rbioapi: user-friendly R interface to biologic web services' API.

SUMMARY: Many packages serve as an interface between R language and the Application Programming Interface (API) of databases and web services. There is usually a 'one-package to one-service' correspondence, which poses challenges such as consistency to the users and scalability to the developers. This, among other issues, has motivated us to develop a package as a framework to facilitate the implementation of API resources in the R language. This R package, rbioapi, is a consistent, user-friendly and scalable interface to biological and medical databases and web services. To date, rbioapi fully supports Enrichr, JASPAR, miEAA, PANTHER, Reactome, STRING and UniProt. We aim to expand this list by collaborations and contributions and gradually make rbioapi as comprehensive as possible. AVAILABILITY AND IMPLEMENTATION: rbioapi is deposited in CRAN under the https://cran.r-project.org/package=rbioapi address. The source code is publicly available in a GitHub repository at https://github.com/moosa-r/rbioapi/. Also, the documentation website is available at https://rbioapi.moosa-r.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Correlation of serum microRNA-122 level with the levels of Alanine aminotransferase and HBV-DNA in Chronic HBV-infected patients.

Background: The microRNA-122 (miR-122) is a liver-specific microRNA that can be used as a potential molecular marker for predicting liver injury. There is a positive correlation between miR-122 level in serum and hepatitis B virus (HBV) replication in patients infected with this virus. The present study was conducted to study the clinical importance and expression of miR-122 in asymptomatic and symptomatic patients with HBV infection in comparison to the healthy group. Methods: This study was performed on 60 samples to examine the presence of HBsAg and total HBc antibody (IgM-IgG) using an enzyme-linked immunosorbent assay. HBV-DNA extraction and real time polymerase chain reaction (PCR) assay were performed on all samples via the Real ART HBV LC PCR kit on a LightCycler instrument. RNA was extracted from the serum of all participants. Next, miRNA expression was assessed using quantitative real time reverse-transcription PCR. Also, ALT levels were measured as a surrogate parameter for liver injury using Pars Azmoon Biochemical assay Kit on Hitachi autoanalyzer. The Levene, Kruskal Wallis, Mann-Whitney and Spearman's correlation tests were used for assessing the differences between the studied groups. Results: Based on the obtained results, miR-122 expression in patients with HBV without clinical symptoms was 1.6 times, while in patients with clinical symptoms was 2.7 times more than the control group (p=0.001). A significant increase was observed in the ALT enzyme of symptomatic patients (p=0.001). HBV DNA in the people with clinical symptoms was higher than 105 copies/mL and in the asymptomatic group was less than 103 copies/mL, suggesting a statistically significant increase in a group with clinical symptoms (p=0.001). Finally, it was found that the miR-122 serum concentration correlated with HBV DNA and serum ALT (p=0.001). Conclusion: Based on the obtained results, measuring the miR-122 levels can serve as a biomarker and an indicator of hepatitis B replication, especially in cases where ALT levels are unchanged; however, more research and more samples are needed.

Doxorubicin-induced senescence through NF-κB affected by the age of mouse mesenchymal stem cells.

The senescence is proposed as a defense mechanism against many anticancer drugs. This complication is marked by differences in cell appearance and inner structures underlying the impairment in function. In this experiment, doxorubicin-induced senescence was assessed in mesenchymal stem cells (MSCs) isolated from the bone marrow of different-aged Balb/c mice (1, 8, and 16 months old). In addition, doxorubicin kinetics in culture medium were investigated to compare the drug absorption rate by different-aged MSCs. Several methods were exerted including Sandwich ELISA for NF-κB activation, propidium iodide staining for cell cycle analysis, Flow-fluorescent in-situ hybridization (Flow-FISH) assay for telomere length measurement, and specific staining for evaluation of ß-galactosidase. Determination of doxorubicin in a medium was performed by high-performance liquid chromatography technique. Following doxorubicin exposure, cells underwent substantial telomere shortening, cell cycle arresting in G2 phase, and increased ß-galactosidase activity. Interestingly, the enhanced level of NF-κB was observed in all age groups. The highest and lowest sensitivity to telomere shortening attributed to 1- and 8-month-old MSCs, respectively. In consistent with Flow-FISH results, the ß-galactosidase activity was higher in young-aged MSCs after treatment. Statistical analysis indicated a correlation between the reduction of telomere length and cessation in G2 phase. Regarding the obtained kinetics equations, the rate of doxorubicin absorption by all aged MSCs followed the same trend. In conclusion, the changing of some elements involved in doxorubicin-induced senescence can be affected by the age of the cells significantly in young MSCs than two other age groups. Hereupon, these changing patterns can open new insights to develop anticancer therapeutic strategies.

Effect of methyl jasmonate and 3-bromopyruvate combination therapy on mice bearing the 4 T1 breast cancer cell line.

Cancer cells apply the Warburg pathway to meet their increased metabolic demands caused by their rapid growth and proliferation and also creates an acidic environment to promote cancer cell invasion. 3-bromopyruvate (3-BrP) as an anti-cancer agent disrupts glycolytic pathway. Moreover, one of the mechanism of actions of Methyl Jasmonate (MJ) is interference in glycolysis. Hence, the aim of this study was to evaluate MJ and 3-BrP interaction. MTT assay was used to determine IC50 and synergistic concentrations. Combination index was applied to evaluate the drug- drug interaction. Human tumor xenograft breast cancer mice was used to evaluate drug efficacy in vivo. Tumor size was considered as a drug efficacy criterion. In addition to drug efficacy, probable side effects of these drugs including hepatotoxicity, renal failure, immunotoxicity, and losing weight were evaluated. Serum alanine aminotransferase and aspartate aminotransferase for hepatotoxicity, serum urea and creatinine level for the possibility of renal failure and changes in body weight were measured to evaluate drug toxicity. IL10 and TGFß secretion in supernatant of isolated splenocytes from treated mice were assessed to check immunotoxicity. 3-BrP synergistically augmented the efficacy of MJ in the specific concentrations. This polytherapy was more effective than monotherapy of 3-BrP, MJ, and also surprisingly cyclophosphamide as a routine treatment for breast cancer in the tumor bearing mice. These results have been shown by decrease in tumor volume and increase of tumor growth inhibition percentage. This combination therapy didn't have any noticeable side effects on kidney, liver, and immune system and body weight.

Investigating the route of administration and efficacy of adipose tissue-derived mesenchymal stem cells and conditioned medium in type 1 diabetic mice.

Type 1 diabetes (T1D) is a chronic autoimmune disease destroying the insulin-producing beta cells. Recently, stem cell therapy has been tested to treat T1D. In the present study, we aim to investigate the effects of intraperitoneal and intravenous infusion of multipotent mesenchymal stem/stromal cells (MSCs) and MSC-conditioned medium (MSC-CM) in an experimental model of diabetes, induced by multiple injections of Streptozotocin (STZ). The adipose tissue-derived MSC and MSC-CM were isolated from C57Bl/6 male mice and characterized. Later, MSC and MSC-CM were injected intraperitoneally or intravenously into mice. The blood glucose, urinary glucose, and body weight were measured, and the percentages of CD4+ CD25+ FOXP3+ T cells as well as the levels of IFN-γ, TGF-ß, IL-4, IL-17, and IL-10 were evaluated. Our results showed that both intraperitoneal and intravenous infusions of MSC and MSC-CM could decrease the blood glucose, recover pancreatic islets, and increase the levels of insulin-producing cells. Furthermore, the percentage of CD4+ CD25+ FOXP3+ T cells was increased after intraperitoneal injection of MSC or MSC-CM and intravenous injection of MSCs. After intraperitoneal injection of the MSC and MSC-CM, the levels of inflammatory cytokines reduced, while the levels of anti-inflammatory cytokines increased. Together current data showed that although both intraperitoneal and intravenous administration had beneficial effects on T1D animal model, but intraperitoneal injection of AD-MSC and AD-MSC-CM was more effective than systemic administration.

Evaluation of effective factors in the acceptance of mobile health technology using the unified theory of acceptance and use of technology (UTAUT), case study: Blood transfusion complications in thalassemia patients.

Background: Mobile health or MHealth refers to the use of mobile phone in healthcare services to enhance the health level of people. Before using MHealth, it is necessary to study the effective factors in physicians' adoption and acceptance of technology in the field of thalassemia. Methods: This cross sectional study was conducted using the survey and correlation methods. The statistical population of the study consisted of hematologists who were selected using the convenience sampling method. In this study, 58 questionnaires along with structural equations modeling based on partial least squares were used. SPSS and SMART PLS2 were used for data analysis. P values less than 0.05 were considered as statistically significant. Results: Based on the outcomes of the model from all theories, the coefficient of variation seems to be positive and the possibility of test is lower than 5%. The results indicated that all factors introduced in the proposed model are significantly effective in MHealth technology adoption. Conclusion: In this study, using the inputs from hematologists in hospitals and clinics in Tehran, it was aimed to find the factors affecting the hematologists' decision to use mobile health technology in reducing the complications of blood transfusion in patients with thalassemia who needed blood transfusion. Thus, plans were made determine to priorities and the existing conditions to implement this new system. Also, the strengths and weaknesses of each factor were measured to improve the weaker factors. UTAUT was used to determine the acceptance factors. After reviewing the results, the use of this model is recommended to physicians.

Effective ways to retain first-time blood donors: a field-trial study.

BACKGROUND: Regular blood donors are the cornerstone of blood safety. Understanding the donors' behavior to donate blood improves blood donor retention programs. The purpose of this study is to evaluate the return rate of first-time blood donors following different interventions to identify effective ways to retain first-time donors. STUDY DESIGN AND METHODS: The study was conducted on 1356 first-time blood donors at four main blood centers in Iran. The donors were randomly assigned based on different interventions (phone calls, educational letter, emotional letter, incentive, motivational meeting, and no intervention) to six groups. The return rate of donors was defined as a second attempt to donate within 6 months after the first donation. Return rate and 95% confidence intervals (CIs) were calculated and compared among different groups. RESULTS: A total of 394 (29%) donors returned within 6 months for a second donation (95% CI, 0.26-0.31). The return rate in the emotional letter group, educational letter, phone reminder, incentives, motivational meeting, and control groups was 36% (95% CI, 0.31-0.42), 33.2% (95% CI, 0.27-0.38), 31.5% (95% CI, 0.25-0.37), 30% (95% CI, 0.22-0.38), 22% (95% CI, 0.17-0.27) and 22.1% (95% CI, 0.17-0.27), respectively. CONCLUSIONS: This study provides evidence supporting the fact that more first-time blood donors can be motivated to donate again by implementing targeted interventions. It demonstrates that emotional letters, educational letters, and phone reminders were effective in improving the return rate of first-time donors.

Implications of immunotherapy with high-dose glatiramer acetate in acute phase of spinal cord injury in rats.

Objective: Recently, many researches with different viewpoints have focused on application of immunotherapy agents in treatment of spinal cord injury (SCI) according to neuroprotective results in some neurodegenerative disease. Glatiramer acetate (GA) is the most commonly used drug for Multiple sclerosis (MS) patients that exerts an immunomodulatory effect against Myelin basic protein (MBP) antigen. Materials and methods: High-dose (2mg/kg) treatment of GA for 28 consecutive days after SCI was compared with its low-dose (0.5 mg/kg) treatment, SCI control and Sham control rat groups. Results: High-dose GA group had significantly worsened outcome in standard functional recovery evaluation test (BBB) 12 weeks after SCI compared to SCI control and low-dose GA groups, which was confirmed by augmented spinal cavity volume and reduced ventral horn motor neurons in high-dose GA group; however, there was no significant difference between low-dose GA and control SCI group. In addition, proliferation test performed on lymphocytes from spleen and lymph nodes one week after SCI showed that high-dose GA injection has more significant effect on Division Index (DI) in response to MBP stimulation compared to low-dose GA and control SCI groups, which was associated with significant increase in IFN-γ, IL-4, and IL-17A secretion. Conclusion: Along with confirmation of deleterious aspects of autoimmunity resulting from autoreactive lymphocytes against myelin antigens in SCI, this study has shown that high-dose immunotherapy using GA, especially in acute phase after SCI, overwhelms any neuroprotective effect of adoptive immune system.

The First Comprehensive Study of H-Deficient Phenotypes in Iran.

BACKGROUND: The lack of correct blood grouping practices can lead to missing of the rare Bombay Oh phenotype and subjecting patients to the risk of severe hemolytic transfusion reaction. In the absence of blood donor registry, transfusion management of patients is a challenge. We performed this study in order to estimate the prevalence of the Bombay blood group (Oh) in Iran and to determine whether consanguinity plays a role in the prevalence of Oh group. METHODS: This is a descriptive study in the Immunohematology Reference Laboratory of the Iranian Blood Transfusion Organization (IBTO) Tehran, Iran, over a period of 7 years. All donor blood samples showing blood group O and a strong initial reaction with blood group O RBC control cells were tested with anti-H lectin. Also blood samples from blood group O patients were tested with anti-H lectin if all cells on both antibody screening tests and antibody identification panels were reactive with negative auto control test. Specialized tests like adsorption/elution technique and inhibition assay for determination of secretor status were performed on Oh cases. Any history of consanguineous marriages were recorded. All variables were categorical variables, and percentage and proportions were calculated manually. RESULTS: Analysis of the results of over 7 million first-time blood donors in Iran showed that the most common ABO blood group was O, with 2,520,000 (36%) subjects. 56 Oh individuals' (donors and patients) phenotypes (0.0008%) were detected. Consanguinity was observed in 50 cases (89%). CONCLUSIONS: This study shows that the prevalence of Bombay blood group in the general population of Iran is relatively high (0.0008%) and associated with consanguineous marriage. Thus, consanguinity is still an important risk factor present.

Enhanced CD40 and ICOSL expression on dendritic cells surface improve anti-tumor immune responses; effectiveness of mRNA/chitosan nanoparticles.

Objective: To improve dendritic cells (DCs) function, we targeted DCs to over express CD40 and inducible costimulator ligand (ICOSL) costimulatory molecules along with total messenger RNA (mRNA) of tumor cells to achieve a safe and effective system for treatment of tumor. Materials and methods: We generated CD40 and ICOSL mRNA in vitro and manipulated DCs using chitosan nanoparticles and also lipofectamine transfection system then examined in vitro and in vivo. Results: Mice bone marrow derived DCs pulsed with total tumor mRNA/CD40 mRNA or ICOSL mRNA showed higher expression of DCs maturation markers (CD40, ICOSL, CD86, and MHC-II) and accelerated secretion of pro-inflammatory cytokines. Co-culture of DCs with T cells enhanced proliferation of T cells and shift toward stronger Th1 cytokine responses especially in presence of CD40 over expressed DCs. Intra-tumor administration of manipulated DCs to 4T1 tumor mice model showed delay in growth of tumor volume, trend to increase in mice survival, and stronger anti-tumor cytokines production in splenocytes of mice model (with higher efficacy of mRNA/chitosan nanoparticle system). Conclusions: Hence, we suggest that targeting intra-tumor DCs to elicit expression of CD40 and ICOSL and present broad range of tumor antigens could yield effective anti-tumor responses. In this regard, CD40 molecule manipulation trigger stronger functions, while mRNA/chitosan nanoparticles system could provide a high potent tool for targeting strategies.

The role of access to affordable and quality assured blood and blood products for achieving Universal Health Coverage (Editorial).

The theme of World Health Day 2018 is 'Universal health coverage: everyone, everywhere' under the slogan 'Health for All'. Universal Health Coverage (UHC), as pertains to blood and blood products, means that all individuals and communities have access to affordable and timely supplies of safe and quality-assured blood and blood products. Blood and blood products are essential components in the proper management of women suffering from bleeding associated with pregnancy and childbirth; children suffering from severe anaemia due to malaria and malnutrition; patients with blood and bone marrow disorders and immune deficiency conditions; victims of trauma, emergencies, disasters and accidents; and patients undergoing advanced medical and surgical procedures.

Seroprevalence and geographical distribution of human T-lymphotropic virus type 1 among volunteer blood donors in endemic areas of Iran.

BACKGROUND: Human T-cell lymphotrophic virus type 1 (HTLV-1) has a worldwide distribution and it is endemic in some regions of Iran. One of the most important routes of HTLV-1 transmission is via transfusion of contaminated blood components. The risk of transmission through asymptomatic blood donors, particularly in endemic areas should be considered and appropriately managed. The main objective of this study was to determine the seroprevalence and description the geographic distribution of HTLV-1 among voluntary blood donors in Iran. METHODS: This retrospective study carried out using the data obtained from the main database of the seven blood transfusion centers of Iranian Blood Transfusion Organization between 2009 and 2013. The presence of anti-HTLV-1/2 antibodies were primarily assessed using Enzyme-linked Immunosorbent Assay. The Ab Kit assay, contain antigens for the screening of antibodies to HTLV type 1 and 2. So, it is expressed as HTLV 1/2 assay. Samples that were positive by the western blot confirmatory test were considered as definite positive HTLV-1 or HTLV-2 cases. The main socio-demographic variables were; age, gender, donation history and marital status. Descriptive and analytical statistics were used to summarize the gathered data. The chi-Square Statistical test was used to test the association between groups, P-value of less than 0.05 was considered significant. RESULTS: A total of 1864489 blood donations were evaluated. There were 1840 confirmed HTLV-1 positive donations (0.098%). None were positive for anti-HTLV-2. The overall HTLV-1 prevalence was 98.7 per 100,000 donations during the 5 year period. Seroprevalence was higher among females, married and older blood donors. The overall seropositivity among first time, regular and lapsed donors was, 0.29% (290/100000), 0.001% (1/100000) and 0.02% (20/100000) respectively. A significant difference was observed between regular and the first time (p <0.0001) and also between lapsed and regular blood donors (p <0.0001). Most of the HTLV-1 seropositive blood donors (175 per 100,000) were from northeastern regions. We observed a gradual decline in overall HTLV-1 prevalence during the course of the study, the prevalence rate decreased from 0.13% (130/100000) in 2009 to 0.07% (70/100000) in 2013. CONCLUSIONS: The Seroprevalence of HTLV-1 among Iranian blood donors in the regions of our study still is considerable, but there is an obvious declining prevalence over the course of present study. Blood transfusion centers should continually evaluate the residual risk of infection in the country, especially in endemic areas.

MicroRNA Modulation during the In vitro Culture of Hematopoietic Stem Cells Prior to Transplantation.

BACKGROUND: Human umbilical cord blood (HUCB) is an acceptable and readily accessible source of stem cells. There is an ongoing interest in cord blood stem cell therapies; however, little is known about the possible unfavorable effects of laboratory modifications on the isolated HUCB cells. The involvement of miRNAs in several biological processes has been shown. The aim of this study was to evaluate the possible changes in miRNA expression profiles in CD133+ hematopoietic cells after in vitro culture. METHODS: HUCBCD133+ hematopoietic stem cells were isolated by magnetic-activated cell sorting, and then the cells were counted using flow cytometry. The cells were divided into 2 groups. In the first group, RNA was extracted and the cells of the second group were cultured in vitro for 12 days and then these cells were used to assay miRNAs expression using real-time qPCR. RESULTS: The results showed that the expression of 349 out of 1,151 screened miRNAs was upregulated following a 12-day in vitro culture of CD133+ cells, whereas the expression of 293 miRNAs was downregulated. In addition, the expression of 509 miRNAs was not significantly altered. Another in-silico analysis involving the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to the selected miRNAs was also conducted. CONCLUSION: Based on our results, the in vitro expansion of HUCB resulted in altered expression levels of miRNAs. This study provides information on the effects of 2-dimensional culture of hematopoietic cells prior to transplantation for more successful transplantation.

