Engineered extracellular vesicles expressing ICAM-1: A promising targeted delivery system for T cell modifications.

BACKGROUND: Extracellular vesicles (EVs) are natural nano-carriers that possess the required crucial features of an ideal biomolecular delivery system. However, using unmodified EVs may have some limitations such as low accumulation in target sites. Studies have established that engineering EVs against different cell surface markers can overcome most of these hurdles. METHODS: In this study, engineered EVs expressing ICAM-1/LAMP2b fusion protein on their surfaces were produced and isolated. The uptake of isolated targeted and non-targeted EVs was evaluated by imaging and flow cytometry. To assess the ability of targeted EVs to be applied as a safe carrier, pAAVS1-Puro-GFP plasmids were encapsulated into EVs by electroporation. RESULTS: The HEKT 293 cell line was successfully modified permanently by a lentiviral vector to express ICAM-1 on the surface of the derived EVs. The ELISA and western blot tests established the binding affinity of targeted EVs for recombinant LFA-1 with a remarkable difference from non-targeted EVs. Furthermore, flow cytometry results revealed noteworthy differences in the binding of LFA-1-positive, non-targeted EVs, and targeted EVs to LFA-1-negative cells. Finally, imaging and flow cytometry indicated that newly produced EVs could efficiently interact with T cells and functionally deliver loaded plasmids to them. CONCLUSION: These LFA-1-targeted EVs were able to interact with T cells as their recipient cells. They can be utilized as an ideal delivery system to transfer various biomolecules to T cells, facilitating immunotherapies or other cell-based treatments.

Fluorescence labeling of anchor-modified Mart-1 peptide for increasing its affinity for HLA-A*0201: Hit two targets with one arrow.

One of the most successful strategies in designing peptide-based cancer vaccines is modifying natural epitope peptides to increase their binding strength to human leukocyte antigens (HLAs). Anchor-modified Mart-1 peptide (ELAGIGILTV) is among the artificial epitope peptides with the highest binding affinity for HLA-A*0201. In this study, by fluorescence labeling of its either C- or N-terminus with Nε -(5-carboxyfluorescein)-l-lysine, we not only made it traceable but also drastically increased its binding strength to HLA-A*0201. HLA streptamer, for the first time, is introduced for measuring the binding constants (Ka ) of the labeled peptides. The affinity of the labeled peptides for the HLA-A*201 of the MCF-7 cells was extraordinarily high and co-incubating them with the highest possible amount of the unlabeled peptide, as a competitor, did not significantly prohibit them from binding to the HLA. The reproducibility of the obtained results was confirmed by using the T2 cell line. The HLA-deficient K562 cell line was used as the negative control. With in silico simulations, we found two hydrophobic pockets on both sides of HLA-A*0201 for anchoring the C- or N-terminal 5-carboxyfluorescein probe, which can explain the extraordinary affinity of the labeled peptides for the HLA-A*0201.

The Effect of Age and Cell Culture Parameters on the Quantity and Function of Bone Marrow-derived Dendritic Cells.

Dendritic cells (DCs) are a group of bone marrow-derived cells that play a crucial role in innate and acquired immune responses. Bone marrow-derived dendritic cells (BMDC) are used in many studies, so the efficiency and purity of the differentiated cells are essential. This study aimed to investigate the effect of several parameters, including the age of mice, cell culture medium, and swirling of the culture plate, to increase the efficiency of the induced cells, considering the standard protocols. Bone marrow-derived dendritic cells were induced from both juvenile and adult mice bone marrow cells. Then, the purity of CD11c+ cells was compared between juvenile mice BMDCs and adult mice BMDCs. Cells were cultured in an enriched and non-enriched medium, and some wells were swirled when changing the medium on the 3rd day. Then the effect of enriched medium and swirling before medium replacement were evaluated based on the expression of the CD11c marker. The efficiency of DCs differentiation (CD11c+ cells) was higher when juvenile mouse bone marrow precursors were used compared to adult mice; using the enriched media with supplements and swirling the well before media replacement significantly affected the purity of immature CD11c+ cells. Due to our results, using juvenile mice, an enriched culture medium, and physical removal of granulocyte cells could significantly improve the purity and efficiency of CD11c+ cells. Therefore, considering these three items in the production protocol of these cells can probably reduce the use of lymphocyte-removing antibodies and purification methods.

