In vitro-derived insulin-producing cells modulate Th1 immune responses and induce IL-10 in streptozotocin-induced mouse model of pancreatic insulitis.

BACKGROUND: Insulitis is defined by the presence of immune cells infiltrating in the pancreatic islets that might progress into the complete ß-cell loss. The immunomodulatory properties of bone marrow-derived mesenchymal stem cells (BM-MSCs) have attracted much attention. This study aimed to evaluate the possible immunomodulatory effects of rat BM-MSCs and MSCs-derived insulin-producing cells (IPCs) in a mouse model of pancreatic insulitis. METHODS: Insulitis was induced in BALB/c mice using five consecutive doses of streptozotocin. MSCs or IPCs were directly injected into the pancreas of mice and their effects on the expression of Th subsets-related genes were evaluated. RESULTS: Both BM-MSCs and IPCs significantly reduced the expression of pancreatic Th1-related IFN-γ (P < 0.001 and P < 0.05, respectively) and T-bet genes (both P < 0.001). Moreover, the expression of IL-10 gene was significantly increased in IPC-treated compared to BM-MSC- or PBS-treated mice (P < 0.001 both comparisons). CONCLUSIONS: BM-MSCs and IPCs could successfully suppress pathologic Th1 immune responses in the mouse model of insulitis. However, the marked increase in IL-10 gene expression by IPCs compared to BM-MSCs suggests that their simultaneous use at the initial phase of autoimmune diabetes might be a better option to reduce inflammation but these results need to be verified by further experiments.

Protective Effect of Edaravone Against Cyclosporine-Induced Chronic Nephropathy Through Antioxidant and Nitric Oxide Modulating Pathways in Rats.

BACKGROUND: Cyclosporine A (CsA) is an immunosuppressant with therapeutic indications in various immunological diseases; however, its use is associated with chronic nephropathy. Oxidative stress has a crucial role in CsA-induced nephrotoxicity. The present study evaluates the protective effect of edaravone on CsA-induced chronic nephropathy and investigates its antioxidant and nitric oxide modulating property. METHODS: Male Sprague-Dawley rats (n=66) were distributed into nine groups, including a control (group 1) (n=7). Eight groups received CsA (15 mg/kg) for 28 days while being treated. The groups were categorized as: Group 2: Vehicle (n=10)Groups 3, 4, and 5: Edaravone (1, 5, and 10 mg/kg) (n=7 each)Group 6: Diphenyliodonium chloride, a specific endothelial nitric oxide synthase (eNOS) inhibitor (n=7)Group 7: Aminoguanidine, a specific inducible nitric oxide synthase (iNOS) inhibitor (n=7)Group 8: Edaravone (10 mg/kg) plus diphenyliodonium chloride (n=7)Group 9: Edaravone (10 mg/kg) plus aminoguanidine (n=7) Blood urea nitrogen and serum creatinine levels, malondialdehyde, superoxide dismutase, and glutathione reductase enzyme activities were measured using standard kits. Renal histopathological evaluations and measurements of eNOS and iNOS gene expressions by RT-PCR were also performed. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test (SPSS software version 18.0). RESULTS: Edaravone (10 mg/kg) significantly attenuated CsA-induced oxidative stress, renal dysfunction, and kidney tissue injury. Aminoguanidine improved the renoprotective effect of edaravone. Edaravone reduced the elevated mRNA level of iNOS, but could not alter the level of eNOS mRNA significantly. CONCLUSION: Edaravone protects against CsA-induced chronic nephropathy using antioxidant property and probably through inhibiting iNOS gene expression.

A1 -Adenosine receptor activation has biphasic roles in development of acute kidney injury at 4 and 24 h of reperfusion following ischaemia in rats.

