Lactobacillus rhamnosus and Lactobacillus delbrueckii Ameliorate the Expression of miR-125a and miR-146a in Systemic Lupus Erythematosus Patients.

The microRNAs are non-coding RNA molecules involved in physiological and pathological processes, causing autoimmune diseases such as systemic lupus erythematosus (SLE). Probiotics are living microorganisms that possess beneficial effects on the host immune system and modulate it. The effect of Lactobacillus rhamnosus and Lactobacillus delbrueckii on the expression of miR-125a and miR-146a was studied in peripheral blood mononuclear cells (PBMCs) from newly diagnosed lupus patients in this in vitro study. During this study, 20 recently diagnosed SLE patients and 20 healthy individuals participated. Ficoll method was used to isolate the PBMCs from whole blood, which were cultured for 48 h with Lactobacillus rhamnosus and Lactobacillus delbrueckii. In the next step, total RNA containing microRNA was extracted. cDNA was synthesized for miR-125a and miR-146a genes and analyzed by real-time PCR. Results were presented as fold changes. As compared to healthy controls, SLE patients expressed lower levels of miR-125a and miR-146a. PBMCs treated with Lactobacillus rhamnosus, Lactobacillus delbrueckii, or both probiotics had significantly higher levels of miR-125a and miR-146a compared to the untreated group. Treatment of PBMCs with both L. rhamnosus and L. delbrueckii upregulated the expression of miR-125a and miR-146a in treated cells compared with untreated cells in SLE patients (p = 0.02, p = 0.001). Lactobacillus rhamnosus and Lactobacillus delbrueckii modify lupus patients' immune responses and disease effects by regulating miR-125a and miR-146a.

The effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on cytokines and their related molecules: An ex vivo study on patients with systemic lupus erythematosus.

Objectives: This study aimed to assess the ex vivo impact of Lactobacillus delbrueckii (L. delbrueckii) and Lactobacillus rhamnosus (L. rhamnosus) on inflammatory and anti-inflammatory cytokines as well as their related molecules on the peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus (SLE) patients. Patients and methods: This study was conducted with 20 newly diagnosed SLE patients (18 females, 2 males; mean age: 33.3±12.4 years; range, 18 to 68 years) between September 2017 and September 2018. Extracted PBMCs from each patient were divided into 4 cell groups in our study. Three cell groups act as treatment groups receiving L. rhamnosus (107 CFU/mL), L. delbrueckii (105 CFU/mL) or a mixture of both, and one group act as our untreated control group in the absence of any probiotic agents. All cell groups were cultured in RPMI 1460 medium for 48 h. Then, total RNA was extracted, and cDNA was synthesized. Results: The gene expression levels of forkhead box P3 (FOXP3), transforming growth factor beta (TGF-ß), interleukin (IL)-6, IL-10, and IL-2 were evaluated by a quantitative real-time polymerase chain reaction. The results revealed that expression levels of FOXP3, TGF-ß, IL-10, and IL-2 increased and the level of IL-6 decreased in probiotics-receiving groups compared to the control group. Lactobacillus delbrueckii and L. rhamnosus enhanced the expression of regulatory T cell-related molecules such as FOXP3 and IL-2 and also increased the expression of IL-10. These probiotics also reduced the expression of IL-6 as proinflammatory cytokines in the PBMCs of SLE patients. Conclusion: The results of the present study show that these probiotics could be effective in regulating the balance of cytokine gene expression ex vivo , and due to their beneficial effects, they can be an intriguing option in the production of new complement drugs for SLE.

The Evaluation of the Effect of Tolerogenic Probiotics on the Maturation of Healthy Dendritic Cells versus Immature Dendritic Cells.

Background: Dendritic cells, (DCs) as one of the important immune cell populations, are responsible for the initiation, development, and control of acquired immune responses. Myeloid dendritic cells can be used as a vaccine for several autoimmune diseases and cancers. Tolerogenic probiotics with regulatory properties can affect the maturation and development of immature dendritic cells (IDC) into mature DCs with certain immunomodulatory effects. Objective: To assess the immunomodulatory effect of Lactobacillus rhamnosus and Lactobacillus delbrueckii, as two tolerogenic probiotics, in the differentiation and maturation of myeloid dendritic cells. Methods: The IDCs were derived from the healthy donors in GM-CSF and IL 4 medium. Mature DCs (MDC) were produced with L. delbrueckii, L. rhamnosus, and LPS from IDCs. Real-Time PCR and flow cytometry were used to confirm the DC maturation and to determine DC markers as well as IDO, IL10, and IL12 expression levels, respectively. Results: Probiotic-derived DCs showed a significant reduction in the level of HLA-DR (P≤0.05), CD86 (P≤0.05), CD80 (P≤0.001), CD83 (P≤0.001), and CD1a. Also, the expression of IDO (P≤0.001) and IL10 increased while IL12 expression decreased (P≤0.001). Conclusion: Our findings revealed that tolerogenic probiotics could induce regulatory DCs by reducing co-stimulatory molecules along with increasing the expression of IDO and IL10 during the differentiation process. Therefore, the induced regulatory DCs probably can be used in the treatment of various inflammatory diseases.