Evaluation of age effects on doxorubicin-induced toxicity in mesenchymal stem cells.

Background: Doxorubicin, by aggregating in bone marrow, causes genotoxic effects, and thus reduces the repair ability of cells. The present study was conducted as an in vitro evaluation of age effects on the cytotoxicity induced by doxorubicin in mesenchymal stem cells (MSCs). Methods: The MSCs of female BALB/c mice aged 1, 8, and 16 months were separated, characterized, and subsequently evaluated in cellular growth media. After 24 hours, exposure of the MSCs of the 3 groups of mice to doxorubicin (25, 50, 100, 200, 400, 800, 1200 nM) and cytotoxicity were assessed, and the sublethal dose was determined using flow cytometry technique and lactate dehydrogenase (LDH) release assay. Results: The IC50 values determined by flow cytometry for the separated MSCs of 1 young, 8 middle- aged, and 16 old mice were and respectively. Interestingly, the results of these 2 methods in determining cytotoxicity were in agreement, and a concentration of approximately 25 nM was considered to be the shared sublethal dose for different ages. Conclusion: The results indicated that MSCs of middle-aged mice were more resistant to the toxic effects of the drug. Besides, MSCs separated from the old mice were the most sensitive to chemotherapy and its side effects such as disruptions of cell proliferation and viability. These disruptions can be ascribed to the alteration of function and physiological processes with age. Determining proper concentration of doxorubicin drug to destruct cancerous cells based on age and individual sensitivity can minimize the amount of toxicity.

Perioperative changes in platelet count and function in patients undergoing cardiac surgery.

Background: Patients undergoing cardiac surgery are at increased risk of bleeding due to multifactorial coagulopathies. In the present study, we aimed at investigating the changes in platelet count and function during and after surgery as well as determining the association of the platelet dysfunction with bleeding and transfusion requirements in these patients. Methods: A total of 40 adult patients scheduled for elective valve coronary cardiac surgery were included in this prospective observational study. Changes in platelet count and function with ADP, acid arachidonic, and collagen (light transmission aggregometry) were analyzed at three time points: before CPB, after CPB, and 24 hours after end of surgery. Postoperative bleeding and intraoperative transfusion requirements were recorded. Results: There were a significant reverse correlation between CPB time and ADP-induced aggregation, particularly after CPB and postoperative AA-induced aggregation. There was not any significant correlation between platelet count and function at all-time points. Both platelet count and platelet aggregation significantly reduced during CPB. While platelet aggregation increased on postoperative Day 1, platelet count reduced by about 40% after CPB, and remained at this level postoperatively. Patients with abnormal ADP-induced aggregation had significant increased postoperative bleeding and transfusion requirements. Conclusion: The results of this study demonstrate that platelet count and platelet aggregation are reduced during CPB. Our results emphasized the effect of platelet dysfunction on increased postoperative bleeding and transfusion requirements. Perioperative monitoring of platelet function can be considered as a bleeding management strategy for implantation of PBM programs.

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.

Key role of Dkk3 protein in inhibition of cancer cell proliferation: An in silico identification.