NEW FINDINGS: What is the central question of this study? A1 -Adenosine receptor (A1 AR) blockade before renal ischaemia aggravated kidney injury after 24 h reperfusion in several studies, whereas we previously observed a renoprotective effect of A1 AR blockade during a 4 h reperfusion period. What are the underlying mechanisms for this biphasic effect of pretreatment with an A1 AR antagonist at 4 and 24 h reperfusion? What is main finding and its importance? A1 -Adenosine receptor blockade protects the kidney against ischaemia-induced injury during the early hours of reperfusion by attenuating the reduction in renal blood flow and lowering energy expenditure, whereas its inflammatory effects gradually dominate over 24 h reperfusion to intensify kidney injury. We previously reported that selective blockade of the A1 -adenosine receptor (A1 AR) with an antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), had protective effects on renal ischaemia-induced structural and functional disruption during a 4 h reperfusion period. In contrast, several studies demonstrated that endogenous and exogenous A1 AR activation before renal ischaemia had a renoprotective role 24 h after reperfusion, through mechanisms that reduced inflammation, necrosis and apoptosis. In this study, we investigated potential mechanisms underlying this biphasic action of A1 AR in renal ischaemia-reperfusion injury. Anaesthetized male Sprague-Dawley rats underwent 30 min of bilateral renal ischaemia, and biphasic effects of pretreatment with DPCPX at 4 and 24 h reperfusion were studied on the kidney injury. Pretreatment with DPCPX attenuated at 4 h but augmented at 24 h reperfusion the renal ischaemia-induced histological damage, reductions in creatinine clearance, urea excretion and free-water reabsorption, and increases in bicarbonate excretion and tissue malondialdehyde. The DPCPX increased tumour necrosis factor-α expression and migration of lymphocytes in the postischaemic kidney at both time points, but with a different pattern; lymphocytes mostly aggregated in cortical periarterial spaces at 4 h reperfusion but had infiltrated into the interstitium at 24 h reperfusion. In conclusion, A1 AR activation contributes to ischaemia-induced acute kidney injury during the early hours of reperfusion by causing a greater reduction in renal haemodynamics and by elevating tubular energy expenditure, which overcome its anti-inflammatory effect. However, its anti-inflammatory actions are exerted by reducing lymphocyte infiltration and cytokine production that begins to dominate from 4 to 24 h of reperfusion, which is reflected in attenuation of renal structural and functional disruption.

Stealth Nanogels of Histinylated Poly Ethyleneimine for Sustained Delivery of Methotrexate in Collagen-Induced Arthritis Model.

PURPOSE: The study aimed to illustrate application of polycation Stealth nanogels for sustained delivery of methotrexate (MTX) in collagen induced arthritis (CIA) model in C57Bl/6 mice. METHODS: Nanogel synthesis involves metal ion coordinated self-assembly of PEGylated poly ethyleneimine (L-histidine substituted), chemical crosslinking and subsequent removal of the coordinated metal. The nanogels were characterized by TEM and DLS-zeta potential. Comparative efficacy and pharmacokinetics of the i.v. administred MTX-loaded nanogels were investigated in the CIA model. Inflammation site passive accumulation of the fluorophore-labeled nanogels was tested using in-vivo imaging of mice paw received unilateral injection of lipopolysaccharide. RESULTS: Uniform nanogels (sizes ~40 nm by TEM) were loaded with MTX (entrapment efficiency = 62% and drug loading = 54% at the MTX feeding ratio of 0.3 relative to total molar concentration of the polymer amines). The nanogels exhibited neutral surface charge and an acceptable biocompatibility in terms of albumin aggregation, hemolysis, erythrocyte aggregation and cytotoxicity. Single dose pharmacokinetics of the MTX-loaded nanogels, unlike free drug, showed a sustained plasma profile. When arthritis established as confirmed by histopathology, a remarkable decline of paw swelling and clinical scores was observed. Fluorescence intensity of the nanogels was enhanced about 2.7 folds at the inflamed than control normal ankle. CONCLUSION: Sustained delivery of MTX and preferential accumulation of the nanogels in inflamed paw might explain the superior clinical outcome of the MTX-loaded nanogels.

Study of the relationships between IL-23R, IL-17, IL-21 polymorphisms and serum level of IL-17, IL-21 with acute graft rejection in iranian liver transplant recipients.