Enhancing the efficacy of Hypericum perforatum in the treatment of an experimental model of multiple sclerosis using gold nanoparticles: an in vivo study.

Objective: Hypericum perforatum is a herbal medicine used in traditional medicine for the treatment of depression due to its antidepressant and anti-inflammatory activities. Therefore, we evaluated the therapeutic efficacy of H. perforatum extract (HPE) in combination with gold nanoparticles (HPE-GNP) against experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Materials and Methods: EAE was induced in C57BL/6 mice with subcutaneous injection of MOG35-55 emulsified in complete Freund's adjuvant, and intraperitoneal pertussis toxin. Mice were treated with drugs in free (HPE) and nano-form (HPE-GNP) preparations. Splenocytes were isolated from all mice and the level of inflammatory and anti-inflammatory cytokines were evaluated by ELISA. The expression of T cells' transcription factors was also assessed using Real-Time PCR. Results: Clinical score was reduced after HPE-GNP treatment. This change was associated with a decrease in the incidence and infiltration of inflammatory cells into the central nervous system. Additionally, treatment with HPE-GNP decreased the level of pro-inflammatory cytokines (IFN-γ, IL-17A and IL-6) and increased anti-inflammatory cytokines (TGF-ß, IL-10 and IL-4). The real-time analysis revealed a decrease in the level of T-bet and ROR-γt but an increase in FoxP3 and GATA3 expression. Conclusion: The current study demonstrated that HPE-GNP could potentially reduce clinical and pathological complications of EAE, but laboratory data showed that HPE-GNP was significantly more effective than HPE in the treatment of EAE.

Evaluation of the immunoregulatory effect of Dicrocoelium dendriticum eggs on inflammatory and anti-inflammatory cytokines in EAE model.

Experimental autoimmune encephalomyelitis (EAE) is an appropriate model for the study of the immunologic and pathologic mechanisms in multiple sclerosis (MS). According to the hygiene hypothesis, helminths can improve immunoregulation and have therapeutic effects on immune-mediated diseases. In this study, we used Dicrocoelium dendriticum (Dicrocoeliidae, Platyhelminthes) eggs for the evaluation of their prophylactic and treatment effects on EAE disease. D. dendriticum eggs were extracted. Female C57BL/6 mice were immunized with the specific antigen MOG35-55 , and then the egg extracts were utilized for prophylaxis and/or treatment. Clinical symptoms and other relevant parameters were assessed daily. The mRNA expression of transforming growth factor-ß (TGF-ß), interleukin-10 (IL-10), IL-6, IL-23 and IL-17 were assessed with a real-time polymerase chain reaction technique. Furthermore, secretion of TGF-ß and IL-17 cytokines were determined by enzyme-linked immunosorbent assay. Data indicated that clinical symptoms in prophylaxis and treatment groups were decreased significantly in comparison with the untreated control group (p < .001). Our results showed a significant decrease in IL-17, as well as an increase in TGF-ß cytokine in the treatment group compared to the EAE control group (p < .01). Furthermore, in the prophylaxis and treatment groups, the mRNA expression of disease-associated cytokines decreased and the mRNA expression of the anti-inflammatory cytokines increased. In this study, the D. dendriticum egg ameliorates the clinical symptoms of the EAE model through the modulation of related cytokines of Th17 and Treg cells. Therefore, using this parasite egg could be a new treatment for MS.

The role of Th22 cells, from tissue repair to cancer progression.

