The implications of exosomes in psoriasis: disease: emerging as new diagnostic markers and therapeutic targets.

As the largest human organ, the skin is continuously exposed to various external and internal triggers that affect body homeostasis. Psoriasis is a persistent inflammatory skin condition that has a major bearing on patients' physiological functioning as well as their mental well-being. It is an autoimmune disorder and has been the focus of extensive research efforts in recent years. Cells secrete exosomes into the environment surrounding them, which comprises a lipid bilayer. The movement of cellular components like microRNAs, mRNAs, DNA, lipids, metabolites, and cell-surface proteins is mediated by exosomes. Exosomes are crucial for inducing communication between cells. There has been extensive study of exosomes, both preclinical and clinical, looking at their potential role in autoimmune diseases. Besides the role that they play in the body's basic processes, exosomes are also considered an increasingly essential part as diagnostic and therapeutic agents. In the following article, we conduct a literature review of current studies related to molecular and structural aspects of exosomes. We emphasis on the function of exosomes in pathogenesis, as well as the possibility of their usage in medicinal applications and as biomarkers.

F1 fraction isolated from Mesobuthus eupeus scorpion venom induces macrophage polarization toward M1 phenotype and exerts anti-tumoral effects on the CT26 tumor cell line.

Scorpion venoms identified as agents with anti-tumor and anti-angiogenic features. Tumor microenvironment (TME) plays a pivotal role in the process of tumorigenesis, tumor development, and polarization of M2 phenotype tumor associated macrophages (TAMs). M2 polarized cells are associated with tumor growth, invasion, and metastasis. The fractionation process was performed by gel filtration chromatography on a Sephadex G50 column. To elucidate whether scorpion venom can alter macrophage polarization, we treated interleukin (IL)-4-polarized M2 cells with isolated fractions from Mesobuthus eupeus. Next, we evaluated the cytokine production and specific markers expression for M2 and M1 phenotype using enzyme linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR), respectively. The phagocytic capacity of macrophages was also assessed. In addition, the migration assay and MTT analysis were performed to investigate the effects of reprogrammed macrophages on the CT-26 colon cancer cells. The results indicated that F1 fraction of venom significantly upregulated the levels and expression of M1-associated cytokines and markers, including tumor necrosis factor-alpha (TNF-α) (p < 0.001), IL-1 (p < 0.01), interferon regulatory factor 5 (IRF5) (p < 0.0001), induced nitric oxide synthase (iNOS) (p < 0.0001), and CD86 (p < 0.0001), and downregulated M2-related markers, including transforming growth factor-beta (TGF-ß) (p < 0.05), IL-10 (p < 0.05), Fizz1 (p < 0.0001), arginase-1 (Arg-1) (p < 0.0001), and CD206 (p < 0.001). The macrophage phagocytic capacity was enhanced after treatment with F1 fraction (p < 0.01). Moreover, incubation of CT-26 cell line with conditioned media of F1-treated macrophages suppressed migration (p < 0.0001) and proliferation (p < 0.01) of tumor cells. In conclusion, our findings demonstrated the potential of Mesobuthus eupeus venom in M2-to-M1 macrophage polarization as a promising therapeutic approach against proliferation and metastasis of colon cancer cells.

Toxocara Canis Increases the Potential of Breast Cancer by Reducing the Expression of the P53 Protein.