Cytokines are important factors determining the outcome of transplantation. Since host ability in cytokine production may be affected by cytokine genes polymorphisms, the aim of the present study was to investigate the effect of IL-17, IL-23R and IL-21 gene polymorphisms in outcome of liver transplantation. A total of 200 liver transplant recipients were included in this study. IL-17 -197 A/G, IL-21+1472 G/T, IL-21 5250 C/T, and IL-23R C/A polymorphisms were evaluated by PCR-RFLP or ARMS-PCR methods. The serum levels of IL-17 and IL-21 in rejected and non-rejected groups were determined by ELISA method. The results showed that IL-23R AC carriers and C allele were significantly more frequent in patients with acute rejection than patients without rejection (p=0.01 and p=0.0005, respectively). After gender classification, IL-23R AA and AC carriers were significantly more frequent in female patients (p=0.01, p=0.01, respectively) and IL-23R AA and AC carriers and A allele were significantly more frequent in male patients (p=0.009, p=0.02, p=0.003, respectively). There is a significant association between CC genotype and C alleles of IL-23R and AR in the patients receiving allograft from living donor (p=0.0003 and p=0.0008, respectively). Also, IL-23R AA and AC genotypes and C alleles showed a significant association with rejection in patients receiving allograft from cadaver donor (p=0.001, p=0.002 and p=0.02). The mentioned results indicate that IL-23R AC carriers and C allele have predictive values for acute rejection. AC genotype and C allele of IL-23R is a genetic risk factor for development of acute rejection. Also, AA and AC genotype of IL-23R is a sex dependent genetic risk factor for development of acute rejection. But this subject needs to be studied in different population.

Alteration in the expression of proteins in unexplained recurrent pregnancy loss compared with in the normal placenta.

The placenta is a unique pregnancy-related tissue and plays a key role in occurrence of unexplained recurrent pregnancy loss (URPL). Abnormal placentation might play a key role in occurrence of URPL. Therefore, the purpose of this study was to compare the human placental proteome between URPL placentas and normal placental matched for gestational week. Total placental proteins were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was used for separation of the placental proteomes. Protein spots differentially expressed between URPL and normal placentas were selected and identified by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF) technique after being digested in the gel. Moreover, quantitative real-time PCR and Western blot techniques were used to confirm the differential expression mass results for some differentially expressed proteins. The results indicated that at least 19 protein spots were differentially expressed between URPL and normal placentas (P < 0.05), and twelve of them were successfully identified. While only two proteins were downregulated (calumenin and enolase 1), the remaining ten spots (actin gamma 1 propeptide, cathepsin D prepropeptide, heat shock protein gp96, tubulin beta, tubulin alpha 1, glutathione S-transferase, vitamin D binding protein, prohibitin, actin beta, apolipoprotein A-I) showed increased expression in URPL cases in comparison with normal placentas. Real-time PCR also confirmed the downregulation of calumenin and upregulation of prohibitin and apolipoprotein A-I at the mRNA levels. In conclusion, the results of the present study showed that alteration in the expression of proteins involved in proliferation and migration of endothelial cells as well as control of coagulation by these cells might play an important role in the pathogenesis of URPL.

Association between Tumor Necrosis Factor- α-308 G/A Polymorphism and Multiple Sclerosis: A Systematic Review and Meta-Analysis.

Multiple sclerosis (MS) is a complex polygenic disease in which gene-environment interactions are important. A number of studies have investigated the association between tumor necrosis factor-α (TNF-α) -308 G/A polymorphism (substitution G→A, designated as TNF1 and TNF2) and MS susceptibility in different populations, but the results of individual studies have been inconsistent. Therefore, performing a systematic review and meta-analysis of the published studies is desirable. We sought to quantitatively summarize the association between TNF-α-308 G/A polymorphism and MS. The Medline and Scopus databases were searched to identify potentially relevant case-control studies published in English journals up to January 2010. A meta-analysis of these studies was performed. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were calculated under fixed and random effects models. Twenty-one eligible studies, comprising 2880 patients with MS and 3579 controls, were included in the meta-analysis. The overall pooled ORs (95%CI) for TNF2 versus TNF1 and TNF2 carriers (2/2+2/1) versus non-carriers (1/1) were 1.02 (0.86-1.21) and 0.99 (0.8-1.24), respectively. In the European populations, the pooled ORs (95%CI) for TNF 2/1 versus 1/1 were 0.85 (0.73-0.98), which was statistically significant. However, the other results did not support this finding. The pooled ORs (95%CI) for TNF 2/1 versus 1/1 and TNF 2/2 versus 2/1 were not statistically significant in the overall population. In addition, the pooled ORs for TNF2/2 versus TNF2/1+1/1 and TNF2/2 versus TNF1/1 were not statistically significant. Our meta-analysis does not support the role of TNF-α -308 G/A polymorphism in developing MS.