CD4+ T helper (Th) cells play a significant role in modulating host defense. In the presence of lineage specific cytokine cocktail, Naive CD4+ T cells can differentiate into several categories with distinct cytokines profile and effector functions. Th22 cells are a recently identified subset of CD4+ T cell, which differentiate from Naive CD4+ T in the presence of IL-6 and TNF-α. Th22 characterized by the production of interleukin-22 (IL-22) and expression of aryl hydrocarbon receptor (AHR). The main function of Th22 cells is to participate in mucosal defense, tissue repair, and wound healing. However, controversial data have shown that overexpression of IL-22 can lead to pathological changes under inflammatory conditions and tumor progression. This review summarizes our knowledge about the role of Th22 and IL-22 cells in tumor progression through induction of inflammation.

Comparative assessment of immunomodulatory, proliferative, and antioxidant activities of crocin and crocetin on mesenchymal stem cells.

Saffron (Crocus sativus L) is a well-known spice with active pharmacologic components including crocin, crocetin, safranal, and picrocrocin. Similar to crocin/crocetin, mesenchymal stem cells (MSCs) have been shown to display immunomodulatory and antioxidant properties, which could be beneficial in treatment of various diseases. In the current study, we have evaluated the effects of crocin and crocetin on the functions of MSCs. We used the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay to evaluate MSCs proliferation, and flow cytometry assay to measure the percentage of apoptotic MSCs and Tregs populations. Furthermore, we used the real-time polymerase chain reaction method to quantify messenger RNA (mRNA) expression of inflammatory and anti-inflammatory cytokines. Antioxidant assay was employed to quantify antioxidant parameters including nitric oxide and malondialdehyde levels besides superoxide dismutase activity. Our findings indicated that both crocin and crocetin at low concentrations (2.5 and 5 µM) exhibited significant effects on increasing MSCs viability and on protecting them against apoptosis-induced death. Furthermore, crocin and crocetin at low concentrations (2.5 and 5 µM) displayed a better antioxidant function. Moreover, increased Treg population was observed at lower doses. In addition, crocin/crocetin at low concentrations caused an elevation in mRNA expression of anti-inflammatory cytokines (transforming growth factor-ß, interleukin-10 [IL-10], and IL-4), while at higher doses (25 and 50 µM) they led to lowering inflammatory cytokines (IL-1ß, IL-6, IL-17, and interferon gamma). Altogether, both crocin and crocetin at lower concentrations exhibited more efficacies on MSCs with a better effect toward crocin. It seems that crocin and crocetin may be considered as complementary treatments for the patients who undergo MSCs transplantation.

Berberine Inhibits the Gene Expression and Production of Proinflammatory Cytokines by Mononuclear Cells in Rheumatoid Arthritis and Healthy Individuals.

OBJECTIVE: Rheumatoid arthritis (RA) is the most prevalent autoimmune arthritis. Berberine is an alkaloid isolated from Berberis vulgaris, and its anti-inflammatory effect has been identified. METHODS: Twenty newly diagnosed RA patients and 20 healthy controls participated. Peripheral mononuclear cells were prepared and stimulated with bacterial lipopolysachharide (LPS,1 µg/ml), exposed to different concentrations of berberine (10 and 50µM) and dexamethasone (10-7 M) as a reference. The toxicity of compounds was evaluated by WST-1 assay. The expression of TNF-α and IL-1ß was determined by quantitative real-time PCR. Protein level of secreted TNF-α and IL-1ß was measured by using ELISA. RESULTS: Berberine did not have any toxic effect on cells, whereas Lipopolysaccharide (LPS) stimulation caused a noticeable rise in TNF-α and IL-1ß production. Berberine markedly downregulated the expression of both TNF-α and IL-1ß, and inhibited TNF-α and IL-1ß secretion from LPS-stimulated PBMCs. DISCUSSION: This study provided a molecular basis for anti-inflammatory effect of berberine on human mononuclear cells through the suppression of TNF-a and IL-1secretion. Our findings highlighted the significant inhibitory effect of berberine on proinflammatory responses of mononuclear cells from rheumatoid arthritis individuals, which may be responsible for antiinflammatory property of Barberry. We observed that berberine at high concentration exhibited anti-inflammatory effect in PBMCs of both healthy and patient groups by suppression of TNF-a and IL-1cytokines at both mRNA and protein levels. CONCLUSION: Berberine may inhibit the gene expression and production of pro-inflammatory cytokines by mononuclear cells in rheumatoid arthritis and healthy individuals without affecting cell viability. Future studies with a larger sample size are needed to prove the idea.

Dicrocoelium ova can block the induction phase of experimental autoimmune encephalomyelitis.