Inhibition of apelin/APJ axis enhances the potential of dendritic cell-based vaccination to modulate TH1 and TH2 cell-related immune responses in an animal model of metastatic breast cancer.

PURPOSE: The immunosuppressive microenvironment of tumors reduces the effectiveness of immunotherapies. Apelin as an immunosuppressor peptide is expressed in the microenvironment of many tumors. Thus, inhibition of apelin-related protumor activities can promote the effectiveness of cancer immunotherapy. Here, we investigated the efficacy of a dendritic cell (DC) vaccine in combination with an apelin receptor antagonist, ML221, to modulate Th1 and Th2 cell-related responses in breast cancer-bearing mice. MATERIALS AND METHODS: Tumor was induced in female BALB/c mice by injecting 7 â€‹× â€‹105 4T1 cells in the right flank. Tumor-bearing mice were then given PBS, ML221, DC vaccine and "ML221 + DC vaccine" for 21 days. On day 37, mice were sacrificed and the frequency of Th1/Th2 cells in spleen and serum levels of IFN-γ/IL-10 were determined using flow cytometry and ELISA, respectively. Lung metastasis was evaluated in lung tissues stained with hematoxylin and eosin. Finally, the obtained data were analyzed using appropriate statistical tests. RESULTS: Combination therapy with ML221 + DC vaccination was more effective in reducing tumor growth (P â€‹< â€‹0.0001), preventing lung metastasis (P â€‹< â€‹0.0001) and increasing survival rate (P â€‹< â€‹0.01) compared to the control group. Moreover, combination treatment substantially increased the frequency of Th1 cells while decreasing the frequency of Th2 cells in the spleen compared to the control group (P â€‹< â€‹0.01). It also reduced serum levels of IL-10 compared with the control group (P â€‹< â€‹0.05). CONCLUSION: Our findings showed that combination therapy using ML221 + DC vaccine can be considered as an effective cancer therapeutic program to potentiate anti-tumor immune responses.

Mesenchymal stem cell therapy attenuates complement C3 deposition and improves the delicate equilibrium between angiogenic and anti-angiogenic factors in abortion-prone mice.

Immunological disorders are one of the main causes of recurrent spontaneous abortions (RSA). A rapidly expanding body of evidence indicates that excessive activation of the complement system is critically involved in the development of miscarriages. In the CBA/J × DBA/2 murine model of recurrent miscarriage, exaggerated and unrestrained complement activation is reported to be the underlying cause of angiogenic factor imbalance and persistent inflammation. We have previously shown that mesenchymal stem cell (MSC) therapy can significantly reduce the abortion rate in abortion-prone mice through regulating the feto-maternal immune response. In the present study, we hypothesized that MSCs might improve the balance of angiogenic factors at the feto-maternal unit of CBA/J × DBA/2 mice by restraining complement activation and deposition. To explore this hypothesis, autologous adipose tissue-derived mesenchymal stem cells (AD-MSCs) were administered intra-peritoneally to abortion-prone mice on the 4.5th day of gestation. Control mice received PBS as vehicle. On day 13.5 of pregnancy, deposition of the complement component C3 and expression levels of Crry, CFD (adipsin), VEGF, PlGF and FLT-1 were measured at the feto-maternal interface by immunohistochemistry and real-time PCR, respectively. Decidual cells were also cultured in RPMI 1640 medium for 48 h and VEGF and sFLT-1 protein levels were quantified in supernatants using enzyme-linked immunosorbent assay (ELISA). Our results indicated that MSC therapy significantly reduced C3 deposition and adipsin transcription in the fetal-maternal interface of abortion-prone mice. Furthermore, administration of MSCs robustly upregulated the mRNA expression levels of Crry, VEGF, PlGF and FLT-1 in the placenta and decidua of CBA/J × DBA/2 mice. Consistently, the in vitro results demonstrated that decidual cells obtained from MSC-treated dams produced increased concentrations of VEGF in culture supernatants, with concomitant decreased levels of sFLT-1 protein. Here, we show for the first time that adoptive transfer of MSCs rectifies the disturbed balance of angiogenic factors observed at the feto-maternal unit of CBA/J × DBA/2 mice, in part at least, through inhibiting excessive complement activation and promoting the production of angiogenic factors. Collectively, these alterations seem to play a pivotal role in reducing the abortion rate and improving the intrauterine condition for the benefit of the fetus.