CD68 Antigen and Cerebral Aneurysms: A Case-Control Study.

BACKGROUND: Intracranial aneurysms are more commonly associated with inflammation as a cause of their development, progression, and rupture. Macrophages and other cells can express the CD68 antigen. The aim of this study was to assess the CD68 antigen levels in cerebral aneurysm (CA) patients compared to a control group at a referral center in Iran. METHODS: A case-control investigation was undertaken on 88 individuals (44 of whom were cases and 44 were controls). Individuals with CA as the case group consisted of 28 ruptured and 16 unruptured subgroups. Clinical, radiographic, and CD68 levels were evaluated and registered. RESULTS: The average age of the participants was 49 years. Males comprised 43.2% of the patients, while 56.8% were females (p = 0.002). There was a statistically significant difference in the CD68 levels between the two groups. There was no significant difference (p = 0.42) between the ruptured and unruptured subgroups (23.66 and 20.47, respectively) in this comparison. No significant correlation was seen between the patients' CD68 and Glasgow Coma Scale (GCS) levels and their aneurysm diameter (p = 0.74 and 0.45, respectively). A link between CD68 levels and age was found, but it was not statistically significant (r = 0.44 and p = 0.002). CONCLUSIONS: A possible involvement of CD68 as an inflammatory agent in the development of CAs but not in aneurysm rupture has been suggested. Inflammation and CD68 were positively associated with age. The CD68 antigen should be studied further in population-based cohort studies.

Generation of large numbers of highly purified dendritic cells from bone marrow progenitor cells after co-culture with syngeneic murine splenocytes.

Dendritic cells (DCs) are called the sentinels of the human immune system because of their function as antigen presenting cells (APCs) that elicit a protective immune response. Given that DCs have been used for many years as target cells in a great number of experiments, it became essential to devise a new method for producing DCs in higher quantities and of greater purity. Here we report a novel technique for obtaining more dendritic cells, and with higher purity, from in-vitro co-culture of bone marrow (BM) cells with splenocytes. From a total of 20 × 10(6) BM cells and 120 × 10(6)splenocytes, 3 × 10(6) BM cells along with 20 × 10(6)splenocytes were co-cultured in petri dishes for DC generation; 120 × 10(6) splenocytes from one C57BL/6 mouse were also co-cultured in petri dishes for DC generation. BM cells were the control group cultured in the same conditions except for the presence of splenocytes. Purity and maturation state of DCs were checked by lineage surface markers (CD11c, CD11b, CD8α, and F4/80) and the expression levels of MHCII as well as co-stimulatory molecules (CD86, CD80, and CD40). Endocytosis and thymidine uptake capacity were also used to test the functionality of DCs. The levels of IL-12p70, IL-23, and IL-10 were also checked in the supernatant of cultured cells by ELISA. The number of DCs derived from co-culture of BM and splenocytes (DCs(TME)) was at least twice that of BM-derived DCs in the absence of splenocytes. In addition, the purity of DCs after co-culture of BM and splenocytes was greater than that of DCs in the control culture (90.2% and 77.2%, respectively; p<0.05). While functional assays showed no differences between co-culture and control groups, IL-10 levels were significantly lower in DCs(TME) compared to BM-derived DCs in the absence of splenocytes (193 pg/ml and 630 pg/ml, respectively; p<0.05). The results of the present study show that the generation of DCs from BM progenitors is accelerated in the presence of syngeneic splenocytes. Given the larger number of generated DCs, and with higher purity, in this technique, DCs(TME) could be more advantageous for DC-based immunotherapy and vaccination techniques.

Association of FCRL3 genotypes with susceptibility of Iranian patients to rheumatoid arthritis.

Rheumatoid arthritis (RA) is a complex disease, the hallmark of which is synovial joint inflammation. The substantial contribution from genetic factors in susceptibility to RA has been well-defined. The Fc receptor-like3 (FCRL3) gene is one of the genes that have recently shown a significant association with RA. To determine the possible role of FCRL3-169 C/T and FCRL3-110 A/G gene polymorphisms in the development of RA in Iranian patients, 320 RA patients and 302 healthy subjects were genotyped by polymerase chain reaction-restriction fragment length polymorphism. No significant difference was found in genotype and allele frequencies of FCRL3-169 C/T between patients and controls. In contrast, at position -110 A/G, the frequency of the AA genotype and A allele was significantly decreased in RA patients compared to controls (p = 0.005). After Bonferroni correction for multiple testing, no significant correlations between FCRL3-169 C/T and -110 A/G polymorphism and laboratory and clinical features of the patients was observed. In conclusion, the results of this study showed a significant association between FCRL3-110 A/G polymorphism and susceptibility to RA.

The association between PD-1 gene polymorphisms and susceptibility to multiple sclerosis.

Programmed cell death 1 (PD-1) is an immune checkpoint and has been reported to be associated with several autoimmune diseases. We aimed to investigate the association between human PD-1 gene (PDCD1) polymorphisms and multiple sclerosis (MS). This case-control study was conducted on 229 MS patients and 246 healthy controls. Genotyping of rs36084323 (PD-1.1 G/A), rs11568821 (PD-1.3 G/A) and rs2227981 (PD-1.5 C/T) polymorphisms was performed by PCR-RFLP technique. The frequency difference of PD-1.1 genotypes and alleles (-536 G/A) between patients and healthy controls was not significant. Regarding PD-1.3, the AA + AG genotype was found to be relatively higher in the control group. Concerning PD-1.5 (+7785 C/T), the frequency of T allele carriers (TT + CT) was relatively higher in MS patients, which was marginally insignificant (p = .07). PD-1 gene polymorphisms may be associated with MS; however, accurate conclusions require further studies with a larger number of samples.

Effect of addition of silymarin to renin-angiotensin system inhibitors on proteinuria in type 2 diabetic patients with overt nephropathy: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: A large proportion of patients with type 2 diabetes mellitus have diabetic nephropathy. Despite current therapies including renin-angiotensin system inhibitors, diabetic nephropathy progresses to end-stage renal disease in most of these patients. Therefore, there is an urgent need to find new treatments for such patients. The aim of this study was to evaluate the efficacy of silymarin, an herbal drug with antioxidant and anti-inflammatory properties, in preventing the progression of diabetic nephropathy. STUDY DESIGN: Randomized, double-blind, placebo-controlled, 2-arm parallel trial. SETTING & PARTICIPANTS: 60 patients with type 2 diabetes with macroalbuminuria (urinary albumin excretion >300 mg/24 h) despite treatment with the maximum dose of a renin-angiotensin system inhibitor for more than 6 months and estimated glomerular filtration rate >30 mL/min/1.73 m(2). INTERVENTION: Patients were randomly assigned to 2 equal groups to receive three 140-mg tablets of silymarin or 3 tablets of placebo daily for 3 months. OUTCOMES: The primary outcome was absolute change in urinary albumin-creatinine ratio (UACR) from baseline to the end of the treatment phase. MEASUREMENTS: UACR and urinary and serum levels of TNF-α (tumor necrosis factor α; an inflammatory marker), malondialdehyde (MDA; an oxidative stress marker), and TGFß (transforming growth factor ß; a marker of fibrosis) at baseline and the end of the treatment phase. RESULTS: Although UACR decreased in both groups, this decrement was significantly higher in the silymarin compared with the placebo group; mean difference in change in UACR between the 2 groups was -347 (95% CI, -690 to -4) mg/g. Urinary levels of TNF-α and urinary and serum levels of MDA also decreased significantly in the silymarin compared with the placebo group. LIMITATIONS: Small sample size and short duration of the treatment phase. CONCLUSIONS: Silymarin reduces urinary excretion of albumin, TNF-α, and MDA in patients with diabetic nephropathy and may be considered as a novel addition to the anti-diabetic nephropathy armamentarium.

Cytokine secretion pattern in treatment of lymphocytes of multiple sclerosis patients with fumaric acid esters.

The present study was performed to investigate the effects of dimethylfumarate (DMF) and methylhydrogen fumarate (MHF) on the cytokine pattern of peripheral blood mononuclear cells (PBMCs) of multiple sclerosis (MS) patients. The PBMCs from patients and healthy controls were stimulated with myelin basic protein (MBP) or phytohemagglutinin (PHA) and cultured in the presence of DMF and MHF. The percentage of CD4+IL-4+ and CD4+IFN-γ+ cells was determined by means of intracellular cytokine staining. CD4+IL-4+ cells were significantly increased in the presence of DMF and MHF when PBMCs were stimulated by MBP (P < 0.003). The same significant result was obtained by PHA stimulation (P < 0.049). In terms of CD4+IFN-γ+ cells, the percentage of cells did not significantly differ between the cultures stimulated with MBP or PHA in the presence and absence of the drugs. Results of MBP stimulation in control group also showed a significant increase in CD4+IL-4+ cells in the presence of DMF and MHF. In comparison between patient and control groups, no statistically significant changes were observed. In conclusion, both DMF and MHF effectively increased IL-4 production, whereas they did not significantly change IFN-γ level, indicating the role of these drugs in increasing the production of beneficial cytokines such as IL-4.

Association between peroxisome proliferator-activated receptor-γ gene polymorphism (Pro12Ala) and Helicobacter pylori infection in gastric carcinogenesis.

OBJECTIVE: Helicobacter pylori infection is accompanied by inflammatory processes leading to peptic ulcer and gastric cancer in the minority of infected individuals. The interaction between H. pylori virulence factors, host defense mechanisms and environmental factors determine the outcome of clinical manifestations. One of the host factors involved in the processes of inflammation and carcinogenesis is the peroxisome proliferator-activated receptor-γ (PPAR-γ) molecule. The present case-control study aimed to determine polymorphism of PPAR-γ gene and its association with H. pylori infection and gastrointestinal diseases (peptic ulcer and non-cardia gastric cancer) in Iranian patients. MATERIALS AND METHODS: One hundred and fifty-five patients with upper gastrointestinal diseases (76 peptic ulcer and 79 non-cardia gastric cancer) and 152 matched controls were genotyped for PPAR-γ gene polymorphism (Pro12Ala) by the PCR-RFLP method. Infection with H. pylori was confirmed by histology, the rapid urease test (RUT) and ELISA assay (IgG anti-H. pylori). RESULTS: The frequency of PPAR-γ G (Ala 12) allele was significantly higher in H. pylori positive patients with non-cardia gastric cancer than in controls (22.8% vs. 3.9%, p = 0.027; OR = 3.28; 95% CI = 1.21-8.89), But there was no significant difference without infection (p = 0.7). Moreover, the PPAR-γ polymorphism was not associated with peptic ulcer in the presence or absence of H. pylori infection. CONCLUSION: Our results indicated PPAR-γ G allele may be an important contributor to non-cardia gastric cancer in Iranian H. pylori infected patients.

Association of CD1A +622 T/C, +737 G/C and CD1E +6129 A/G genes polymorphisms with multiple sclerosis.

Antibodies and specific T cells to glycolipids have been found in MS patients. CD1 molecules are involved in presentation of lipid antigens to T-cells. Therefore, functional polymorphisms in two CD1 genes (+622 T/C and +737 G/C in CD1A along with +6129 A/G in CD1E) might be associated with susceptibility to MS. First, 351 MS patients and 342 controls were enrolled in this study. Allele-specific oligonucleotide polymerase chain reaction and PCR-RFLP methods were used for genotyping. The frequency of CD1A genotypes was not different between cases and controls. However, investigating females, the frequency of CD1A*01 allele was significantly higher in patients with PP-MS compared to controls (p = 0.028) as well as to RR-MS and SP-MS (p = 0.042 and 0.021, respectively). The distribution of CD1E +6129 A allele (CD1E*01) and CD1E*01/01 genotype is more frequent in normal controls in comparison with MS patients (p = 0.001 and p = 0.0003, respectively). In addition, after categorization of study groups according to disease types, differences between alleles and genotypes of CD1E gene polymorphism remained significant for RR-MS patients compared to those of normal controls (p = 0.0001 and p = 0.0003, respectively). CD1E and CD1A genes may be involved in networks which determine susceptibility to RR-MS and PP-MS, respectively.