AIMS: This study aimed at investigating the impact of Dicrocoelium ova on experimental autoimmune encephalomyelitis (EAE) treatment in C57BL6 mice. METHODS AND RESULTS: Twenty-eight C57BL/6 mice were assigned into four groups as PBS, prophylaxis (P), treatment1 (T1) and treatment2 (T2). Prior to induction of EAE in prophylaxis group and on days 7 and 18 in T1 and T2 groups, respectively, Dicrocoelium eggs were injected intraperitoneally to each mouse. The clinical score, weight changes and incidence time of EAE were recorded. IFN-γ and IL-4 expression is quantified on spleen cells. Also, histopathological study by (H&E) and Toluidine-Blue (TB), and Luxol Fast Blue (LFB) were performed. The data were analysed using SPSS version 21. Mean disease scores were significantly lower in P and T1 groups than the PBS group (P = .01). IFN-γ was lower in P and T1 groups than the PBS group. The highest level of IL-4 was observed in T1 group. The total number of neuroglia cells of corpus callosum was similar in all groups, but the density increased in T1 group compared to the PBS group (P = .03). CONCLUSIONS: Dicrocoelium eggs have a great potential to stimulate immunomodulation towards treatment of EAE during the initial phase.

Evaluation of 1,25(OH)2D3 Effects on FOXP3, ROR-γt, GITR, and CTLA-4 Gene Expression in the PBMCs of Vitamin D-Deficient Women with Unexplained Recurrent Pregnancy Loss (URPL).

Background: Vitamin D insufficiency and deficiency can be associated with adverse effects on fetus and pregnancy outcomes. This study aimed at evaluating the effect of 1,25VitD3 on specific transcription factor and markers of Tregs and T helper 17 (Th17) cells in peripheral blood mononuclear cells (PBMCs) of women with unexplained recurrent pregnancy loss (URPL) as a case group and PBMCs of healthy women as a control group. Methods: Samples from 20 non-pregnant patients with a history of URPL were compared to 20 normal non-pregnant women. PBMCs were divided into three wells for each subject in the presence of 1,25VitD3 (50 nM, for 16 hours), phytohemagglutinin (10 µM; positive control), and without any treatment (negative control). By Real-time PCR (Taqman assay), specific transcription factors of Tregs and Th17 cells, forkhead box P3 (FOXP3), retinoic acid-related orphan receptor γt (ROR-γt), glucocorticoid-induced tumor necrosis factor receptor-related (GITR), and CTLA-4 mRNA expressions in two groups were measured. Results: FOXP3/ROR-γt mRNA expression in PBMCs decreased significantly in women experiencing URPL compared to the control group (p = 0.0001). Although 1,25VitD3 (50 nM) increased FOXP3 gene expression (p = 0.0001), it did not significantly affect ROR-γt gene expression. Besides, 1,25VitD3 treatment significantly increased FOXP3/ROR-γt mRNA expression from baseline in PBMCs of the fetal loss group compared to that of the control group (p = 0.01). The 1,25VitD3 also increased GITR gene expression (p = 0.017) in PBMCs of URPL women compared to the controls. Conclusion: Vitamin D deficiency may be a contributor to recurrent pregnancy loss and suggests that the supplementation of women with Vitamin D pre-pregnancy may be protective against URPL via affecting Tregs signature genes, FOXP3 and GITR.

Evaluation of the Effects of 1,25 Vitamin D3 on Regulatory T Cells and T Helper 17 Cells in Vitamin D-deficient Women with Unexplained Recurrent Pregnancy Loss.

BACKGROUND: Vitamin D insufficiency and deficiency can be associated with adverse effects on pregnancy outcomes, which may include recurrent pregnancy loss through the mechanisms that are yet unknown. The aim of this study was to evaluate the effect of 1,25VitD3 on regulatory T cells (Tregs) and T helper17 (Th17) cell populations In vitro in unexplained recurrent pregnancy loss (URPL) patients and healthy women. METHODS: Samples from 20 non-pregnant women with a history of URPL were compared to 20 normal non-pregnant women. Peripheral blood mononuclear cells (PBMC) were divided into 3 wells for each subject: in the presence of 1, 25 VitD3 (50 nM, for 16 hours), PHA (positive control) (10µM), and without any treatment (as a baseline or negative control). The percentage of regulatory T cells and Th17 cells was measured by flow cytometry at baseline and then after cell culture experiments. RESULTS: Our study indicated that the percentage of Tregs in patients with URPL was significantly lower than the control group (2.42 ± 0.27 vs. 3.41 ± 0.29, P= 0.01). The percentage of Th17 cells was significantly greater in URPL patients compared to the control group (2.91 ± 0.33 vs. 1.18± 0.15, P=0.001). 1, 25VitD3 treatment significantly increased the percentage of Tregs from the baseline in the URPL group compared to that in the control group (1.23 ± 0.03 vs. 1.00 ± 0.03, P= 0.01). CONCLUSION: Vitamin D deficiency may be a contributor to recurrent pregnancy loss and suggests supplementation of women with Vit D pre-pregnancy may be protective against URPL.