Simultaneous transduction of dendritic cells with A20 and BTLA genes stimulates the development of stable and efficient tolerogenic dendritic cells and induces regulatory T cells.

BACKGROUND: This study investigated the potential of simultaneous overexpression of A20 and B- and T-lymphocyte attenuator (BTLA) genes in dendritic cells (DCs) to develop a tolerogenic phenotype in DCs and investigate their capabilities for induction of immunosuppression. METHODS: Plasmid vectors were designed harboring A20, BTLA, and A20 + BTLA genes and were transfected to HEK 293T cells to produce lentiviruses. DCs were transduced by the gene carrying viruses and evaluated for the surface expression of MHCII, CD40, and CD86 molecules by flow-cytometry. The mRNA expression of A20, BTLA, and CCR7 were determined. Mixed-lymphocyte reaction was conducted to evaluate the T cell stimulation potency and ELISA was used to measure the production of IL-10, TGF-ß, and TNF-α. The potential of DCs for migration to lymph nodes and Treg induction were assessed by in vivo experiments. RESULTS: Transduction of DCs resulted in significantly decreased surface expression of CD40 and CD86 co-stimulators and upregulated A20, BTLA, and CCR7 mRNA expression. The IL-10 and TGF-ß levels were enhanced significantly in the supernatant of LPS-treated DCs transduced with A20 + BTLA-containing virus group relative to the DCs transduced with pCDH vectors. DCs transduced with A20 + BTLA harboring vectors had higher migratory potential to mouse lymph nodes and caused the development of higher numbers of Treg cells compared with the DCs transduced with pCDH vectors. CONCLUSIONS: Simultaneous overexpression of A20 and BTLA genes in DCs caused development of tolerogenic DCs with a promoted potential in induction of Treg cells, accompanied by remarkable stability after inflammatory stimulation. All these offer a promising potential of such DCs in treating autoimmune and inflammatory disorders.

Recombinant CD137-Fc, its synthesis, and applications for improving the immune system functions, such as tumor immunotherapy and to reduce the inflammation due to the novel coronavirus.

CD137 (ILA/4-1BB), a member of tumor necrosis factor receptor superfamily, is one of the most important T cell costimulatory molecules. Interaction of this molecule with its ligand transmits a two-way signal that activates both T lymphocyte and antigen presenting cells. The soluble form of CD137 (sCD137) reduces the activity of its membrane isoform and is associated with T lymphocyte activation-induced cell death. Recombinant CD137-Fc may be used to treat cancers, autoimmune disorders and viral infections. It may also be useful for management of coronavirus infection. The 1276 bp DNA sequence encoded CD137-Fc recombinant protein was prepared and subcloned into lentiviral vector and expressed in transduced CHO-K1 eukaryotic cells. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay analysis results demonstrated that the expression of the 70-kDa CD137-Fc molecule was detectable without any degradation. This study helps to confirm previous research suggesting the use of this recombinant protein as a promising solution for the treatment of virus infections. CD137-Fc fusion protein could also make immunotherapy more effective for some diseases. This product is widely used in novel medical treatments, including cell-based immunotherapy such as dendritic cell, CAR T and CAR NK therapy. Its production and usage in research and treatment is noticeable also in current coronavirus disease 2019 pandemic.

Mesenchymal stem cells induce expansion of regulatory T cells in abortion-prone mice.