In Vitro Effects of Sodium Benzoate on the Expression of T Cells-related Cytokines and Transcription Factors in Adjuvant-induced Arthritis Model.

Though the exact etiology of rheumatoid arthritis (RA) is unknown, the contribution of immune cells in the disease process is completely acknowledged. T helper (Th) 1 and Th17-related cytokines are required for the disease development and progression, while Th2 and regulatory T cells (Tregs)-derived cytokines are protective. Studies have shown that sodium benzoate (NaB) can switch the balance of Th cell subsets toward Th2 and Tregs. The present study aimed to evaluate the possible effects of NaB on the expression of CD4+T cells-related cytokines and transcription factors in splenocytes derived from an animal model of RA, adjuvant-induced arthritis (AIA). AIA was induced in rats by injection of Freund's adjuvant containing mycobacterial antigens (Mtb). Splenocytes were isolated from AIA rats and restimulated ex vivo with Mtb in the presence or absence of NaB for 24 h. To determine the effects of NaB on the expression of T cells-related cytokine and transcription factor genes, real-time PCR was performed. NaB treatment of Mtb-stimulated splenocytes derived from arthritic rats resulted in significant increases in the gene expressions of Tregs-related cytokines (IL-10 and TGF-ß) and Foxp3 transcription factor, and significant decreases in the expression of Th1-related cytokines (TNF-α and IFN-γ) and the T-bet transcription factor. The ratios of Th1/Th2 (IFN-γ/IL-4), Th1/Treg (IFN-γ/TGF-ß and IFN-γ/IL-10) and Th17/Treg (IL-17/IL-10 and IL-17/IL-10+TGF-ß)-related cytokines were also significantly decreased. In conclusion, NaB can potentially be considered as a useful therapeutic agent for the treatment of RA and other Th1 and Th17-mediated diseases.

Bone marrow dendritic cells deficient for CD40 and IL-23p19 are tolerogenic in vitro.

OBJECTIVES: In addition to pro-inflammatory role, dendritic cells (DCs) can also be anti-inflammatory when they acquire tolerogenic phenotype. In this study we tested the role of CD40 and IL-23p19 in antigen presenting function of bone marrow-derived DCs (BMDCs) by comparing BMDCs derived from CD40 knockout (CD40KO-DCs) and IL-23p19 (IL-23p19KO-DCs) knockout mice with those from C57BL/6 mice (Cont-DCs). We have focused on CD40 and IL-23, as these molecules have well established pro-inflammatory roles in a number of autoimmune and inflammatory diseases. MATERIALS AND METHODS: The expression of maturation markers MHC II and co-stimulatory molecules CD40, CD80, and CD86 was analyzed by flow cytometry, while the expression of CD40 and IL-23p19 was measured by RT-PCR. The capacity of BMDCs to activate CD4+ T cells was evaluated by 3H-thymidine incorporation, and the capacity of BMDCs to uptake antigen was evaluated using fluorescent OVA and flow cytometry. RESULTS: The lack of CD40 or IL-23p19 had no effect on uptake of FITC-OVA by the DCs, confirming their immature phenotype. Moreover, CD40KO-DCs had significantly reduced capacity to stimulate proliferation of CD4+ T cells. CD4+ T cells activated by CD40KO-DCs and IL-23p19KO-DCs produced significantly less IFN-γ (P-value ≤0.05), while CD4+ T cells stimulated by IL-23p19KO-DCs produced less GM-CSF and more IL-10 than Cont-DCs. CONCLUSION: This study shows that CD40KO-DCs and IL-23p19KO-DCs have a marked tolerogenic potency in vitro. Future in vivo studies should determine if and to what extent DCs lacking CD40 or IL-23 have a potential to be useful in therapy of autoimmune inflammation.

