Therapy with regulatory T-cell infusion in autoimmune diseases and organ transplantation: A review of the strengths and limitations.

In the last decade, cell therapies have revolutionized the treatment of some diseases, earning the definition of being the "third pillar" of therapeutics. In particular, the infusion of regulatory T cells (Tregs) is explored for the prevention and control of autoimmune reactions and acute/chronic allograft rejection. Such an approach represents a promising new treatment for autoimmune diseases to recover an immunotolerance against autoantigens, and to prevent an immune response to alloantigens. The efficacy of the in vitro expanded polyclonal and antigen-specific Treg infusion in the treatment of a large number of autoimmune diseases has been extensively demonstrated in mouse models. Similarly, experimental work documented the efficacy of Treg infusions to prevent acute and chronic allograft rejections. The Treg therapy has shown encouraging results in the control of type 1 diabetes (T1D) as well as Crohn's disease, systemic lupus erythematosus, autoimmune hepatitis and delaying graft rejection in clinical trials. However, the best method for Treg expansion and the advantages and pitfalls with the different types of Tregs are not fully understood in terms of how these therapeutic treatments can be applied in the clinical setting. This review provides an up-to-date overview of Treg infusion-based treatments in autoimmune diseases and allograft transplantation, the current technical challenges, and the highlights and disadvantages of this therapeutic approaches."

Cytokine single nucleotide polymorphisms in patients' with gallstone: dose TGF-ß gene variants affect gallstone formation?

Gallstone is a common biliary disorder with several risk factors. Immune responses and inflammatory cytokines are important in this disease; as a result, some cytokines can be detected in bile fluid. In this research, cytokine gene polymorphisms were studied, and their effects on gallstone formation were evaluated. On 158 gallstone patients and 254 normal subjects, by PCR- RFLP method, IL-4-C590T polymorphism and by ARMS-PCR method, IFN-γ T+874A, TNF-α-A308G, IL-6 G-174C and TGF-ß T+869C variants were studied. Pathologic evaluations were done on surgical specimens. There were no significant differences in distribution of evaluated polymorphisms between patient group and normal control group (P > 0.05), except TGF-ß +869T allele (P = 0.04, OR = 1.23, 95 % CI = 1-1.79) which was higher in patients with gallstone. Although the pro-inflammatory cytokines such as TNF-α and IL-6 may promote gallstone formation, in this study no significant correlation between TNF-α and IL-6 polymorphisms and gallstone formation was seen. It is taught that TGF-ß may affect gallbladder cells to promote gallstone formation and higher producer TGF-ß +869T allele can be a risk factor of gallstone disease, so further studies would be more elucidative

Association of MicroRNA146a G>C and MicroRNA196a-2 C>T Gene Polymorphisms with Outcome of Kidney Transplantation in Iranian Patients.

Acute organ rejection remains a serious clinical challenge. Novel accessible biomarkers of acute rejection could easily enable us to detect the rejection earlier and make more fine-tuned calibration of immunosuppressive or new target treatment possible. Control of gene expression by microRNAs influences many cellular functions, including cellular differentiation, cell proliferation, cell development, and functional regulation of the immune system. Therefore, this study was aimed to investigate if miRNA146a G>C and miRNA196a-2 C>T gene polymorphisms are associated with kidney transplant rejection in Iranian patients. Tissue samples were collected from 100 renal transplant patients between the years 2009 and 2013. The miRNA146a G>C (rs2910164) and miRNA196a-2 C>T (rs11614913) gene polymorphisms were evaluated in kidney transplant patients; using the in-house-polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In this study, we found that the CC genotype, C and G alleles of the miRNA146a G>C polymorphism was associated with increased risk of transplant rejection in kidney transplant patients (p=0.003, p=0.01 and p=0.01), respectively. The CC genotype, T, and C alleles of the miRNA196a-2 C>T were also significantly more frequent in transplanted patients compared to healthy controls (p=0.02, p=0.05, and p=0.05), respectively. However, significant associations were not found between miRNA196a-2 C>T polymorphisms and kidney transplant rejection. The CC genotype, G, and C allele of the miRNA146a G>C and also, the CC genotype, T and C alleles of the miRNA196a-2 C>T may be genetically susceptible factors for transplant rejection and development of kidney disorders, especially in Iranian patients. Further studies are required to validate these findings in a larger population, as well as in patients with different ethnic origins.