Recurrent pregnancy loss (RPL) is one of the most common complications of early pregnancy associated in most cases with local or systemic immune abnormalities such as the diminished proportion of regulatory T cells (Tregs). Mesenchymal stem cells (MSCs) have been shown to modulate the immune responses by de novo induction and expansion of Tregs. In this study, we analyzed the molecular and cellular mechanisms involved in Treg-associated pregnancy protection following MSCs administration in an abortion-prone mouse mating. In a case-control study, syngeneic abdominal fat-derived MSCs were administered intraperitoneally (i.p) to the DBA/2-mated CBA/J female mice on day 4.5 of pregnancy. Abortion rate, Tregs proportion in spleen and inguinal lymph nodes, Ho1, Foxp3, Pd1 and Ctla4 genes expression at the feto-maternal interface were then measured on day 13.5 of pregnancy using flow cytometry and quantitative RT-PCR, respectively. The abortion rate in MSCs-treated mice reduced significantly and normalized to the level observed in normal pregnant animals. We demonstrated a significant induction of Tregs in inguinal lymph nodes but not in the spleen following MSCs administration. Administration of MSCs remarkably upregulated the expression of Ho1, Foxp3, Pd1 and Ctla4 genes in both placenta and decidua. Here, we show that MSCs therapy could protect the fetus in the abortion-prone mice through Tregs expansion and upregulation of Treg-related genes. These events could establish an immune-privileged microenvironment, which participates in the regulation of detrimental maternal immune responses against the semi-allogeneic fetus.

A Randomized, Triple-blind Placebo-controlled Trial to Determine the Effect of Saffron on the Serum Levels of MMP-9 and TIMP-1 in Patients with Multiple Sclerosis.

Matrix metalloproteinases (MMP)-9 facilitates the migration of T-cells to central nervous system (CNS), while tissue inhibitor of metalloproteinases-1(TIMP-1) inhibits the function of MMP-9. This study aimed to determine the appropriate treatment option for multiple sclerosis (MS). Forty-three relapsing-remitting MS (RRMS) patients were randomly divided into two groups of 22 (group A, placebo) and 21 (group B, Saffron pill) individuals. Serum samples were collected from patients' blood before using the Saffron pills/placebo pills and then after 12 months. The serum level of MMP-9 and its inhibitor, as well as TIMP-1, were measured by ELISA kits. MMP-9 serum levels noticeably decreased in patients with MS following 12 months of treatment with Saffron pills (p=0.006) while the changes were not significant before and after 12 months of treatment with placebo pills. Although the levels of TIMP-1 increased significantly after one year treating with Saffron pills (p=0.0002), a considerable difference was not observed before and after taking the placebo pills. The study finding revealed that 12-months treatment with Saffron could have a significant role in reducing the serum level of MMP-9 and increasing the serum level of TIMP-1 in RRMS patients. Therefore, modulating the serum levels of MMP-9 as an important regulator of T cell trafficking to the CNS might be a promising strategy in the treatment of MS patients.

Mesenchymal stem cells alter the frequency and cytokine profile of natural killer cells in abortion-prone mice.

Natural killer cells, which play a pivotal role in the establishment and maintenance of normal pregnancy, are the most abundant leukocytes at the fetomaternal interface that their subsets frequencies and cytokine profile are influential factors in the preservation of the decidual tolerogenic microenvironment. Any imbalance in NK cells' frequency and functions could be associated with pregnancy failure. Mesenchymal stem cells (MSCs) are shown to have immunomodulatory effects on NK cells and their cytokine profile. The purpose of this study is to evaluate the impact of MSCs therapy on the cytokine profiles and subpopulations of NK cells in a murine model of recurrent pregnancy loss. Adipose-derived MSCs were injected intraperitoneally to the abortion-prone mice on Day 4.5 of gestation. The abortion rate was determined after MSCs administration and the frequency and cytokine profiles of the different subsets of NK cells were determined using the flow cytometry. Our results showed that, in abortion-prone mice, the frequency of CD49b+ NK cells was significantly higher than normal pregnant mice that decreased after therapy. We also demonstrated that MSCs downregulated the production of IFN-γ and upregulated IL-4 and IL-10 production by uNK cells. These findings indicate that MSCs can decrease the infiltration of CD49b+ NK cells to the fetomaternal interface and modulate the cytokine profile of NK cells from inflammatory to tolerogenic profile and thereby improve the tolerogenic microenvironment at the fetomaternal interface in benefit of pregnancy maintenance.

New Insights Into Implementation of Mesenchymal Stem Cells in Cancer Therapy: Prospects for Anti-angiogenesis Treatment.

Tumor microenvironment interacts with tumor cells, establishing an atmosphere to contribute or suppress the tumor development. Among the cells which play a role in the tumor microenvironment, mesenchymal stem cells (MSCs) have been demonstrated to possess the ability to orchestrate the fate of tumor cells, drawing the attention to the field. MSCs have been considered as cells with double-bladed effects, implicating either tumorigenic or anti-tumor activity. On the other side, the promising potential of MSCs in treating human cancer cells has been observed from the clinical studies. Among the beneficial characteristics of MSCs is the natural tumor-trophic migration ability, providing facility for drug delivery and, therefore, targeted treatment to detach tumor and metastatic cells. Moreover, these cells have been the target of engineering approaches, due to their easily implemented traits, in order to obtain the desired expression of anti-angiogenic, anti-proliferative, and pro-apoptotic properties, according to the tumor type. Tumor angiogenesis is the key characteristic of tumor progression and metastasis. Manipulation of angiogenesis has become an attractive approach for cancer therapy since the introduction of the first angiogenesis inhibitor, namely bevacizumab, for metastatic colorectal cancer therapy. This review tries to conclude the approaches, with focus on anti-angiogenesis approach, in implementing the MSCs to combat against tumor cell progression.

Uterine Dendritic Cells Modulation by Mesenchymal Stem Cells Provides A Protective Microenvironment at The Feto-Maternal Interface: Improved Pregnancy Outcome in Abortion-Prone Mice.

OBJECTIVE: Dendritic cells (DCs) as major regulators of the immune response in the decidua play a pivotal role in establishment and maintenance of pregnancy. Immunological disorders are considered to be the main causes of unexplained recurrent spontaneous abortions (RSAs). Recently, we reported that mesenchymal stem cells (MSCs) therapy could improve fetal survival and reduce the abortion rate in abortion-prone mice, although the precise mechanisms of this action are poorly understood. Since MSCs have been shown to exert immunomodulatory effects on the immune cells, especially DCs, this study was performed to investigate the capability of MSCs to modulate the frequency, maturation state, and phenotype of uterine DCs (uDCs) as a potential mechanism for the improvement of pregnancy outcome. MATERIALS AND METHODS: In this experimental study, adipose-derived MSCs were intraperitoneally administered to abortion-prone pregnant mice on the fourth day of gestation. On the day 13.5 of pregnancy, after the determination of abortion rates, the frequency, phenotype, and maturation state of uDCs were analyzed using flow cytometry. RESULTS: Our results indicated that the administration of MSCs, at the implantation window, could significantly decrease the abortion rate and besides, increase the frequency of uDCs. MSCs administration also remarkably decreased the expression of DCs maturation markers (MHC-II, CD86, and CD40) on uDCs. However, we did not find any difference in the expression of CD11b on uDCs in MSCs-treated compared to control mice. CONCLUSION: Regarding the mutual role of uDCs in establishment of a particular immunological state required for appropriate implantation, proper maternal immune responses and development of successful pregnancy, it seems that the modulation of uDCs by MSCs could be considered as one of the main mechanisms responsible for the positive effect of MSCs on treatment of RSA.

Leukemia inhibitory factor (LIF) modulates the development of dendritic cells in a dual manner.