Association of TNFA -308 G/A and TNFRI +36 A/G gene polymorphisms with glaucoma.

OBJECTIVE: TNF-alpha is one of the most important factors recognized in the pathogenesis of open-angle glaucoma. Therefore, the association of single nucleotide polymorphisms in the TNFRI gene and the promoter region of the TNFA gene with glaucoma susceptibility was investigated in the present study. METHOD: In total, 223 patients with glaucoma and 202 unrelated controls were genotyped by allele-specific oligonucleotide PCR and PCR- restriction fragment length polymorphism to determine TNFA -308 G/A and TNFRI +36 A/G polymorphisms, respectively. RESULTS: In contrast to TNFRI polymorphisms, the genotypes of TNFA -308 G/A polymorphisms showed a significant difference between patients and controls (p = 0.0025). Of interest, the distribution of TNFA genotypes was significantly different between patients with primary open-angle glaucoma (p = 0.001) or pseudoexfoliative glaucoma (p = 0.001) and controls, while no difference was found when chronic angle-closure glaucoma patients were compared to controls (p = 0.72). CONCLUSION: In line with studies showing the role of TNF-alpha in open-angle glaucoma, the results of the present study showed that inheritance of the high producer TNFA -308 A allele might be a susceptibility factor for the development of open-angle glaucoma.

A simple method for the generation of insulin producing cells from bone marrow mesenchymal stem cells.

To produce insulin-producing cells (IPCs) from bone marrow mesenchymal stem cells (BM-MSCs) using a simple and cost effective method. During the initial 7 days of three-dimensional (3D) culture, BM-MSCs were cultured on 1% agar or agarose to form multicellular spheroids. Spheroids and spheroid-derived single cells (SS and SSC, respectively) were cultured in the absence of any proteinaceous growth factor in a simple specific medium for a further 7 d. The insulin content of the differentiated cells was evaluated at the mRNA and protein levels. Furthermore, the expression of pancreatic beta cells-related genes other than INS as well as the in vitro responses of IPCs to different glucose concentrations were investigated. Cellular clusters generated on agar and SS conditions (agar+SS-IPCs) stained better with beta cell specific stains and were more reactive to serum-containing insulin reactive antibodies compared with agarose-SS-IPCs. Gene expression analysis revealed that in comparison to agarose + SS-IPCs, agar+SS-IPCs expressed significantly higher levels of INS-1, INS-2, PDX-1, NKX6.1, and XBP-1. Of interest, agar+SS-IPCs expressed 2215.3 ± 120.8-fold more INS-1 gene compared to BM-MSCs. The expression of ß-cell associated genes was also higher in agar+SS-IPCs compared to the agar+SSC-IPCs. Moreover, the expression of INS-1 gene was significantly higher in agar+SS-IPCs compared with agar+SSC-IPCs after culture in media with high concentration of glucose. Compared to the most expensive and time-consuming protocols, 3D culture of MSCs on agar followed by 2D culture of cellular clusters in a minimally supplemented high glucose media produced highly potent IPCs which may pay the way to the treatment of diabetic patients.

Leishmania major: identification of developmentally regulated proteins in procyclic and metacyclic promastigotes.

The differentiation from procyclic to metacyclic promastigotes (metacyclogenesis) has been correlated with an increased infectivity in a number of Leishmania species. We compared the proteomes of procyclic and metacyclic promastigotes of L. major. Lysates from either life cycle stage were resolved by 2D-PAGE, followed by Coomassie brilliant blue staining. Spots were analyzed by MALDI-TOF MS. 25 protein spots were found to be differentially expressed during metacyclogenesis. We found that proteins involved in protein synthesis were less abundant in metacyclic promastigotes, while proteins involved in motility, including paraflagellar rod protein 1D, alpha-tubulin and beta-tubulin were more abundant. Also, two mitochondrial enzymes (succinyl-CoA synthetase beta subunit and cytochrome c oxidase subunit IV) were differentially expressed in both life cycle stages. Down-regulation of proteins related to synthetic pathway in metacyclic promastigotes is consistent with the arrested growth in this life cycle stage, while up-regulation of proteins related to motility in metacyclic promastigotes is in agreement with the high motility observed in this stage.