Dkk3 is a member of Dkk family proteins, regulating Wnt signaling. Dkk3 plays different roles in human and mouse tumors. Dkk3 predominantly act as a tumor suppressor, however several reports revealed that Dkk3 could accelerate cancer cell proliferation. Herein, we aimed at launching an in silico study to determine Dkk3 structure and its interactions with Kremen and LRP as Wnt signaling receptors as well as EGF receptor. Using various softwares a model was built for Dkk3 molecule. Different protein modeling approaches along with model refinement processes were employed to arrive at the final model. To achieve the final complex of Dkk3 with Kremen, LRP and EGFR molecules protein-protein docking servers were employed. Model assessment softwares indicated the high quality of the finally refined Dkk3 3D structure, indicating the accuracy of modeling and refinement process. Our results revealed that Dkk3 is capable of interacting with Kremen, LRP and EGFR with comparable binding energies. Dkk3 efficiently interacts with LRP, Kremen and EGF receptor and may be a promising protein in cancer therapy by blocking Wnt and EGFR downstream signaling.

Increasing proliferation of murine adipose tissue-derived mesenchymal stem cells by TNF-α plus IFN-γ.

The objective of the current study was to assess the effects of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) along with their simultaneous application on proliferation and pluripotency genes of murine adipose tissue-derived mesenchymal stem cells (AT-MSCs). The proliferation, doubling time (DT), colony-forming unit-fibroblast (CFU-F), pluripotency genes expression, and proliferation-related immunomodulatory markers of MSCs were analyzed upon activation with TNF-α (10 ng/ml), IFN-γ (10 ng/ml) and both TNF-α and IFN-γ (5 ng/ml + 5 ng/ml). Pluripotency genes including Oct-4, Sox-2, and Nanog as well as proliferation-associated immunomodulatory cytokines such as insulin-like growth factor 1 (IGF-1) and transforming growth factor-ß (TGF-ß) expression were evaluated using real-time PCR. Surface expression of Qa2 (HLA-G) was analyzed by flow cytometry. Pretreatment of MSCs with TNF-α plus IFN-γ led to significantly increased proliferation, DT and CFU-F as well as expression of pluripotency genes in AT-MSCs (p < 0.01). MSCs expressed more IGF-1, TGF-ß, and Qa2 upon activation with TNF-α plus IFN-γ and IFN-γ. MSCs expressed significantly decreased amounts of TGF-ß and Qa2 in presence of TNF-α. TNF-α combined with IFN-γ may be improved the proliferation of AT-MSCs. Conversely, expanded MSCs pointed out low levels of the immunomodulatory marker, s especially Qa2 in the presence of TNF-α. In conclusion, we showed that TNF-α together with IFN-γ increased the proliferation of MSCs and slightly enhanced the expression of pluripotency genes.

Microvesicles preparation from mesenchymal stem cells.

BACKGROUND: Extracellular vesicles are particles ranged from 30 nm to 5µm and subcategorized into three groups; exosomes, microvesicles and apoptotic bodies, each of which have different biological impact. Lack of a standard method for the detection and isolation of MVs has led to a challenging issue that is a worth considering. In this study, we isolated MVs from the conditioned medium of UC-MSCs by four different schemes of ultracentrifugation. METHODS: We examined the efficacy of differential centrifugation ranging from 10,000×g to 60,000×g on UCMSCs- derived microvesicles yield and purity. The fractions were evaluated by Dynamic Light Scattering (DLS) method, total protein quantification and flow cytometry. RESULTS: UC-MSCs were spindle cells that adhered to plastic culture flasks. These cells expressed MSC markers such as CD44 and CD73, whereas were negative for hematopoietic markers CD45 and CD34. UC-MSCparticles were successfully isolated. Particles were heterogeneous vesicles of approximately 50 to 1250 nm in diameter that bear the surface-expressed molecules UC-MSCs such as; CD90, CD106, CD166 and CD44, and negative for CD34, CD63, and CD9. According to the results of DLS method, centrifugation at 10,000, 20,000, 40,000 and 60,000 ×g, all gave MVs of less than 1000 nm. It is of notion that only at the centrifugation rates of 40,000 and 60,000×g, particles of less than 100 nm in diameter were also obtained. CONCLUSION: The choice of exact speed greatly influences the purity of MVs and their yield. Our findings indicate that centrifugation at 20,000×g is appropriate for the purification of UC-MSC-MVs.