Objective: Dendritic cells (DCs) are professional antigen presenting cells majorly modulated by various environmental factors. Leukemia inhibitory factor (LIF) is a pleiotropic cytokine from interleukin-6 family. Previous studies demonstrate that LIF is associated with several tolerogenic events; yet the exact effect of this cytokine on the generation and function of DCs was not explicitly identified. Materials and methods: To clarify the role of LIF in DCs development, immature DCs were differentiated from mouse bone marrow (BM) in a GM-CSF and IL-4 containing medium with or without LIF. Afterwards, in maturation process, the differentiated DCs were exposed to TNF-α in the presence or absence of LIF. Results: Immature DCs differentiated in the presence of LIF, proved a significant enhancement in the expression of MHCII, CD40, or CD86 molecules and in the antigen uptake function. LIF treatment of normal DCs while stimulating for maturation, caused a significant decrement in the expression of phenotypic markers as well as an increment in the antigen uptake function in comparison with TNF-α-only stimulated cells; however, the reduced ability for induction of allogenic T-cell proliferation proved no statistical significance. Conclusions: Our results can reflect a role for LIF in the generation and particularly maturation of DCs. It can be assumed that LIF rather modulates the maturation level, leading to the development of semi-mature and tolerogenic DCs. According to the high levels of LIF in immune-privileged sites like brain and uterine, it seems that the cytokine may account for the formation of local DCs that help the establishment of immunosuppressive environments.

Engineered Exosomes for Targeted Transfer of siRNA to HER2 Positive Breast Cancer Cells.

Exosomes are the best options for gene targeting, because of their natural, nontoxic, non-immunogenic, biodegradable, and targetable properties. By engineering exosome-producing cells, ligands can be expressed fusing with exosomal surface proteins for targeting cancer cell receptors. In the present study, HER2-positive breast cancer cells were targeted with a modified exosome producing engineered HEK293T cell. For this purpose, the HEK293T cells were transduced by a lentiviral vector bearing-LAMP2b-DARPin G3 chimeric gene. Stable cells expressing the fusion protein were selected, and the exosomes produced by these cells were isolated from the culture medium, characterized, and then loaded with siRNA for subsequent delivery to the SKBR3 cells. Our results showed that stable HEK293T cells produced DARPin G3 on the surface of exosomes. These exosomes can bind specifically to HER2/Neu and are capable of delivering siRNA molecules against TPD52 gene into SKBR3 cell line down-regulating the gene expression up to 70%. Present approach is envisaged to facilitate genes and drugs transfer to HER2 cancer cells providing additional option for gene therapy and drug delivery.

Comparison of The Therapeutic Effect of Syngeneic, Allogeneic, and Xenogeneic Adipose Tissue-Derived Mesenchymal Stem Cells on Abortion Rates in A Mouse Model.

OBJECTIVE: Mesenchymal stem cells (MSCs), due to their immunomodulatory functions, are an ideal candidate for the treatment of immune-related diseases. Recurrent spontaneous abortion (RSA) is one of the most common complications of pregnancy which in many cases is related to the immune system disorders. Our previous study has shown that the abortion rate was decreased following the syngeneic MSCs therapy in abortion-prone mice. In this study, the therapeutic effect of syngeneic, allogeneic, and xenogeneic MSCs was compared in a mouse model of RSA. MATERIALS AND METHODS: In this experimental study, MSCs were isolated from adipose tissue (ASCs) of CBA/J and BALB/c mice and human. After characterization, ASCs were injected (IP) at day 4 of gestation to female CBA/J mice following their mating with DBA/2 male mice. In the control group, phosphate-buffered saline (PBS) was injected and CBA/J×BALB/c mating was also used as the normal pregnancy control. On day 14.5 of pregnancy, embryo resorption rate was determined. RESULTS: The abortion rate significantly decreased following the ASCs therapy from syngeneic (6.31%), allogeneic (6.54%), and xenogeneic group (12.36%) compared to ASCs non-treated group (34.4%). There was no statistical difference between ASCs treated groups, however syngeneic and allogeneic ASCs reduced the abortion rate more efficiently than xenogeneic ASC. CONCLUSION: The abortion rate was significantly decreased following the intraperitoneal administration of ASCs from various donated sources in abortion-prone mice. These results indicated that the immunogenicity of allogeneic and xenogeneic ASCs is not a contradictory problem for their therapeutic effects on RSA.