INTRODUCTION: Breast cancer is considered the most frequent type of cancer in women with high mortality worldwide, and most importantly, it is the second most common cancer. However, some breast cancer-related risk factors remain unknown. So, the current study was designed to evaluate the effect of Toxocara canis on the biomarkers correlated with proliferation, apoptosis, inflammation, and angiogenesis in 4T1 tumor-bearing mice infected with Toxocara canis for the first time. METHODS: Mice were categorized into four groups: A) control, B) treated with 4T1+ Toxocara canis, C) treated with Toxocara canis, and D) treated with 4T1. The expression of Ki-67 and P53 was then evaluated by using the immunohistochemical technique. In addition, the levels of transforming growth factor-ß, Interferon gamma-γ, Interleukin 10, tumor necrosis factor-α and vascular endothelial growth factor as well as anti- Toxocara canis IgG were determined using the enzyme-linked immunosorbent assay method. RESULTS: The expression of Ki-67 was significantly increased in the 4T1+ Toxocara canis group than control and Toxocara canis groups (P < 0.001 and P < 0.001, respectively). Moreover, a significant decrease in P53 was found in the 4T1+ Toxocara canis group than in the control and Toxocara canis groups (P < 0.001 and P < 0.001, respectively). Also, the 4T1+ Toxocara canis group significantly reduced the expression of P53 more than 4T1 tumor-bearing mice (P = 0.005). In addition, the 4T1+ Toxocara canis group had an increasing tumor necrosis factor-α and vascular endothelial growth factor than controls (P = 0.004 and P = 0.002, respectively). Furthermore, a significant reduction in Interleukin 10 was found in the 4T1+ Toxocara canis group than in the control group (P = 0.004). CONCLUSION: Our findings showed that Toxocara canis could probably increase the potential of breast cancer by reducing P53 in 4T1 tumor-bearing mice infected with Toxocara canis more than other groups.

Sublingual prophylactic administration of OVA-loaded MSC-derived exosomes to prevent allergic sensitization.

AIM: This study evaluated the immunomodulatory and delivery potential of adipose tissue-isolated MSC-derived exosomes as a prophylactic regimen through a sublingual route in the ovalbumin (OVA)-induced allergic asthma murine model. MATERIAL AND METHODS: Balb/c mice received 10 µg/dose of OVA-enriched MSC-derived exosomes as a prophylactic regimen in six doses during three weeks, and then OVA sensitization was conducted through intraperitoneal and aerosol administration of allergen. The total cells and eosinophils counted in nasal lavage fluid (NALF) and lung tissues were assessed for histopathological analysis. In addition, the secretion of IFN-γ, IL-4, and TGF-ß by spleen cells and serum OVA-specific IgE levels were measured via ELISA. RESULTS: Significant reduction in the IgE levels and IL-4 production, along with elevated TGF-ß levels, were observed. Also, limited cellular infiltrations and perivascular and peribronchiolar inflammation in the lung tissues and normal total numbers of cells and eosinophils in the NALF were reported. CONCLUSION: Prophylactic regimen using OVA-enriched MSC-derived exosomes modulated immune responses and inhibited allergic OVA sensitization.

miR-155 rs767649 T>A gene polymorphism is associated with downregulation of miR-155 expression, suppressor of cytokine signaling-1 overexpression, and low probability of metastatic tumor at the time of breast cancer diagnosis.

Background: MicroRNA-155 is a key player in inflammatory reactions, carcinogenesis, and tumor development. In this study, polymorphism of miRNA-155 rs767649 T>A and its gene and suppressor of cytokine signaling-1 (SOCS-1) expression were investigated in relation to cancer susceptibility and development in breast cancer (BC) patients. Materials and Methods: Polymorphism of miRNA-155 rs767649 T>A was evaluated between a population of 174 patients with BC and 129 controls using restriction fragment length polymorphism and the expression of miR-155 and SOCS-1 were examined in peripheral blood mononuclear cells (PBMCs) by real-time polymerase chain reaction. Results: TT genotype of miR-155 rs767649 T>A was associated with higher level of miR-155 in PBMCs of BC patients relative to AT and AA genotypes (21.76 ± 4.4, 4.046 ± 1.35, 2.56 ± 0.81, respectively; P < 0.001) and increased lymph node metastasis (r = 0.292, P = 0.001), not BC susceptibility (P = 0.402 and P = 0.535; respectively). TT genotype of miR-155 rs767649 T>A was associated with less gene expression of SOCS-1 in PBMCs of BC patients compared to AT and AA genotypes (1.173 ± 0.57, 0.92 ± 0.827, 5.512 ± 0.92, respectively; P = 0.003). Conclusion: This study demonstrated for the first time the association between the T allele of the rs767649 T>A polymorphism in the pre-MIR155 gene and higher expression of miR-155, lower expression of SOCS-1, and swift latent progression in newly diagnosed BC patients. Thus, miR-155 may play a critical role in BC pathogenesis.

Evaluation of CD3+ T Cell Percentage, Function and its Relationship with Serum Vitamin D Levels in Women with Recurrent Spontaneous Abortion and Recurrent Implantation Failure.

BACKGROUND: Women afflicted with recurrent spontaneous abortion (RSA) and repeated implantation failure (RIF) may have immune abnormalities. The role of vitamin D has been demonstrated in the function of the immune system. OBJECTIVE: To assess the percentage and function of CD3+ T cells and their relationship with the level of the serum vitamin D or 1,25-dihydroxy vitamin D3 (the active form of the vitamin) in women with RSA and RIF. METHODS: In this case-control study, peripheral blood was obtained from the patient and the healthy control groups. The ratio of CD3+T cell and activated CD3+ CD69+T cell was investigated using flow cytometry. The serum levels of Interferon-γ (IFN-γ) and vitamin D were measured by ELISA. RESULTS: The mean proportion of CD3+T cells in women with RSA increased significantly compared with the healthy control group (p<0.04). However, no significant difference was observed in RIF women compared with the control group. There was no significant difference in the ratio of activated CD3+CD69+T cells between the patient and the healthy control groups. Serum IFN-γ levels in women with RSA showed a significant increase compared to the control group (p<0.031); however, no significant difference was observed between women with RIF and the control group. Serum levels of vitamin D showed a significant reduction in both RSA (p<0.01) and RIF (p<0.04) groups in comparison with the control. CONCLUSION: An increase in the percentage and inflammatory function of T cells was associated with RSA. Decreased vitamin D levels may contribute to immune dysfunction and pregnancy loss.

Altered Expression Levels of MicroRNA-155 and SOCS-1 in Peripheral Blood Mononuclear Cells of Newly Diagnosed Breast Cancer Patients.

MicroRNA-155 (miR-155) has a critical role in pro-inflammatory activation and tumor progression. In addition, miR-155 has various oncogenic effects in the tumor microenvironment by targeting the suppressor gene of cytokine signaling-1(SOCS-1) and interleukin-6 (IL-6). This study investigated the association of inflammatory changes with the variations of miR-155 expression in newly diagnosed breast cancer (NDBC) patients. Seventy NDBC patients were categorized as lobular and ductal subgroups and forty healthy individuals participated in this study. The expression rate of miR-155 and its downstream target gene, SOCS-1, as well as the plasma levels of IL-6, were evaluated in peripheral blood mononuclear cells of NDBC patients; using real-time PCR and enzyme-linked immunosorbent assay, respectively. Our results indicated an over-expression of miR-155 in the PBMCs of NDBC patients which was significantly associated with the tumor grade and the type of ductal carcinoma. In contrast, a significant downregulation of SOCS-1 was observed in NDBC patients compared to control group, however, there was no significant difference between two subtypes of BC. Furthermore, a higher concentration of plasma IL-6 was detected in NDBC patients compared to the healthy control group which had an inverse correlation with the SOCS-1 levels. According to the potential effects of miR-155 on regulating the expression of SOCS-1 and IL-6, we suggest this small transcript as a promising diagnostic marker for various types of BC patients.

Is Immuno-PCR Better than ELISA Test for Detection of Toxoplasma gondii IgG Antibody?

INTRODUCTION: IgG antibodies against T. gondii persist for years, and can act as a reliable serological biomarker for the diagnosis of previous exposure to this parasite. Hence, the current investigation was designed to compare diagnostic power of immuno-polymerase chain reaction (iPCR) and enzyme-linked immunosorbent assay (ELISA) methods for detection of T. gondii IgG antibody. METHODS: Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against T. gondii were measured by the ELISA method in 81 participants. In addition, detection of acute and chronic toxoplasmosis was performed via the ELISA IgG avidity. The set-up of iPCR was carried out and then, serum IgG of subjects were detected using the iPCR method. RESULTS: Of 81 samples, 4 (4.9%) and 30 (37%) cases were be found positive for IgM and IgG against T. gondii in the ELISA method, respectively. Moreover, of 81 specimens, 42 (51.9%) and 39 (48.1%) samples had low-avidity IgG and high-avidity IgG by the IgG avidity kit, respectively. While, 59 (72.8%) of 81 samples were detected positive using the iPCR technique. Kappa (κ) value coefficient, between the iPCR and ELISA (for IgG) showed a strong agreement (0.360, p value < 0.001). A value of 0.25 I.U./ml for serum IgG [area under curve (AUC) = 0.720 (CI = 0.613-0.827); p = 0.002] was the cut-off value to differentiating between positive and negative toxoplasmosis (with sensitivity 66.0% and specificity 60.0%). CONCLUSION: Our findings indicated despite a strong agreement shown between iPCR and ELISA methods, the diagnostic power of iPCR technique was more sensitive than ELISA test for detection of T. gondii IgG antibody. However, more complementary investigations are widely needed in this regard.

Cytokine profiles in peripheral blood mononuclear cells from patients with cystic echinococcosis.

INTRODUCTION: Cystic echinococcosis (CE) is a chronic zoonotic disease caused by the larval stage of Echinococcus granulosus (E. granulosus), which affects domestic and wild carnivores as the definitive host and ungulates as intermediate hosts. In intermediate hosts, both Th1 and Th2 cells are involved in the immune responses to an echinoccocal infection. This study aimed to investigate production of IL-4, IL-10, and IFN-γ cytokines in peripheral blood mononuclear cells (PBMCs) of CE patients before and after surgical treatment. METHODS: To evaluate cytokine production in response to E. granulosus antigens, we investigated IL-4, IL-10, and IFN-γ production in PBMCs of 20 CE patients in response to hydatid cyst fluid antigen (HCF-Ag) before and after surgical treatment using ELISA. RESULTS: The mean IL-4 production from HCF-Ag stimulated PBMCs was significantly decreased (p < 0.05), while IFN-γ was significantly increased in HCF-Ag stimulated PBMCs in patients after surgery (p = 0.005). Furthermore, our results showed that there is no significant difference between IL-10 production in patients before and after treatment (p = 0.562). CONCLUSIONS: Our data Indicated production of IL-4 in cultured PBMCs of CE patients stimulated with HCF-Ag was decreased significantly. While, production of IFN-γ was increased significantly in responses to HCF Ag after surgery. We concluded that the evaluation of IL-4 and IFN-γ in HCF-Ag stimulated PBMCs of CE patients should be considered as a useful marker in the follow up of patients with cystic echinococcosis.

Evaluation of Exhausted Regulatory T Cells in Preeclampsia.

BACKGROUND: The development of a maternal immune response to fetal antigens and deficiency in regulatory T-cells (Tregs) may lead to preeclampsia. A plausible explanation for the reduced Treg cell function in women with preeclampsia is the presence of exhausted Treg cells which express CD279 or programmed cell death receptor-1 (PD-1), a negative regulatory molecule associated with limited proliferative capacity and reduced immune suppression. OBJECTIVE: To assess the number of Treg CD4+ CD25 high and exhausted Treg CD4+ CD25 high CD279+ cells in women with preeclampsia (PE group) and healthy pregnant women (HP group) during the third trimester of pregnancy. METHODS: Three-color flow cytometry was used to determine the proportion of Treg and exhausted Treg cells in 40 women in the PE group and 37 women in the HP group. Participants' blood samples were placed in EDTA blood collection tubes. Peripheral mononuclear cells were separated from the samples and stained with flurochrome-conjugated antibodies against human CD4, CD25 and CD279 markers, and subsequently analyzed by flow cytometry. RESULTS: The PE group had fewer Tregs compared to the HP group (p=0.011). There was a significant increase in the percentage of exhausted PD-1+(CD279) Tregs (p=0.035) in the PE group comparisons with the HP group. CONCLUSION: The increased number of PD-1 (CD279) molecules on the Treg cells may play a role in preeclampsia, hence it recommendation as a therapeutic target for the disease.

Increased frequency of CD4+CD25high CD127low/ - regulatory T cells in patients with multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS), one of the most common diseases of the central nervous system (CNS), is characterized by demyelination and chronic inflammation of the CNS. Failure of immune tolerance and induced autoimmune processes are involved in MS immunopathogenesis. Regulatory T (Treg) cells play an important role in maintaining peripheral tolerance and immune homeostasis. OBJECTIVE: The aim of this study was to evaluate the frequency of CD4+CD25highCD127low/ -Treg cells in MS patients. METHODS: The study population was composed of 25 healthy controls (HCs), 35 patients with relapsing remitting multiple sclerosis (RRMS) and 25 patients with progressive multiple sclerosis (PMS). Frequency of CD4+CD25highCD127low/ - Treg cells in RRMS and PMS patients was compared with HC by flow cytometry. RESULTS: Treg cells frequency in PMS patients was significantly higher compared to RRMS patients (P < 0.001) and HCs (P < 0.001). It was lower in RRMS patients than HCs (P = 0.005). A Significant direct correlation between Treg cells frequency and expanded disability status scale (EDSS) in PMS patients (P = 0.001, r = 0.6) was observed. Reverse correlation between Treg cells frequency and EDSS in RRMS patients was found (P = 0.01, r = -0.4). CONCLUSION: More de-tailed clarification of the role of Treg cells in MS patients could provide a basis for development of Treg cells-mediated therapeutic strategies.

Enhanced Cardiac Differentiation of Human Cardiovascular Disease Patient-Specific Induced Pluripotent Stem Cells by Applying Unidirectional Electrical Pulses Using Aligned Electroactive Nanofibrous Scaffolds.

In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus venosus and appear to be involved in cardiogenesis. In this work, aligned and random polyaniline/polyetersulfone (PANI/PES) nanofibrous scaffolds doped by Camphor-10-sulfonic acid (ß) (CPSA) were fabricated via electrospinning and used to conduct electrical impulses in a unidirectional and multidirectional fashion, respectively. A bioreactor was subsequently engineered to apply electrical impulses to cells cultured on PANI/PES scaffolds. We established cardiovascular disease-specific induced pluripotent stem cells (CVD-iPSCs) from the fibroblasts of patients undergoing cardiothoracic surgeries. The CVD-iPSCs were seeded onto the scaffolds, cultured in cardiomyocyte-inducing factors, and exposed to electrical impulses for 1 h/day, over a 15-day time period in the bioreactor. The application of the unidirectional electrical stimulation to the cells significantly increased the number of cardiac Troponin T (cTnT+) cells in comparison to multidirectional electrical stimulation using random fibrous scaffolds. This was confirmed by real-time polymerase chain reaction for cardiac-related transcription factors (NKX2.5, GATA4, and NPPA) and a cardiac-specific structural gene (TNNT2). Here we report for the first time that applying electrical pulses in a unidirectional manner mimicking the unidirectional wave of electrical stimulation in the heart, could increase the derivation of cardiomyocytes from CVD-iPSCs.

The effect of transplanted human Wharton's jelly mesenchymal stem cells treated with IFN-γ on experimental autoimmune encephalomyelitis mice.

Interferon gamma (IFN-γ) increases the immunosuppressive property of human Wharton's jelly mesenchymal stem cells (hWJ-MSCs). In this study, we evaluated the therapeutic effects of IFN-γ primed WJ-MSCs in EAE mice. IFN-γ primed WJ-MSCs were injected on days 3 and 11 after EAE induction. 21 days after EAE induction, splenocytes and cervical lymph node cells were isolated and cell proliferation, secretion of inflammatory cytokines and frequency of regulatory T-cells was measured. On day 50 of the study, cell infiltration and gene expression of inflammatory cytokines in brain of mice were studied. Leukocyte infiltration and symptoms were significantly reduced in IFN-γ primed WJ-MSCs treated group compared to other groups. These cells showed significantly reduced proliferation and increased Treg cells as well as decreased secretion and gene expression of inflammatory cytokines in EAE mice. Our data suggest that IFN-γ may be used to stimulate the immunomodulatory property of WJ-MSCs in clinical situations.

Evaluation of AD-MSC (adipose-derived mesenchymal stem cells) as a vehicle for IFN-ß delivery in experimental autoimmune encephalomyelitis.

Interferon-ß (IFN-ß) is commonly used as a disease modifying drug for the treatment of relapse-remitting multiple sclerosis (RR-MS). However, the underlying mechanism by which IFN-ß mediate this immunosuppressive effect is still unknown. In this study, we analyzed the effects of genetically modified adipose-derived mesenchymal stem cells (AD-MSCs) expressing murine interferon beta (MSCs-VP/IFN-ß) on the animal model of MS, experimental autoimmune encephalomyelitis (EAE). Lymph node mononuclear cells and serum were examined by using RT-PCR and ELISA methods to measure the production of IL-10 and IL-17 gene and protein expression, respectively. Our results indicated that in the MSCs-VP/IFN-ß treated group induction of Tregs and IL-10 and reduction of IL-17 were significant. Taken together, we showed that using AD-MSCs expressing IFN-ß as an anti-inflammatory agent, offer evidence supporting that the stem cell therapies in EAE conceivably will improve the valuable effects of IFN-ß in this autoimmune disease.

Altered microRNA Expression and Immunosuppressive Cytokine Production by Regulatory T Cells of Ulcerative Colitis Patients.

Regulatory T (Treg) cells are essential for maintenance of peripheral tolerance and prevention of autoimmune diseases in part by producing immunosuppressive cytokines. Recently, microRNAs (miRNAs) have also been involved in autoimmune disorders, not least for their crucial role in the regulation of Treg biology and function. We simultaneously investigated the concentration of IL-35, IL-10, TGF-ß, and sCD25 in supernatant of cell culture and the expression patterns of several miRNAs in CD4(+)CD25(+) CD127(-/low) FoxP3(+) Tregs of ulcerative colitis (UC) patients. Significantly lower levels of IL-10 and IL-35 were observed in Treg cultures of UC patients. miR-21, miR-146a, and miR-155 levels were downregulated and miR-31 level was upregulated in Tregs of patients. Our results suggest that microRNAs may serve as a novel regulator in function and homoeostasis of UC Treg cells, providing a key role for them in pathophysiology of UC.

The Opposite Effects of DNA and Protein Components of Listeria Monocytogenes and Toxoplasma gondii on Immunologic Characteristics of Dendritic Cells.

The innate immune system utilizes pattern recognition receptors (PRRs) to recognize microbes. Pathogens contain various molecules with diverse effects on immune response. In this study, we evaluated the effect of DNA and protein components derived from two intracellular microorganisms including Listeria monocytogenes (L. monocytogenes) and Toxoplasma gondii (T. gondii) on dendritic cells (DCs) activation and ensuing adaptive immune responses. DNA and protein components of L. monocytogenes and T. gondii were prepared using relevant kits. DNA and protein components of these two pathogens were added to immature DCs (iDCs). Subsequently, co-stimulatory expression and cytokine production by DCs were measured. Finally, we evaluated the stimulatory capacity of mature DCs (mDCs) in DC-T cells co-culture. The results showed that protein matured-DCs produced higher level of IL (Interleukin)-12p70. There was also a significant increase in Interferon-Gamma (IFN-γ) production and proliferative capacity in T cells co-cultured with protein matured-DCs. On the other hand, DNA matured-DCs produced significantly higher amounts of Transforming growth factor-beta (TGF-ß). Collectively, these results imply a regulatory nature for DNA and potent stimulatory character for protein components of these two intracellular microorganisms.

In Vitro Generation of IL-35-expressing Human Wharton's Jelly-derived Mesenchymal Stem Cells Using Lentiviral Vector.

Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are easily available cells without transplant rejection problems or ethical concerns compared to bone-marrow-derived MSCs for prospective clinical applications. These cells display immunosuppressive properties and may be able to play an important role in autoimmune disorders. Regulatory T-cells (Treg) are important to prevent autoimmune disease development. Interleukin 35 (IL-35) induces the proliferation of Treg cell populations and reduces the activity of T helper 17 (Th17) and T helper 1 (Th1) cells, which play a central role in initiation of inflammation and autoimmune disease. Recent studies identified IL-35 as a new inhibitory cytokine required for the suppressive function of Treg cells. We created IL-35-producing hWJ-MSCs as a good vehicle for reduction of inflammation and autoimmune diseases. We isolated hWJ-MSCs based on explant culture. HWJ-MSCs were transduced at MOI=50 (Multiplicity of Infection) with lentiviral particles harboring murine Interleukin 35 (mIL-35). Expression of IL-35 in hWJ-MSCs was quantified by an IL-35 ELISA kit. IL-35 bioactivity was analyzed by inhibiting the proliferation of mouse splenocytes using CFSE cell proliferation kit. Frequency of CD4+CD25+CD127 low/neg Foxp3+ Treg cells was measured by flow cytometry. There was an up to 85% GFP positive transduction rate, and the cells successfully released a high level of mIL-35 protein (750 ng/ml). IL-35 managed to inhibit CD4+ T cell proliferation with PHA, and improved the frequency of Treg cells. Our data suggest that transduced hWJ-MSCs overexpressing IL-35 may provide a useful approach for basic research on gene therapy for autoimmune disorders.

