = 27) at clinical onset. Soluble RAGE (sRAGE), HMGB1, S100 calcium-binding protein A12 (S100A12), interleukin (IL)-1β and tumor necrosis factor (TNF)-α were measured in the CSF and plasma by enzyme-linked immunosorbent assay. Gene expression in PBL mononuclear cells (PBMCs) was detected by quantitative PCR for RAGE, HMGB1, S100A12 and several proinflammatory/immunoregulatory cytokines. Results: We found a significantly lower expression of IL-10 (p = 0.031) in the PBMCs of MS patients. The level of sRAGE in the CSF of MS patients was lower (p = 0.021), with the ability to discriminate between MS patients and control subjects. Moreover, PBMC gene expression for HMGB1 and S100A12 positively correlated with IL-6. Conclusions: Our study confirmed that the cytokine network is disturbed in PBL and CSF at MS clinical onset. The deregulated HMGB1/RAGE axis found in our study may present an early pathogenic event in MS, proposing sRAGE as a possible novel therapeutic strategy for MS treatment.

Up-regulation of inducible heat shock protein-70 expression in multiple sclerosis patients

Inducible heat shock protein (HSP)70 (HSP70-1A and HSP70-1B proteins) is a chaperone responsible for assisting proper protein folding. Following stress conditions, HSP70 is highly up-regulated to mediate cytoprotective functions. In addition, HSP70 is able to trigger innate and adaptive immune responses that promote the immune recognition of antigens and to act as a cytokine when it is released. The data in the literature are controversial with regard to expression studies in peripheral blood mononuclear cells (PBMCs). In the present study, we aimed to examine if alterations of HSP70-1A/B expression are involved in the autoimmune pathogenesis of multiple sclerosis (MS). We determined both mRNA and protein expression in PBMCs of MS patients and healthy donors (HDs). We found a baseline increased expression of the HSPA1A gene in PBMCs from MS patients compared with HDs. Gene expression findings were associated with an increased protein expression of HSP70-1A/B in T lymphocytes (CD4+ and CD8+) and monocytes from MS patients under basal conditions that may reflect the immunological activation occurring in MS patients. We also provided evidence that heat shock (HS) stimulus induced HSP70-1A/B protein expression in HDs and MS patients, and that HS-induced HSP70-1A/B protein expression in monocytes correlated with the number of T2 lesions at baseline in MS patients. However, after lipopolysaccharide inflammatory stimulus, monocytes from MS patients failed to induce HSP70-1A/B protein expression. Our data hint at altered immune responses in MS and may indicate either a state of chronic stress or increased vulnerability to physiological immune responses in MS patients.

Impact of Vitamin A Supplementation on RAR Gene Expression in Multiple Sclerosis Patients

Vitamin A and its derivatives have been shown to modulate the immune system via retinoic acid receptor (RAR). This study explored the impact of retinyl palmitate supplementation on RAR subtype gene expression in peripheral blood mononuclear cells (PBMCs) in multiple sclerosis (MS) patients. The study designed as a double-blind randomized clinical trial in which relapsing remitting multiple sclerosis patients were evaluated. Both groups received one capsule 50,000 IU vitamin D3 per 2 weeks and one intramuscular injection interferon beta-1a per week. The intervention group received one 25,000 IU retinyl palmitate capsule daily for 6 months and the placebo group received one placebo capsule daily. The PBMCs were isolated from participants and the expression level changes of RAR-α and RAR-γ genes were determined by real-time PCR. After supplementation, in the intervention group, the RAR-α gene expression level was significantly decreased compared to the placebo group (p = 0.03); however, the expression of RAR-γ gene did not significantly change (p = 0.10). These results show that vitamin A supplementation can significantly downregulate the expression of RAR-α gene in PBMCs of MS patients that suggest the presence of in vivo regulatory mechanisms for the action of vitamin A on the immune system.

Comparison of the immunomodulatory effects of the plant sterol β-sitosterol to simvastatin in peripheral blood cells from multiple sclerosis patients

Statins as hypocholesterolimic drugs have recently shown to have ant-inflammatory properties and thus are being assessed for the treatment of multiple sclerosis (MS). Dietary phytosterols such as β-sitosterol (SIT) are also hypocholesterolemic compounds and from preliminary studies they appear to have also anti-inflammatory properties. In this communication, we report on studies to investigate the immunomodulatory effects of SIT on proliferation and release of key cytokines from peripheral blood mononuclear cells (PBMC) of MS patients. In PBMC of MS patients, 16 μM SIT had no effect on cell proliferation; however simvastatin (SV) at 10 and 20 μM reduced cell proliferation by as much as 60%. SIT (4 μM) reduced TNF-α release by 24% in PBMC of MS patients whereas 10 μM SV reduced TNF-α release by 94%. SIT reduced IL-12 release in MS patients at 4 and 16 μM by 27% and 30%, respectively. In healthy subjects, 16 μM SIT increased the anti-inflammatory cytokine IL-10 by 47% whereas 10 μM SV decreased IL-10 by 30%. In PBMC of MS patients, SIT had no effect on IL-10 release whereas 10 μM SV reduced IL-10 by 62%. SIT (4 μM) reduced IL-5 release by 47% in MS patients while 10 μM SV reduced IL-5 by 89%. The results show that SIT is effective in modulating the secretion of pro/anti-inflammatory cytokines and suggest a potential beneficial effect of SIT in MS management without the side effects associated with statin therapy.