Mesenchymal stem cells therapy protects fetuses from resorption and induces Th2 type cytokines profile in abortion prone mouse model.

The imbalance of Th1/Th2 cytokines is well known in recurrent spontaneous abortion (RSA) mouse model. Mesenchymal stem cells (MSCs) possess potent immunoregulatory properties that could modulate the Th1 cytokine responses in benefit of Th2 types. In this study, we aimed to analyze the local and systemic balance of Th1/Th2 cytokines following MSCs therapy. Syngeneic adipose derived MSCs were administered to abortion prone mice during the implantation window. The abortion rate was determined and IL-4, IL-6, IL-12, IL-2, IFN-γ and GM-CSF gene expression was evaluated by Real-Time-PCR in decidual and placental tissues of pregnant mice at day 13.5 of pregnancy. Splenocytes of pregnant mice were co-cultured with mitomycin C treated paternal splenocytes and IL-2, IL-4, IL-10 and IFN-γ cytokines were measured in co-cultures supernatants by ELISA method. Proliferation response of female splenocytes to paternal antigens was also evaluated using the CFSE method. Our results showed a significant reduction in abortion rate following MSCs administration in abortion prone mice. We also observed a significant down-regulation of IL-2 and IFN-γ as well as up-regulation of IL-4 and IL-10 production from pregnant mouse splenocytes following MSCs therapy along with a significant reduction of splenocytes proliferation against paternal antigens. Our findings revealed that MSCs therapy increased the IL-4, IL-6, IL-10 and GM-CSF and at the same time decreased the IL-12, IL-2 and IFN-γ gene expression at feto-maternal interface. Here, we showed that MSCs therapy could modulate the systemic as well as local Th1/Th2 cytokines production along with protection of fetus from resorption in abortion prone mice. The fine balance of Th1/Th2 cytokine response could be considered as one of the possible mechanisms for fetal protection following MSCs therapy.

Mesenchymal Stem Cell Therapy Prevents Abortion in CBA/J × DBA/2 Mating.

Immunological disorders are among the main causes of recurrent spontaneous abortions (RSAs). Mesenchymal stem cells (MSCs) have been shown to modulate various aspects of immune responses. It seems that MSCs may improve the immunological conditions in immune-mediated RSA. The aim of this study is the reduction of resorption in RSA mouse model through MSCs therapy. The adipose-derived MSCs were administered intraperitoneal to pregnant CBA/J mice on day 4.5 of gestation in abortion-prone matting. On day 13.5 of pregnancy, abortion rates were calculated and transforming growth factor-ß (TGF-ß), interleukin 10 (IL-10), interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α) gene expression in deciduas were evaluated by real-time polymerase chain reaction (PCR). The level of TGF-ß in serum was also determined by enzyme linked immunosorbent assay (ELISA) method. The obtained results showed that MSCs therapy could reduce the abortion rate significantly in test group compared to controls. MSCs therapy also caused a significant upregulation of TGF-ß and IL-10 and downregulation of IFN-γ and TNF-α genes expression in deciduas. However, the levels of TGF-ß didn't change in mice sera. Due to the significant decrease in abortion rate, we concluded that MSCs therapy could modulate the immune responses in fetomaternal interface and protect fetus from undesirable immune responses. So, these cells might be considered as a new therapeutic for spontaneous pregnancy loss. The local upregulation of TGF-ß and IL-10 and downregulation of IFN-γ and TNF-α gene expression in decidua could be considered as one possible mechanism of immune regulation, which could protect the fetus.

CD40 Knocked Down Tolerogenic Dendritic Cells Decrease Diabetic Injury.

