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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.

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., APC and T cell use the CD137 receptor/ligand interaction to co‐stimulate T cell activity as well as cytokine production in APCs. We have developed an efficient method to produce stable recombinant CD137‐Fc protein. This recombinant protein is capable of acting either in membrane (through the Fc receptor) or in soluble form. The soluble form of CD137 (sCD137) reduces the activity of its membrane isoform, therefore be ideal for the treatment of autoimmune diseases and uncontrolled immune responses. One of the well‐known properties of sCD137 is activation of antiviral immunity to halt viral replication. It may reduce the inflammation of the lung caused by the coronavirus disease 2019 (COVID‐19) and improve the condition of patients. Generally, interrupting the activation of lymphocytes by sCD137 may be helpful in reducing the harmful inflammatory conditions and cytokine storm. Therefore, its use along with other antiviral therapy should be a considerable solution for the treatment of patients with COVID‐19 virus infection.

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.

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.

Application of “bioactive materials”, as a modified version of biomaterials, can optimize the response of the biological system due to their surface reactivity and formation of strong interactions with the adjacent tissue upon implantation. However, choosing an appropriate bioactive material that suits to the application and provides the desired mechanical, physical, chemical and biological functionality, as well as understanding the aspects of biological reaction to the biomaterial, in particular immune response, it plays a key role in successful integration of the implant. In this review, we will discuss different bioactive materials including bioactive ceramics, polymers and composites and their applications in drug delivery and scaffold preparation in order to provide an adequate introduction to the recent studies. Considering the necessity of regulation of implant fate for higher biocompatibility, the comprehensive overview to the immune response will be reviewed with the focus on representing the cell-biomaterial interactions and more importantly, the inflammatory responses. Ultimately, we will also discuss about different approaches namely as immunomodulation to elicit the desired physiochemical properties and mimicking native cellular response using bioactive compounds, functionalizing the implant surface with active molecules and alteration of the surface morphology. With better understanding of bioactive materials and their interactions with body, more novel biomaterials representing desired properties can be designed.

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.Tumor was induced in female BALB/c mice by injecting 7 ​× ​10Combination therapy with ML221 + DC vaccination was more effective in reducing tumor growth (P ​ ​0.0001), preventing lung metastasis (P ​ ​0.0001) and increasing survival rate (P ​ ​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 ​ ​0.01). It also reduced serum levels of IL-10 compared with the control group (P ​ ​0.05).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.

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.

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-β, 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-β 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.

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.

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)-β 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 TGFβ 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-β, -6, and -23, with or without TGFβ), 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 TGFβ 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-1β, -6, and -23, but not TGFβ, could be used as a cytokine combination to induce development of human TH17 cells. It was also shown that TGFβ 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 TGFβ signaling pathways by selective inhibitors up-regulated TH17 cell differentiation. From the data here, we concluded that TGFβ 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.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), leading to high mortality worldwide. It is well-established that cellular immunity plays a critical role to control Mtb infection. Dendritic Cells (DCs) are potent antigen presenting cells, which play an important role to prime cell-mediated immune responses. In vivo targeting of DCs has been shown to induce both strong cellular immunity and protection against tumor challenges. The aim of the present study was not only to assess the immunizing potential of a novel DC-targeted recombinant lentivirus expressing fusion antigen Ag85A-ESAT6 of Mtb, but also to compare it with a recombinant lentivirus with broad cellular tropism expressing the same antigen in mice. The findings demonstrated that our novel recombinant DC-targeted lentivector was able to successfully transduce and express the fusion antigen Ag85A-E6 in vitro and in vivo. Moreover, a single footpad injection of targeted lentivectors could elicit strong T-helper 1 (Th1) immunity against the above mentioned antigen, as indicated by the specific high-level production of IFN-γ and IL-2 using spleen lymphocytes and lymphoproliferative responses. Despite of these promising results, more attempts are required to elucidate the protective and therapeutic efficacy of this approach in future.

Poly lactic-co-glycolic acid (PLGA), a biodegradable polymer, can effectively protect encapsulated peptides from enzymatic degradation. PLGA was approved by FDA as a safe drug delivery system suitable for inhalation administration. Vasoactive intestinal peptide (VIP), a 28-amino-acid peptide, displays anti-inflammatory and anti-spasmodic effects, which can be considered as a new therapeutic option to control and treat asthma. Because of in vivo enzymatic degradation of VIP including in the lung, there is a need for an applicable delivery system. In light of this, the purpose of this study was to prepare VIP-loaded PLGA microspheres as a drug delivery system, assuming that the newly-introduced model has the ability to persist for a longer time in respiratory tracts. The PLGA microsphere was produced, and loaded with VIP as an applicable nanodrug system. A series of physiochemical properties were determined, including the morphological characteristics, average size of nanoparticles, electric charge distribution, FTIR spectroscopy absorption, and loading and releasing percentage of the nanodrug. VIP-loaded PLGA exhibited an average size of approximately 550 ± 50 nm. Additionally, the produced microsphere showed 78 % VIP release after 10 h at the pH value corresponding to bronchioalveolar microenvironment (approximately 6.5). In the present study, PLGA was formulated and used as a delivery system for VIP. Taken together, the newly-introduced nanodrug seems to be helpful for the clinical treatment of allergic asthma. PLGA nanoparticles can be considered as a potential efficient delivery system for VIP in the respiratory system.

Dendritic cells (DCs) can acquire immunogenic or tolerogenic properties depending on intrinsic and tissue environmental factors. We aimed to determine the immunomodulatory effects of decidual soluble factors from abortion- and non-abortion-prone mice on DC functions. The decidual cell supernatants (DS) were obtained from abortion-prone and non-abortion-prone mice. Splenic DCs were treated with DS and conalbumin (as an antigen) and injected into the palms of the mice. After five days, regional lymph node cells were collected and cultured in the presence and absence of conalbumin. The proliferation of lymphocyte cells, the frequency of regulatory T cells (Tregs), and the production of IL-4 and IFN-γ were measured by [ 3 H]thymidine incorporation, flow cytometry, and ELISA respectively. Our results indicated that DS from both abortion- and non-abortion-prone mice decreased the ability of DCs to induce lymphocyte proliferation and IFN-γ production, while enhanced their capacity to induce Tregs compared with non-treated DCs. Another important finding was that the immunosuppressive effects of DS from abortion-prone mice on DCs for inducing proliferative responses, developing Tregs, and producing IFN-γ by primed lymphocytes was less than DS from non-abortion-prone mice. We also found that only DS from non-abortion-prone mice could enhance the capacity of DCs to induce IL-4 production by primed lymphocytes. It can be concluded that decidua-secreted factors, by altering DC functions, can determine the pattern of immune responses at the fetomaternal interface and, subsequently, pregnancy outcome.

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.

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.

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-β (TGF-β), 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-β 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-β and IL-10 and downregulation of IFN-γ and TNF-α genes expression in deciduas. However, the levels of TGF-β 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-β 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.

Objective To explore the effects of induction of Gamma-interferon(IFN-γ) production by adenoviral vectors (AdVs) on dendritic cells (DCs) viability and activity. Methods AdVs were propagated in human embryonic kidney 293 cell lines and DCs were derived from mouse bone marrow using GM-CSF-IL-4-enriched media. The DCs were infected by either AdVs-green fluorescent protein or AdVs-IFN-γ during a 48 h culture. Phenotypic and functional characteristics of AdV-infected DCs were measured by flowcytometry-based methods. Results Our results displayed that AdVs-IFN-γ could significantly induce DCs necrosis. Furthermore, AdVs-IFN-γ-infected DCs efficiently expressed the CD86 molecules. Conclusions According to our finding, AdV-IFN-γ is able to affect murine bone marrow-derived DC viability and activity.

Nanoparticles can decrease the defects of usual drug and gene delivery systems. Chitosan is a low-toxicity, biodegradable, biocompatible, and safe polymer which is used in the production of nanoparticles. Nanoparticles including chitosan-tripolyphosphate (TPP) can affect the immune cells including dendritic cells (DCs) as the most potent antigen-presenting cells with a pivotal role in both humoral and cellular immunity. For efficient antigen presentation, DCs need to become mature and express high levels of class II major histocompatibility complex (MHC-II) as well as costimulatory molecules, including CD40 and CD86 molecules. In this study, we investigated the effects of chitosan-TPP nanoparticles on DC maturation and function. Chitosan-TPP was synthesized by ionotropic gelation methods from chitosan and TPP salt, and DCs were isolated from mouse spleen using the magnetic cell separation method. DCs were treated with chitosan-TPP nanoparticles during an overnight cell culture and phenotypic and functional characteristics of chitosan-TPP-treated DCs were evaluated by flow cytometric analysis. Our results showed that chitosan-TPP induced DC maturation. Moreover, chitosan-TPP-treated DCs were shown to be efficient inducers of T cell proliferation in allogenic mixed lymphocyte reaction. Indeed, chitosan-TPP as an adjuvant is able to induce DC maturation which is a vital step in efficient antigen presentation and activation of the immune system. Thus, chitosan-TPP nanoparticles can be used as a dual-function agent in clinical immunotherapy methods aiming at using them in drug and gene delivery as well as in potentiating immune responses.

Common variable immunodeficiency (CVID) is a heterogeneous group of disorders characterized by hypogammaglobulinemia and recurrent bacterial infections. Impaired antibody production in these patients is due to defect in B-cell differentiation into specific plasma cells. Class switch recombination (CSR), which plays a critical role in the production of different immunoglobulin isotypes, may be defective in a group of CVID patients. The aim of this study was to investigate the CSR process in B cells of CVID patients, by evaluating the expression of IgE mRNA and production of its protein in an IgE inductive medium. Peripheral blood mononuclear cells (PBMCs) from 29 CVID patients and 21 healthy controls were isolated and cultured in the presence of rhIL-4 and CD40L. IgE mRNA and IgE protein were measured by RT-PCR and ELISA techniques, respectively. Normal production of IgE mRNA was recorded in 23 out of 29 patients (79.31%) as well as all controls; while the remaining 6 patients (20.69%) were unable to express IgE mRNA indicating a defect in CSR. PBMCs of 16 out of 29 patients (55.2%) could not produce normal amounts of IgE compared with controls, after being stimulated by IL-4 and CD40L. Post B cell stimulation IgE production was impaired in about half of studied CVID patients. Defects in processes occur following the CSR process such as IgE mRNA transcription, protein production, and secretion can be the causative mechanism of CVID in patients with normal mRNA expression of the immunoglobulin but impaired protein production. Determination of these defects can help to clarify the various underlying mechanisms responsible for the development of CVID.

Dendritic cells (DCs) are recognized as key regulators of the immune system. Active DC immunization protocols are quickly obtaining interest as an alternative therapeutic approach in acute myeloid leukemia patients. Despite apparent progress in DC-based immunotherapy, some discrepancies were reported in generating potent DCs and their source. In addition to monocytes, DCs can be differentiated from leukemic blasts of acute myeloid leukemia (AML) patients (AML-DC) possessing the ability of stimulating anti-leukemic immune response. In this study, we differentiated peripheral blood blasts of 16 out of 20 AML patients in vitro in the presence of GM-CSF and IL-4 into immature AML-DC. Then, DCs matured using different combinations of Toll-like receptor (TLR) ligands to obtain functional DCs as demonstrated by cell morphology, immunophenotype, and functional activity. Autologous cytotoxic T cell induction of matured DCs was evaluated in four patients and compared with immature counterparts. Our results showed that although the TLR3 agonist (Poly I:C) has a synergistic effect on the TLR4 agonist (lipopolysaccharide, LPS), the addition of the TLR7/8 agonist (R848) is necessary to reinforce the effect of LPS or LPS + POLY(I:C) to produce efficient DCs with the higher level of IL-12 (30 to 90 times). Such DCs activate allogeneic T cells and effectively prime autologous cytotoxic T cells in vitro. In contrast, FSL-1 as a TLR2/6 agonist has a negative effect on LPS + Poly(I:C) and LPS + R848 to produce IL-12. Thus, DCs prepared using a maturation mixture including a TLR7/8 agonist may be used as a potential tool for DC-based immunotherapy purposes in leukemic patients.

Objectives There are few studies with conflicting results on the effects of in vivo administration of opioids on immune function. The aim of this study was to evaluate the serum levels of interferon (IFN)-γ, interleukin (IL)-4, IL-10, IL-17, and hs-C-reactive protein (hs-CRP) in opium smokers. Methods The study was conducted between 44 male opium addicts and 44 controls aged 20 to 40 years. The control group was healthy individuals with no lifetime history of substance abuse. All the opium abusers were selected from those who had a history of use of opium, as a regular habit, at least for 1 year, with a daily opium dosage of not less than 2 g. Addicts known to abuse alcohol or other drugs were excluded. Serum samples were collected from all participants and tested for the cytokine and hs-CRP levels by ELISA (enzyme-linked immunosorbent assay) method. Statistical analysis was performed using the Student t test. Results The mean serum levels of IFN-γ, IL-10, and IL-17 in the opium addicts were significantly higher than those observed in the control group. The mean concentration of serum IL-4 in opium addicts did not differ from that in the control group. Systemic IL-10 levels correlated positively and significantly with CRP in opium addicts. Conclusions Long-term, daily use of opium is associated with higher Th1 (IFN-γ), Tr1 (IL-10), and Th17 (IL-17) cytokines concentration in serum. Interferon-γ and IL-17 are involved in inducing and mediating proinflammatory responses. Our data suggest that an immunoregulatory response is occurring with the upregulation of IL-10.

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of children diarrhea in the world. Adhesion of ETEC to small intestine is an important virulence trait. One of the most prevalent colonization factors (CFs) in human is CFA/I fimbriae and CfaE which is the required binding factor for adhesion of ETEC to intestinal mucosa. We optimized cfaE gene codons according to codon bias of E. coli to achieve a high level of recombinant protein expression. The optimized gene was expressed in E. coli and rCFaE protein was used for mice immunization. Blocking activity of the obtained antibody was examined by microplate agglutination inhibition test. SDS-PAGE analysis indicated that the optimized sequence of cfaE produces a suitable amount of rCFaE in comparison with native gene sequence. This optimized rCFaE protein could induces strong humoral response in mice and the antibody obtained against rCFaE inhibited the adhesion of ETEC to human group A erythrocytes. It is concluded that codon optimization is a useful approach for obtaining large quantities of recombinant rCFaE protein. With regard to the results of hemagglutination inhibition test, codon optimization and increased production of recombinant protein expressed in E. coli did not affect the immunogenicity potential of CFaE.

The Helicobacter pylori outer membrane proteins play an important role in pathogenesis; the outer inflammatory protein A (OipA) is one of these proteins which play the main role in the development of inflammation. In this study, purification of recombinant H. pylori OipA was performed by Ni-NTA affinity chromatography. Gastric carcinoma epithelial cells (AGS cell) were treated by different concentrations of recombinant OipA for various lengths of time and cell viability was evaluated by the viability assay. Statistical analysis showed that OipA had toxic effects on AGS cells in a concentration of 500 ng/ml after 24 and 48 h, and this toxic dose was 256 ng/ml after 72 h. OipA had direct toxic effects on gastric epithelial cells and the toxicity was observed to depend on time and dose of H. pylori exposure. Attachment of H. pylori to gastric epithelial cells is a key part in the pathogenesis and enables H. pylori to damage the epithelial cells with OipA.

BACKGROUND: Immunotherapy with paternal lymphocytes plays an important role in preventing recurrent spontaneous abortion (RSA) and is an effective treatment for it. This kind of treatment is performed as an immunotherapy method in several centers in the world. It attributes to the production of anti-paternal cytotoxic antibodies (APCAs) in women with RSA. Production of APCA after lymphocyte immunotherapy (LIT) in RSA patients gives them a better chance for successful pregnancy. Regarding the important effect of trace elements on the function of the immune system, we tried to investigate the correlation between serum zinc level and the success of LIT in RSA. MATERIALS AND METHODS: Serum zinc concentration was determined in two groups of RSA patients using atomic absorption spectrophotometer systems. Group (a) that responded to the paternal lymphocytes and their cross-match test was positive, and group (b) that had no response to the paternal lymphocytes immunizations and their cross-match test was negative. RESULTS: Serum zinc levels in group (a) patients were 74.98 ± 11.88 μg/dl, which was significantly higher than those in group (b) with the zinc concentration of 64.22 ± 9.22 μg/dl. CONCLUSIONS: Zinc deficiency may be one of the substantial causes of negative results for LIT in RSA patients. Therefore, compensation of zinc defect before LIT can be a promising approach to improve the immune response in patients.

T-Helper 17 (T

Aim: Tolerance to the semi-allogenic fetal graft by the maternal immune system is a medical enigma. Many aspects of immunoregulation at the feto–maternal interface have been clarified, but systemic effects of pregnancy on the immune system are still elusive. The present study was undertaken to determine whether mid-pregnancy mouse serum has an inhibitory effect on dendritic cells (DC) function. Material and Methods: Mid-gestational sera were obtained from allogenic pregnant Balb/c mice (Balb/c × C57BL/6) on days 9–11 of gestation. Splenic DC were purified from Balb/c mice, and treated with mid-pregnancy mouse serum. Antigen pulsed DC were injected into mice palms. After 5 days, draining lymph nodes were removed, cultured in the presence of cognate antigen, and proliferation of responding cells was measured by 3H-thymidin incorporation. Interleukin (IL)-10 and interferon-gamma (IFN-γ) production by stimulated lymph node antigen-specific cells was also measured in culture supernatants using sandwich ELISA. Results: Treatment of DC with pregnant mouse serum markedly blocked their ability to induce antigen-specific lymphocyte proliferation and IFN-γ and IL-10 production by primed lymph node cells in comparison with non-pregnant serum-treated DC. Conclusion: Pregnant mouse serum has an inhibitory effect on DC capacity to induce antigen-specific proliferation and cytokine secretion by lymph node cells. The suppressive effects of pregnant serum on DC could be considered as one of the mechanisms responsible for the systemic immunomodulation observed during pregnancy.

In this In vitro study, T cell responses induced by breast tumor cell lysate pulsed monocyte-derived DCs were analyzed in terms of proliferation, specific cytotoxicity and cytokine-release in order to use in immunotherapeutic settings. Nylon wool enriched T lymphocytes from 5 patients with breast cancer stimulated In vitro with tumor cell lysate pulsed monocyte-derived DCs and their proliferation response were analyzed by [3H] thymidine uptake test. Specific cytotoxic activity of tumor antigen primed T cells after three rounds weekly stimulation was evaluated by flow cytometry, and interferon-γ (IFN-γ) and interleukin-4 (IL-4) cytokines release assay was carried out 24 hours after last stimulation in the supernatant of primed T cells using commercially available ELI-SA kits. T cell proliferation assay revealed that tumor cell lysate pulsed DCs could stimulate autologous T cell proliferation response with stimulation indices 4.9 - 30. T cell mediated cytotoxicity assay demonstrated that tumor antigen primed T cells could significantly kill autologous tumor cells more than normal cells (P γ and IL-4 in response to restimulation by antigen pulsed DCs which were dominated by IFN-γ production in 2 and IL-4 production in 3 out of 5 patients. Our result suggested that breast tumor antigen pulsed DCs could elicit effective specific antitumor T cell responses In vitro, therefore, tumor antigen pulsed DC vaccination may be considered as a novel strategy for immunotherapy of patients with breast cancer refractory to standard modalities.

IVIG can be prepared from fractionation of normal human plasma and it is used as a therapeutic drug for treatment of various diseases. IVIG has been for some time the high-growth product within the plasma derived products, at both a global and a national country level. Fractionation was performed according to Cohn method with some modifications. Fraction II was first produced and then it was used for purification and virus inactivation steps. Two methods of virus inactivation (pasteurization at 60 degrees C for 10 hours and solvent/detergent treatment with TnBP and Tween 80) were used and validated. A chromatography method (cation exchange chromatography on CM Sepharose FF) was also added to obtain high purity. The final product (in liquid and freeze dried formulation) meets European Pharmacopeias requirements. The amount of PKA and aggregates was beyond the acceptance limit. The intactness of the IVIG was also examined by circular dichroism (secondary and tertiary structure). It was stable after 6 months of storage. Since Iran market is completely dependant on importation of plasma derived products, it is important to develop such methods for production of IVIG to obtain regional demands.

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.