Investigation of immunomodulatory properties of human Wharton's Jelly-derived mesenchymal stem cells after lentiviral transduction.

Human Wharton's Jelly-derived Mesenchymal Stem Cells (hWJ-MSCs) are considered as an alternative for bone-marrow-derived MSCs. These cells have immunosuppressive properties. It was unclear whether the WJ-MSCs would sustain their immunomodulatory characteristics after lentiviral transduction or not. In this study, we evaluated immunomodulatory properties of WJ-MSCs after lentiviral transduction. HWJ-MSCs were transduced with lentiviral particles. Expression of transduced and un-transduced hWJ-MSCs surface molecules and secretion of IL-10, HGF, VEGF and TGF-ß was analyzed. Cell proliferation and frequency of CD4(+)CD25(+) CD127(low/neg) Foxp3(+) T regulatory cells was measured. There was no difference between the surface markers and secretion of IL-10, HGF, VEGF and TGF-ß in transduced and un-transduced hWJ-MSCs. Both cells inhibited the proliferation of PHA stimulated PBMCs, and improved the frequency of T regulatory cells. These findings suggest that lentiviral transduction does not alter the immunomodulatory function of hWJ-MSCs. However, lentiviral transduction may have a wide range of applications in gene therapy.

Fibroblasts feeder niche and Flt3 Ligand as a novel inducer of plasmacytoid dendritic cells development in vitro.

Plasmacytoid dendritic cell (pDC), plays central role in antiviral immunity. The aim of this study was to assess the effect of Flt3 ligand (FL) alone or with L929 fibroblast feeder or L929 conditioned media on differentiation of mouse bone marrow (BM) cells into pDC in vitro. Murine BM cells were cultured with FL or with L929 or conditioned media for 9days. The differentiated cells were analyzed using flow cytometry for PDCA-1, B220 and CXCR4. The relative expression of Stat3, CXCR4, CXCR7, IFN-ß, TGF-ß and Runx2 in differentiated cells determined by real time PCR. The development of pDC showed up to 19% increase after co-culture of BM cells with fibroblast feeder. Upregulation of Stat3, Runx2 and CXCR4 due to the presence of fibroblast feeder with FL in culture results in improved pDC development. Furthermore, 30% L929 supernatant along with Flt3 ligand was able to derive pDC up to 8.9% in comparison with FL alone, which was 6.6% in vitro. Thus, for the first time we introduced L929 fibroblast feeder as a niche producer of M-CSF and probably other growth factors and chemokines, which promotes the development of pDC in vitro along with FL, similar to in vivo niche.

Reduced efficacy of multiple doses of CpG-matured dendritic cell tumor vaccine in an experimental model.

CpG motifs have been advanced as agents that stimulate the maturation of DCs for immunotherapy. The present study tested the hypothesis that multiple doses of CpG-matured DC vaccine would be efficacious for complete eradication of experimentally-induced tumor. Accordingly, WEHI164 cells were implanted subcutaneously in the flank of BALB/c mice. During DC culture, tumor lysate was added to immature DCs followed by addition of CpG or non-CpG control 4-6h later. A total of three doses of CpG or non-CpG control-matured DCs were injected around tumors. The results showed that multiple doses of CpG-matured DCs led to considerable decrease in cytotoxicity of lymphocytes and significantly increased tumor growth rate compared to a single dose. Further, mice which received three doses of the vaccine also displayed significant FoxP3 in tumor tissue. In conclusion, multiple doses of CpG-matured DCs exhibited decreased anti-tumor immunity in association with increased expression of FoxP3.