BACKGROUND: Type-1 diabetes (T1D) is an autoimmune disease in which T lymphocytes destroy insulin-producing ß-cells. Control of self-reactive T lymphocytes and recovery of diabetic injury is the end point of T1D. OBJECTIVE: To investigate generation of tolerogenic dendritic cells (tolDCs) as an innovative method of diabetes therapy. METHODS: Lentivirus vector production was achieved by GIPZ mouse CD40 shRNA, psPAX2 and pMD2G plasmids DNA. Purified bone marrow derived DCs were treated with CD40 shRNA, and expression of CD40 and mRNA level were evaluated by flow cytometry and Real-Time PCR, respectively. CD40 knock-down DCs were injected into STZ-induced diabetic mice. Blood glucose; glucose tolerance test and weight were analyzed in different groups. RESULTS: Mice treated with CD40 shRNA transfected DCs showed considerable differences in blood glucose, glucose tolerance, and weight compared to other groups. Also cytokine assays indicated an increase in IL-13 production in the CD40 shRNA group. CONCLUSION: In streptozotocin-induced diabetic mice model, administration of tolerogenic dendritic cells could improve diabetic parameters.

Is TGFß as an anti-inflammatory cytokine required for differentiation of inflammatory TH17 cells?

T-Helper 17 (TH17) cells are a CD4+ TH subset that plays a critical role in the pathophysiology of inflammatory disorders, especially chronic forms. It seems that the derivation of TH17 cells from their precursors take place in inflammatory microenvironment. The role of transforming growth factor (TGF)-ß as an anti-inflammatory cytokine in TH17 cell differentiation is controversial. To address some of the discrepancies that exist among different studies, this study was undertaken to more clarify the TGFß role in human TH17 cell differentiation. Here, CD4+ T-cells were isolated from peripheral blood samples and cultured in X-VIVO 15 serum-free medium. Purified cells were then treated with different combinations of polarizing cytokines (interleukin [IL]1-ß, -6, and -23, with or without TGFß), neutralizing anti-interferon (IFN)-γ and anti-IL-4 antibodies and polyclonal stimulators anti-CD3 and -CD28 antibodies, and then analyzed for IL-17, IFNγ, Foxp3, and CD25 expression by flow cytometry and for release of IL-17, -21, -22, and -10 into culture media by ELISA. The effects of selective inhibition of TGFß signaling pathway on TH17 cell polarization were also determined by using small molecules SB-431542 and A83-01. The current study found that a combination of pro-inflammatory cytokines, including IL-1ß, -6, and -23, but not TGFß, could be used as a cytokine combination to induce development of human TH17 cells. It was also shown that TGFß acted as a negative regulator in this regard and also led to reduced IL-17 and IL-22 production while inducing Foxp3 expression. Indeed, blocking of TGFß signaling pathways by selective inhibitors up-regulated TH17 cell differentiation. From the data here, we concluded that TGFß down-regulates human TH17 cell differentiation and that a presence of pro-inflammatory cytokines (along with IFNγ and IL-4 neutralizing antibodies) is sufficient for optimal differentiation of human TH17 cells.

Conjugated Alpha-Alumina nanoparticle with vasoactive intestinal peptide as a Nano-drug in treatment of allergic asthma in mice.

Asthma is a chronic respiratory disease characterized by airway inflammation, bronchoconstriction, airway hyperresponsiveness and recurring attacks of impaired breathing. Vasoactive intestinal peptide (VIP) has been proposed as a novel anti-asthma drug due to its effects on airway smooth muscle relaxation, bronchodilation and vasodilation along with its immunomodulatory and anti-inflammatory properties. In the current study, we investigated the therapeutic effects of VIP when conjugated with α-alumina nanoparticle (α-AN) to prevent enzymatic degradation of VIP in the respiratory tract. VIP was conjugated with α-AN. Balb/c mice were sensitized and challenges with ovalbumin (OVA) or PBS and were divided in four groups; VIP-treated, α-AN-treated, α-AN-VIP-treated and beclomethasone-treated as a positive control group. Specific and total IgE level, airway hyperresponsiveness (AHR), bronchial cytokine expression and lung histology were measured. α-AN-VIP significantly reduced the number of eosinophils (Eos), serum IgE level, Th2 cytokines and AHR. These effects of α-AN-VIP were more pronounced than that seen with beclomethasone or VIP alone (P<0.05). The current data indicate that α-AN-VIP can be considered as an effective nano-drug for the treatment of asthma.