The safety of plasma derived medicinal products, such as immunoglobulin, depends on viral inactivation steps that are incorporated into the production process. Several attempts have been made to validate the effectiveness of these inactivation methods against a range of physio-chemically diverse viruses. Treatment with solvent/detergent (S/D) and pasteurization (P) has been continuously used in our IgG production and these methods were analysed in this study as models of viral inactivation. Bovine Viral Diarrhoea Virus (BVDV), Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) were employed as models of HCV, HBV and HIV respectively. Polio and Reo viruses also were used as stable viruses to chemical substances. The infectivity of a range of viruses before and after treatment with two methods of viral inactivation was measured by end point titration and their effectiveness expressed as Logarithmic Reduction Factors (LRF). Solvent/detergent treatment reduced the amount of enveloped viruses by 5-6 logs. The reduction factor was between 5-6 logs for all viruses used in the pasteurization process. A final log reduction factor was obtained as the sum of the two individual methods. Both inactivation methods have advantages and disadvantages with respect to their ability to inactivate viruses. Thus,combination of two robust virus inactivation steps, solvent/detergent and pasteurization, increases the safety margin of immunoglobulin preparations.

Background Monoclonal antibody against horseradish peroxidase (HRP) has many applications which peroxidase anti-peroxidase. Peroxidase-antiperoxidase (PAP) complex formation is its most known and important usage. This complex is used in many immunohistochemical and immunocytochemical staining techniques. Objective The aim of this study was the preparation of anti-HRP monoclonal antibody through hybridoma technology. Methods The BALB/c mice were immunized by repeated injections of HRP. After the confirmation of their immunization by ELISA test, the spleen lymphocytes and SP2/0 myeloma cells were hybridized using PEG as fusing agent. The hybridoma cells were then selected by culturing in HAT medium. Identification and selection of anti-HRP producing clones were done by ELISA test on culture supernatants of the obtained clones. To acquire the monoclones, limiting dilution was performed twice and the effect of finally obtained antibodies on enzyme activity was investigated by a specific ELISA test. In vivo tumor induction method was used for production of concentrated antibody. At last class and subclass of the obtained antibodies were determined by Isostrip Kit. Results After seven rounds of cell fusions, 224 clones were obtained, from which, six ones were anti-HRP producers. Two clones (P1F11 and P2F6) with higher antibody secretion were selected and subcloned. Both derived hybridoma monoclones (P1F11D2 and P2F6F3) were producing antibodies from IgG1 subclass with kappa (Kappa) light chains which didn't affect the enzyme activity. The electrophoresis of ascetic fluid of tumor induced mice showed an obvious band in gamma (gamma) position. Conclusion The obtained monoclonal antibodies are from IgG class and don't affect the enzyme activity, therefore it seems that they are suitable for PAP complex production.

Objective To investigate the immunomodulatory activity of decidual culture supernatant on dendritic cell (DC) functions. Design In vivo and in vitro experimental study using mice. Setting Academic research laboratory. Animal(s) C57BL/6-mated female Balb/c mice. Intervention(s) Culture supernatants of decidual cells obtained from the uteri of allogenic pregnant mice (Balb/c × C57BL/6) were collected. Dendritic cells were purified from Balb/c mice spleens and pulsed with antigen during overnight culture. In some cultures, decidual supernatant was added at 5%, 10%, or 20% final concentration. Endometrial culture supernatant-treated DCs served as a control. Antigen-pulsed DCs were injected into the front footpads of syngeneic mice. Main Outcome Measure(s) Lymph nodes of primed mice were removed 5 days after DC injection. Antigen-specific proliferation and interleukin-10 and interferon gamma production by lymphocytes were measured by 3 H-Thymidine incorporation and ELISA, respectively. Result(s) The results showed that decidual culture supernatant markedly blocked in vivo antigen presentation by DCs and inhibited their capacity to induce interferon gamma (but not interleukin-10) production by primed lymphocytes. Conclusion(s) It seems that soluble factors produced by decidual cells are important mediators of immunoregulation at the feto–maternal interface, which provide the two fundamental requirements for protection of the semiallogenic fetus, namely immunologic tolerance and predominance of T helper 2 immunity, through modulation of DCs function.