Alteration in inflammatory mediators in seriously eye-injured war veterans, long-term after sulfur mustard exposure.

BACKGROUND: Sulfur mustard (SM) exposure produces extensive systemic and ocular adverse effects on the victims. One of the most important effects is immunological insults that can lead to other organ damages, including the eyes. METHODS: In this descriptive study, 128 SM-exposed veterans with severe eye injury were compared with 31 healthy controls. Tear levels of tumor necrosis factor (TNF)-α and serum concentrations of interleukin (IL)-1α, IL-1ß, IL1Ra, IL-6, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Fas Ligand (FasL) were compared between the two groups. RESULTS: Meibomian gland dysfunction (MGD); tear breakup time (TBUT < 10″); and conjunctival, limbal, and corneal abnormalities were more frequent among the cases (MS-exposed veterans) than the controls. Ocular involvement was mild in 14.8%, moderate in 24.2%, and severe in 60.9% of the cases. Serum levels of IL-1α and FasL were significantly higher among the cases than among the controls (P < 0.001 and P = 0.037, respectively). Also, a significant decrease was observed in serum and tear levels of TNF-α in the cases as compared with controls (P < 0.001, P < 0.001, respectively). Serum levels of FasL were significantly higher in cases with severe ocular involvement than in the controls (P = 0.03). Nonetheless, serum levels of IL-1ß, IL-1Ra, IL-1α/IL-1Ra, and IL-6 were not significantly different between the two groups. CONCLUSION: Serum levels of IL-1α and FasL may cause different ocular surface abnormalities in SM-exposed patients. Lower tear TNF-α concentration may be due to lower serum levels of this cytokine in these patients.

Tear and serum MMP-9 and serum TIMPs levels in the severe sulfur mustard eye injured exposed patients.

INTRODUCTION: Sulfur mustard (SM) intoxication produces local and systemic changes in the human body. In this study, the relationship between tear and serum matrix metalloproteinase (MMP)-9 and serum tissue inhibitors of metalloproteinases (TIMPs) are assessed in serious eye-injured SM-exposed casualties. METHODS: A group of 128 SM-exposed patients with serious ocular injuries in three subgroups (19 mild, 31 moderate, and 78 severe cases) is compared with 31 healthy controls. Tear and ocular status and serum MMPs and MMP-9/TIMPs complex levels were evaluated using enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum level of MMP-9 was significantly higher in the SM-exposed group compared to the control group (P = 0.009). Mean serum MMP-9 level in the SM-exposed group with ocular abnormalities was significantly higher than that in the SM-exposed group without ocular abnormalities. SM-exposed people with corneal calcification had significantly higher serum MMP-9/TIMP-1 level compared to the SM-exposed ones without this problem (P = 0.045). The SM-exposed group with severe ocular injuries had significantly higher MMP-9/TIMP-1 than the controls (P = 0.046). The SM-exposed group had significantly lower levels of MMP-9/TIMP-4 complex than the controls (P < 0.001). The SM-exposed group with tear meniscus and fundus abnormality had significantly higher MMP-9/TIMP-4 levels than the SM-exposed group without these problems (P = 0.009 and P = 0.020). CONCLUSION: Serum MMP-9 level had increased in SM-exposed groups with ocular problems, while TIMP-1 and TIMP-2 levels had remained unchanged. Serum TIMP-4 drastically decreased in SM-exposed group, which clearly explains the severity of the systemic and ocular damages.

Immunoregulatory, proliferative and anti-oxidant effects of nanocurcuminoids on adipose-derived mesenchymal stem cells.

Curcuminoids are dietary complexes extracted from the seeds of Curcuma longa L. that contain curcumin, bisdemethoxycurcumin and desmethoxycurcumin. Curcuminoids are popular for their pleiotropic therapeutic functions, such as their anti-inflammatory and anti-oxidant effects. Nonetheless, their clinical use is associated with poor systemic bioavailability and insolubility. The nano-formulation of curcuminoids eliminates these shortcomings. In the present study, we explored immunoregulatory, proliferative and anti-oxidant effects of nanocurcuminoids on adipose-derived mesenchymal stem cells (AT-MSCs). Flow cytometry analysis and MTT assay were employed to explore the effects of nanocurcuminoids on the apoptosis and proliferation of adipose-derived MSCs (AT-MSCs). The anti-oxidant effect of nanocurcuminoids on AT-MSCs also was examined. The immune regulatory effect of nanocurcuminoids was evaluated by the flow cytometric measurement of the T regulatory (Treg) population. The expression of inflammatory and anti-inflammatory cytokines was quantified using real-time PCR. Our findings demonstrate that low concentrations of nanocurcuminoids are beneficial for MSC proliferation, protection of MSCs from apoptosis, reducing inflammatory cytokines and SOD activity. A high concentration of nanocurcuminoids increases the population of Tregs and elevates the expression of TGFß and FOXP3 genes. The beneficial effects of nanocurcuminoids on AT-MSCs were mainly observed at low doses of nanocurcuminoids.

Acylated and deacylated quillaja saponin-21 adjuvants have opposite roles when utilized for immunization of C57BL/6 mice model with MOG35-55 peptide.

BACKGROUND: The majority of patients with multiple sclerosis (MS) suffer from central neuropathic pain (CNP). Using experimental autoimmune encephalomyelitis (EAE) model, only a few experiments were performed to assess pain behaviors in MS. To address this issue, complete Freund's adjuvant (CFA) was replaced with an acylated triterpene glycoside saponin adjuvant named quillaja saponin-21 (QS-21) to develop CNP in the EAE mouse model. The deacylated form of QS-21, named QT-0101, has been suggested to have an immunomodulatory effect. Thus, QT-0101 was used as a vaccine adjuvant to modulate the immune system against myelin oligodendrocyte glycoprotein (MOG35-55) antigen. METHODS: In this study, C57BL/6 mice, except for mice in the negative control (PBS) and MOG groups, were divided into three groups and immunized by MOG35-55 emulsified with CFA, QS-21, or QT-0101 adjuvants, respectively. Thermal hyperalgesia, as a CNP clinical manifestation, through the Hot Plate test and the clinical signs, was assessed for 60 days after immunization. On days 21 and 60, mice were sacrificed and the frequency of TCD4+, TCD8+, IL-17+, IL-4+, and CD25+/FoxP3+ cells population in the total splenocytes population was assessed by flow cytometry. Infiltration of Leukocytes into the brain and demyelination of white matter were also evaluated by histopathologic studies. RESULTS: Our results revealed that unlike the MOG+QT-0101 group, the MOG+QS-21 and MOG+CFA groups represented clinical symptoms that mimic the mild relapsing-remitting and monophasic models, respectively. Thermal hyperalgesia, as a CNP clinical manifestation, developed in the bilateral hind paws in the MOG+CFA and MOG+QS-21 mice groups during the onset of neurologic deficits, but it is maintained until completion of the study only in MOG+QS-21 mice group. The frequency of TCD4+, TCD8+ and IL-17+ cells population in the MOG+QS-21 and MOG+CFA mice groups, as well as IL-4+ and CD25+/Foxp3+ cells population in the MOG+QT-0101 mice group, significantly increased in comparison with the PBS mice group. Infiltration of inflammatory cells increased significantly in the MOG+QS-21 and MOG+CFA mice groups compared with the PBS mice group. Demyelination of white matter was identified significantly only in the MOG+CFA mice group compared with the PBS mice group. CONCLUSION: These results showed that QS-21 is a suitable adjuvant for the establishment of a mild relapsing-remitting EAE model for CNP development and open a new avenue to future pre-clinical and clinical research studies related to CNP treatment. Nevertheless, QT-0101 seems to have the potential to act as a vaccine adjuvant with immunomodulatory property against auto-antigens.

Subtypes of Antiphospholipid Antibodies in Neurologic Disorders: An Observational Study.

BACKGROUND AND OBJECTIVES: Concomitant neurologic manifestations and positive antiphospholipid antibodies (APAs) have been investigated in different manners. The present study aimed to investigate the association between neurologic manifestations and APAs. MATERIALS AND METHODS: This cross-sectional descriptive study was conducted on 100 consecutive patients with selected neurological manifestations and at least one positive APAs within the age range of 20-50 years, referred to the Rheumatic Diseases Research Center from the Northeast Central Neurology Department of Iran during August 2012 to March 2014. RESULTS: According to the results, 89% of the participants were persistently positive for APAs, including lupus anticoagulant, IgG anticardiolipin (aCL), IgM aCL, IgG ß-2 glycoprotein 1 (ß2- GP1), and IgM ß2-GP1, observed in 16%, 41%, 42%, 17%, and 15% of the patients, respectively. Furthermore, 10% of the patients had concomitant lupus manifestations, and 37% of them showed anti-DNA. The IgG and IgM aCL were the most prevalent antibodies. Cerebral vascular accident (33%), retinal artery/vein occlusion (21%), and seizure (20%) were the most frequent presentations among the patients. In addition, the patients with multiple sclerosis (composing 3% of the subjects) were 100% positive for IgG and IgM aCL, as well as lupus anticoagulant. In addition, IgM anti-ß2- GP1 was 100% positive in optic neuritis patients (composing 5% of the subjects) and was significantly associated with this neurologic disorder. IgM anti-ß2-GP1 was also prevalent in the cases with Guillain-Barré syndrome. The most prevalent persistently positive antibody in the patients with cerebrovascular accident was IgM aCL. CONCLUSION: The findings of this study revealed some associations between the subtypes of APAs and incidence of neurologic disorders. However, the exact correlation between those symptoms and APAs needs further investigations.

Amelioration of regulatory T cells by Lactobacillus delbrueckii and Lactobacillus rhamnosus in pristane-induced lupus mice model.

Regulatory T cells (Tregs) play an indispensable role in the control of immune responses and induction of peripheral tolerance. Dysregulation of Tregs is involved in the pathogenesis of systemic lupus erythematosus (SLE). Tolerogenic probiotics have shown beneficial effects in the control of autoimmune diseases. We evaluated the prophylactic and therapeutic effects of Lactobacillus delbrueckii and Lactobacillus rhamnosus on Tregs and their related molecules in pristane-induced lupus mice model. Fifty-four female BALB/c mice (3-5 weeks) were randomly divided into nine groups. Lupus was induced in all groups using pristane. Prophylactic groups were treated from Day 0 (at the time of pristane injection) and treatment groups were treated 2 months later with L. rhamnosus, L. delbrueckii, mix of both probiotics, and prednisolone. One group was considered as SLE-induced control group without any treatment. Presence of antinuclear antibodies (ANA), antidouble-stranded DNA (anti-dsDNA), antiribonucleoprotein (anti-RNP), proteinuria, and serum level of creatinine, urea, the expression of forkhead box P3 (Foxp3), interleukin 6 (IL-6), IL-10, transforming growth factor ß, and the number of Tregs were determined. SLE induction by pristane led to the formation of lipogranuloma, presence of ANA, anti-dsDNA, and anti-RNP. Probiotics consumption decreased the level of lipogranuloma, ANA, and anti-dsDNA. In addition, in probiotics receiving groups, Tregs and the expression level of Foxp3 increased, while IL-6 decreased. The effect of probiotics in the prophylactic group was more prominent. The results may indicate the effectiveness of L. delbrueckii and L. rhamnosus in the enhancement of Tregs and the decrease of inflammatory cytokines and disease severity in SLE-induced mice.

In vitro modulatory effect of dehydroepiandrosterone sulfate on apoptosis and expression of apoptosis-related genes in patients with systemic lupus erythematosus.

OBJECTIVES: Systemic lupus erythematosus| (SLE) is an autoimmune disease characterized by hyperactive B cells that produce various autoantibodies. Sex hormones have been documented to influence the development of SLE, in which women with SLE have low plasma level of dehydroepiandrosterone sulfate (DHEAS). A strong conclusion about the effect of DHEAS on apoptosis in SLE patients has not been provided. The aim of this study was to assess apoptotic effects of DHEAS on peripheral blood lymphocytes (PBLs) from SLE patients. METHODS: Twenty SLE patients and 20 age- and sex-matched healthy controls were included into this study. Concentration of DHEAS was measured using enzyme-linked immunosorbent assay in serum from all participants. Freshly isolated PBLs from each individual were treated with 7.5-µmol of DHEAS for 24 hr in cell culture medium to assess the effect of DHEAS on apoptosis using fluorescein isothiocyante-conjugated annexin V and propidium iodide. The messenger RNA (mRNA) expression level of apoptosis-related genes (Fas, Fas-L, Bcl-2, and Bax) in PBLs was measured using real-time PCR before and after treating with DHEAS. RESULTS: Level of DHEAS was low in SLE patients compared with healthy controls (p < 0.05). After treating with DHEAS, the percentage of apoptotic cells in SLE patients was decreased in comparison with healthy controls. DHEAS treatment increased the mRNA expression level of Bcl-2 in PBLs from SLE patients. CONCLUSIONS: DHEAS reduced the apoptosis rate in PBLs from SLE patients and may decrease the load of autoantigens. Therefore, DHEAS might be considered as a therapeutic tool in SLE patients.