Gene Expression of Toll-Like Receptors 2 and 4 in Renal Transplant Rejection.

OBJECTIVES: Toll-like receptors are a crucial part of the innate immune system and have a pivotal role in the acquired immunity system. Studies have shown that Toll-like receptors 2 and 4 are important during the transplant process. Therefore, we analyzed the gene expression of Toll-like receptors 2 and 4 in cases of renal transplant rejection. We measured the messenger RNA expression levels of Toll-like receptors 2 and 4 in renal transplant rejection recipients compared with nonrejection recipients. MATERIALS AND METHODS: We enrolled 151 deceased-donor kidney transplant recipients, whom we divided into 2 groups: 101 nonrejection recipients and 50 recipients with acute allograft rejection. We collected 3 mL of blood (treated with ethylenediaminetetraacetic acid) from each patient. Ribonucleic acid extraction and complementary DNA synthesis were conducted for all samples, and the constructed complementary DNAs were used for real-time polymerase chain reaction analysis. RESULTS: We measured gene expression levels of Toll-like receptors 2 and 4 in renal transplant recipients with acute allograft rejection and in recipients who did not experience acute renal allograft rejection, and the results showed that messenger RNA expression levels for both Toll-like receptors 2 and 4 were significantly increased in the acute rejection group compared with the nonrejection group. CONCLUSIONS: Toll-like receptors 4 and 2 could increase the risk of acute rejection after renal transplant and could be defined as a risk factor for rejection. Further studies are recommended.

Immune modulation through RNA interference-mediated silencing of CD40 in dendritic cells.

RNA interference (RNAi) is an exciting mechanism for knocking down any target gene in transcriptional level. It is now clear that small interfering RNA (siRNA), a 19-21nt long dsRNA, can trigger a degradation process (RNAi) that specifically silences the expression of a cognate mRNA. Our findings in this study showed that down regulation of CD40 gene expression in dendritic cells (DCs) by RNAi culminated to immune modulation. Effective delivery of siRNA into DCs would be a reasonable method for the blocking of CD40 gene expression at the cell surface without any effect on other genes and cell cytotoxicity. The effects of siRNA against CD40 mRNA on the function and phenotype of DCs were investigated. The DCs were separated from the mice spleen and then cultured in vitro. By the means of Lipofectamine2000, siRNA was delivered to the cells and the efficacy of transfection was estimated by flow cytometry. By Annexine V and Propidium Iodide staining, we could evaluate the transfected cells viability. Also, the mRNA expression and protein synthesis were assessed by real-time PCR and flow cytometry, respectively. Knocking down the CD40 gene in the DCs caused an increase in IL-4 production, decrease in IL-12 production and allostimulation activity. All together, these effects would stimulate Th2 cytokines production from allogenic T-cells in vitro.

Evidence for CEA release from human colon cancer cells by an endogenous GPI-PLD enzyme.

Elevated carcinoembryonic antigen (CEA) blood levels are found in a wide variety of epithelial neoplasms. The precise mechanism of the spontaneous CEA release from normal and cancer cells has not been established yet. In this study we investigated 'in vitro' the role of an endogenous glycosylphosphatidyl inositol phospholipase D (GPI-PLD) in spontaneous CEA release from human colon carcinoma cells. We detected GPI-PLD-specific transcript expression in four human colorectal tumor cell lines, LS180, HT29, HT29/219, and SW742 by RT-PCR. Furthermore, CEA release could be activated and inhibited by incubation of LS180 cells with suramin and 1,10-phenanthroline, compounds known to activate and inhibit GPI-PLD activity, respectively. The results suggest a mechanism for the involvement of an endogenous GPI-PLD in spontaneous CEA release from human colon cancer cells.

Plant components for immune modulation targeting dendritic cells: implication for therapy.

Medicinal plant utilization is as old as human life. There are thousands of herbs consumed for medicinal purposes all over the world, especially in east. Their value has not decreased over time and many modern pharmaceuticals have originated from traditional medicinal plants. Studying the reason for their influence is an attractive field of medicine. Among various types of herbs, some function via their immunomodulatory effects. Experiments have shown the regulatory influences of several plants on each type of immune cell, including T cells, B cells, dendritic cells (DCs), macrophages and NK cells. Because of the prominent role of DCs in antigen presentation as the major APC, this review summarizes the immunomodulatory effects of some plants performed through DC effects.