β-Endorphin Concentrations in Peripheral Blood Mononuclear Cells of Patients With Multiple Sclerosis

It has been reported that the opioid peptide beta-endorphin (BE) has immunosuppressive effects. Interferon beta (IFN-beta) is a well-established therapy for multiple sclerosis (MS), but immunological mechanisms underlying its beneficial effects in MS are partially undefined. To determine BE levels in peripheral blood mononuclear cells (PBMCs) of patients with relapsing-remitting MS during different phases of disease activity and the possible modulating effects of IFN-beta treatment on PBMC BE synthesis in patients with MS. We measured BE levels in blood samples collected from 6 patients with MS who had not experienced clinical changes during the previous 3 months (patients with stable MS) and from 7 patients with MS during a clinical relapse. We also surveyed BE levels in PBMC samples from 8 patients with MS before treatment and for 6 months after the beginning of IFN-beta administration. The control group was 13 healthy subjects. Low PBMC BE levels were detected in patients with stable MS and in those entering IFN-beta treatment compared with control subjects. Increased BE concentrations were observed in MS patients experiencing a clinical relapse compared with patients with stable MS. During IFN-beta treatment, the levels of BE in PBMC samples from patients with MS increased significantly (after 1 month, P =.02; after 3 months, P =.007; and after 6 months, P =.16). A reduction of BE levels was present in patients with clinically inactive MS. Treatment with IFN-beta seems to induce an increase of this opioid in PBMCs of MS patients. The increase of BE concentration during a clinical relapse may represent a possible control mechanism aimed at counterbalancing the inflammatory phase of the disease. Arch Neurol. 2000;57:1178-1181

Absence of HHV-6 and HHV-7 in cerebrospinal fluid in relapsing-remitting multiple sclerosis.

To contribute to clarifying the controversy on the association between Human Herpesviruses 6 and 7 (HHV-6, HHV-7) and multiple sclerosis (MS) studying patients with relapsing-remitting MS (RRMS) with or without evidence of disease activity (clinically or radiologically evaluated). In 25 RRMS patients, 7 suspected MS patients and 9 patients with other neurological diseases, the following parameters were analysed: i) antibody titers (IgM and IgG) against HHV-6 by indirect immunofluorescence both in serum and cerebrospinal fluid (CSF) samples; ii) PCR-detection of HHV-6 DNA and HHV-7 DNA in CSF and HHV-6 DNA in peripheral blood mononuclear cells (PBMCs). MS patients in remission underwent a gadolinium-enhanced magnetic resonance imaging in proximity of sample collections. No viral DNA was found in any CSF sample, HHV-6 DNA frequency in PBMCs of MS patients and controls was not statistically different. Antibody titers against HHV-6 were comparable to those of the general population. Some 30.4% of MS patients were seronegative to HHV6. Our data suggest that there is no relationship between HHV-6 or HHV-7 and MS.

B7-1 (CD80), B7-2 (CD86), interleukin-12 and transforming growth factor-β mRNA expression in CSF and peripheral blood mononuclear cells from multiple sclerosis patients

Costimulatory molecules B7-1 (CD80) and B7-2 (CD86) are differently involved in T cell stimulation. In chronic experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), B7-1 was preferentially involved in pathophysiology of relapses. We used reverse transcription polymerase chain reaction (RT-PCR) to amplify the mRNA coding for these molecules in cerebrospinal fluid (CSF) cells and peripheral blood mononuclear cells (PBMC) from 18 MS patients and 21 other neurological patients. In CSF cells of MS cases, B7-1 mRNA was only detected in some patients who showed clinical signs of acute relapse at the time of the spinal tap, while B7-2 mRNA was widely detectable without difference between active or stable MS and controls. mRNA coding for transforming growth factor-β (TGF-β) was detectable in the majority of cases, with higher expression in CSF cells of MS and other inflammatory neurological diseases (OIND) than in noninflammatory controls, and higher expression in PBMC of MS patients than in all other cases. Finally, mRNA coding for interleukin (IL)-12p40 was only detected in a very few number of MS and inflammatory cases. These findings were related to previous detection of other cytokines in the same cases, showing relationships in CSF cells between high expression of B7-1, IL-12p40 and TNF-α.

Lack of detectable human T-cell lymphotropic virus type I sequences in samples from multiple sclerosis patients.

Recently, inconclusive results have followed the early data on the possible association between multiple sclerosis (MS) and human T-cell lymphotropic virus type I (HTLV-I) infection. For this reason, we examined this hypothesis using the polymerase chain reaction (PCR) to study samples of differing origin from Italian MS patients. In particular, we developed a systematic analysis of paraffin-embedded brain white matter from histologically defined lesions of 14 MS patients using PCR and primer sets specific for HTLV-I sequences; additionally, cerebrospinal fluids (CSFs) from 12 patients and peripheral blood mononuclear cells (PBMCs) from subjects at the early and late phase of the disease were investigated for free HTLV-I virions and specific proviral sequences, respectively. In agreement with some groups who reported lack of HTLV-I sequences in PBMCs of MS patients but in clear contrast with others, we failed to detect specific viral sequences using this broad approach.