In vivo study: Th1-Th17 reduction in pristane-induced systemic lupus erythematosus mice after treatment with tolerogenic Lactobacillus probiotics.

Uncontrolled inflammation in systemic lupus erythematosus (SLE) could cause dysfunction in multiple organs. T helper 17 (Th17) cells are a main branch of inflammatory responses in the pathogenesis of SLE, and by producing interleukin 17 (IL-17), represent a major functional tool in the progression of inflammation. Animal models provide a special field for better studies of the pathogenesis of diseases. Tolergenic probiotics could decrease inflammation in autoimmune diseases by modulating the immune system and maintaining homeostasis. The aim of this project was to evaluate the effects of Lactobacillus rhamnosus and Lactobacillus delbrueckii on Th17 cells and their related mediators in a pristane-induced BALB/c mice model of SLE. The mice were divided into pretreatment groups, which received probiotics or prednisolone at Day 0, and treatment groups, which received probiotics and prednisolone 2 months after injection. The presence of antinuclear antibody (ANA), anti-double-stranded DNA (anti-dsDNA), and anti-ribonucleoprotein (anti-RNP) and lipogranuloma was evaluated; also, the population of Th1-Th17 cells as well as interferon Î³ (IFN-γ), IL-17, and IL-10 levels, and the expression of RAR-related orphan related receptor gamma (RORγt) and IL-17 were determined. We observed that probiotics and prednisolone could delay SLE in pretreatment and treatment mice groups, with a reduction in ANA, anti-dsDNA, anti-RNP, and mass of lipogranuloma. Probiotics and prednisolone decreased the population of Th1-Th17 cells and reduced IFN-γ and IL-17 as inflammatory cytokines in the pretreatment and treatment groups in comparison with SLE-induced mice. Our results indicated that, due to their anti-inflammatory properties and reduction of Th17, Th1, and cytotoxic T lymphocyte (CTL) cells, the use of these probiotics could probably represent a new tool for the better management of SLE.

Generation of tolerogenic dendritic cells using Lactobacillus rhamnosus and Lactobacillus delbrueckii as tolerogenic probiotics.

Systemic lupus erythematosus (SLE) concurs with excessive uncontrolled inflammatory immune responses that lead to the loss of immune tolerance. Dendritic cells (DCs) are important and determinant immune cells that regulate immune responses. Tolerogenic DCs with regulatory markers and cytokines could induce regulatory immune cells and responses. Tolerogenic probiotics are capable of producing regulatory DCs from monocytes in in vitro conditions. The purpose of this study was to evaluate the effect of Lactobacillus delbrueckii and Lactobacillus rhamnosus on the production of DCs in an in vitro condition. Peripheral blood mononuclear cells were isolated from the healthy and SLE donors. Monocytes were cultured with optimized concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) to produce immature DCs (IDCs). An IDC uptake assay was performed, and IDCs of healthy and SLE donors were divided into three subgroups following 48 hours of treatment with GM-CSF and IL-4, along with L. delbrueckii, L. rhamnosus, and mixed probiotics for the production of tolerogenic DCs. The surface expression of Human Leukocyte Antigen-antigen D Related (HLA-DR), CD86, CD80, CD83, CD1a, and CD14 was analyzed using flow cytometry, and the gene expression levels of indoleamine 2,3-dioxygenase (IDO), IL-10, and IL-12 were measured using real-time polymerase chain reaction. We observed significantly reduced expression of costimulatory molecules and other surface markers in the probiotic-induced mature DCs (MDCs) in both healthy and SLE donor groups in comparison with lipopolysaccharide (LPS)-induced MDCs. In addition, the expression of IDO and IL-10 increased, whereas IL-12 decreased significantly in probiotic-induced MDCs compared with LPS-induced MDCs. IDCs and especially mature tolerogenic DC of SLE patients highly expressed IDO. The results of the current study suggested that live probiotics could modify properties of DCs to modulatory cells, which might contribute to the induction of tolerance and renovation of immune hemostasis.