Association of cytokine/costimulatory molecule polymorphism and allograft rejection: a comparative review.

One reason for genetic variations among human individuals is SNP which may confer diverse disease susceptibility or resistance in a population. Genetic variations in a key immunoregulatory agent can manifest various immunological responses, such as graft rejection. In fact, the outcome of organ transplantation can be impacted by several genetic causes including polymorphisms in genes encoding cytokines and costimulatory molecules in the donor or recipient. Thus, it can be helpful to contemplate the SNPs relating to these immunological determinants in order to achieve an improved transplantation therapy.

Tolerance Induction by CD40 Blocking through Specific Antibody in Dendritic Cells.

Blocking antibodies are valuable tools for inhibiting the specific receptor- ligand interactions. The interaction of co-stimulatory molecules on the antigen presenting cells with their ligands on T cells is an essential step for T cell activation. In the present study, the effect of blocking antibody against CD40 on its T cell stimulatory potential is investigated.The DCs (dendritic cells) were collected from the mice spleens and then cultured in vitro. We used purified rat anti-mice CD40 (Clone HM40-3) (BD USA) as a blocking antibody and the appropriate titer of the blocking antibody was determined by flow cytometry. The DCs were then treated by antibody and used in MLR assay. The results of these experiments showed that CD40 blockade were associated with the increase in the of IL-4 secretion, shifting the DCs to stimulate Th2 cytokine production by the allogenic T cells, while the secretion of IL-12 by DCs decreased. Similarly, the DCs with reduced CD40 expression poorly responded to alloantigen stimulation in the MLR. Collectively, these results emphasize the importance of CD40 pathway in tolerogenic DCs generation and also support the idea that downregulation of CD40 is effective in inhibiting the allostimulatory function.

The IFN-gamma allele is correlated to moderate-to-severe acute graft-versus-host disease after allogeneic stem cell transplant.

OBJECTIVES: Analysis of nonhistocompatibility leucocyte antigen functional genomics in stem cell transplant can lead to prediction of clinical outcomes in histocompatibility leucocyte antigen-matched sibling-transplant recipients. Some of the cytokine gene polymorphisms might be associated with severe, acute graft-versus-host disease after allogeneic stem cell transplant. We evaluated gene polymorphisms of IL-6 G-174C, TGF-beta T+869C, IL-4 C-590T, and IFN-alpha T+874A cytokines in bone marrow transplant patients. MATERIALS AND METHODS: The amplification refractory mutation system-polymerase chain reaction ARMSPCR method was used to characterize IL-6 G-174C, TGF-beta T+869C, and IFN-alpha T+874A polymorphisms, and PCR-RFLP, using AvaII restriction enzyme, was done for IL-4 C-590T characterization in 35 bone marrow transplant patients. Acute graft-versus-host disease episodes were diagnosed according to EMBT criteria. RESULTS: Analysis showed that IFN-alpha +874T allele (P = .027, OR=0.198, 95% CI=0.049-0.801) was correlated to moderate-to-severe graft-versus-host disease. TGF-beta T+869C, IFN-alpha T+874A, IL-6 G-174C and IL-4 C-590T frequencies were not significantly different in the 2 graft-versus-host disease severity groups (P > .05). CONCLUSIONS: According to the results, we concluded that the IFN-alpha T+874A gene polymorphism has a predictive value for severity of graft-versus-host disease after bone marrow transplant. High producer genotypes of IFN-alpha are genetic risk factors for development of graft-versus-host disease.

CD40 expression in Wehi-164 cell line.

CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body's defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system.

Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense, and antibody blocking.

In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.

The efficiency of CD40 down regulation by siRNA and antisense ODN: comparison of lipofectamine and FuGENE6.

BACKGROUND: Dendritic cells (DCs) are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs (siRNAs) and antisense oligodeoxynucleotides (ODN)s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. OBJECTIVE: We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. METHODS: Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. RESULTS: CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. CONCLUSION: Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs.