Exosomes derived from rapamycin-treated 4T1 breast cancer cells induced polarization of macrophages to M1 phenotype.

M2 macrophages are the most prevalent type in the tumor microenvironment and their polarization to M1 type can be used as a potential cancer immunotherapy. Here, we investigated the role of tumor microenvironment and particularly purified exosomes in M2 to M1 macrophage polarization. Rapamycin treatment on triple-negative breast cancer cells (TNBC) was performed. Tumor cells-derived exosomes (called texosomes) were isolated and characterized using scanning electron microscopy, transmission electron microscopy, dynamic light scattering, high-performance liquid chromatography, Fourier transform infrared, and Western blot assays. M2 mouse peritoneal macrophages were treated with rapamycin or rapamycin-texosome. Then, M1/M2 phenotype-specific marker genes and proteins were measured to assess the degree of M2 to M1 polarization. Finally, nitric oxide (NO) production, phagocytosis, and efferocytosis assays were assessed to verify the functionality of the polarized macrophages. Purified rapamycin-texosomes significantly increased the expression of the M1 markers (Irf5, Nos2, and CD86) and decreased M2 markers (Arg, Ym1, and CD206). In addition, the levels of M1-specific cytokines tumor necrosis factor alpha and interleukin 1ß (IL-1ß) were increased, whereas the levels of M2 specific cytokines IL-10 and transforming growth factor beta were declined. Furthermore, texosome treatment increased NO concentration and phagocytosis and decreased efferocytosis indicating M1 polarization. These findings suggest rapamycin-texosomes can induce M2 to M1 macrophages polarization as a potential immunotherapy for TNBC.

MicroRNA-122 Is More Effective than Rapamycin in Inhibition of Epithelial-mesenchymal Transition and mTOR Signaling Pathway in Triple Negative Breast Cancer.

The fundamental mechanism responsible for the aggressiveness of metastatic cancers such as triple-negative breast cancer (TNBC) is the epithelial-mesenchymal transition (EMT). In cancer microenvironments, the Phosphoinositide 3-kinases (PI3K)-Akt- mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in regulating the EMT mechanism. The current study focuses on the impacts of rapamycin, a newly retargeted chemotherapeutic agent against mTOR, and MicroRNA (miR)-122 on the aggressive behavior of TNBC.  The half-maximal inhibitory concentration (IC50) of rapamycin on 4T1 cells was determined using an MTT assay. Also, miR-122 was transiently transfected into 4T1 cells to study its effect on the pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to assess the expression level of central mTOR and EMT-related cascade genes. Moreover, cell mobility and migration were evaluated using scratch and migration assays, respectively. Both rapamycin and miR-122 significantly decreased the expression levels of PI3K, AKT, and mTOR, as well as ZeB1 and Snail genes. However, no significant change was observed in Twist gene expression. Furthermore, scratch and migration assays revealed that the migration of 4T1 cells was markedly reduced, especially following miR-122 induction. Our experimental findings and gene enrichment studies indicated that miR-122 mainly operates on multiple metabolic pathways, as well as EMT and mTOR, while rapamycin has restricted targets in cancer cells.  Consequently, miR-122 can be considered a potential cancer microRNA therapy option, which can be validated in the future in animal studies to demonstrate its efficacy in cancer control.

Effect of Calcitriol Treated Mesenchymal Stem Cells as an Immunomodulation Micro-environment on Allergic Asthma in a Mouse Model.

BACKGROUND: Allergic asthma is a chronic inflammatory illness of the respiratory system characterized by an increase in the number of inflammatory cells in the airways and trouble breathing. Mesenchymal stem cells (MSCs) have the potential to be used in inflammatory diseases as a cellular immunosuppressive treatment. They express calcitriol receptors and communicate with other immunocytes, which increases their anti-inflammatory activity. This study aimed to determine the effects of calcitriol-treated MSC treatment on allergic asthma pathways in a mouse model. METHODS: To generate a mouse model of asthma, the mice were sensitized intraperitoneally with ovalbumin (OVA) and aluminum hydroxide emulsion and then challenged intra-nasally with OVA. On day 14, experimental mice received tail vein injections of calcitriol-treated MSCs in PBS prior to allergen exposure. The cytokines assays including IL-4, 10, 12, 17, TGF-ß and IFN-γ, splenocytes proliferation, and histological examination of lungs samples were performed. The mice were sensitized with OVA and the response to dexamethasone treatment was compared. RESULTS: Calcitriol-treated MSCs significantly increased the levels of IL-12, TGF-ß, and IFN-γ compared to non-treated MSCs groups. Moreover, calcitriol-treated and non-treated MSCs significantly decreased IL-4 and IL-17 compared to asthmatic groups. The results of the histopathological examination showed that calcitriol-treated MSCs reduced the accumulation of inflammatory cells and bronchial wall thickening in comparison with the asthma group. CONCLUSION: Using the allergic asthma model, we were able to show that calcitriol-treated MSCs had an inhibitory impact on airway inflammation. Our findings suggest that the injection of calcitrioltreated MSCs may be a viable treatment option for allergic asthma.

Impact of the covid-19 pandemic on the psychological status and cortisol level of multiple sclerosis patients.

This study aimed to compare the changes in psychological status and cortisol level between multiple sclerosis (MS) patients and a healthy control group (HC). One hundred and fifty-five MS patients and 165 HC subjects had completed questionnaires consisting of 36-Item short health survey (SF-36), Hamilton Anxiety Rating Scale (HAM-A), Beck Depression Inventory-II (BDI-II), and fatigue severity score (FSS) before and after (one year from onset) COVID-19 pandemic. The salivary cortisol level was also measured again in 26 MS patients and 14 control individuals. MS patients had lower scores of mental and physical components of quality of life (MCS and PCS), but higher HAM-A, FSS, and BDII scores than HC Before and after COVID-19. There were significant changes in scores of MCS, BDI-II, HAM-A, and FSS after the COVID-19 outbreak in MS patients, but not in PCS score. In HC group, we observed significant changes in scores of MCS, BDI-II, and FSS, but not in scores of PCS and HAM-A. Compared to HC, the MS patients reported greater deterioration in the overall mental health component of their health-related quality of life, and their levels of anxiety and fatigue over the study period. The change of cortisol levels was non-significant with a small effect size.

A comparative study of laboratory findings in PCR-positive and PCR-negative COVID-19 hospitalized patients.

INTRODUCTION: Given the many misconceptions in terms of both diagnosis and treatment, SARS-CoV-2 continues to infect and victimize. Notwithstanding molecular testing is the gold standard method of in vitro diagnostic, the often long-waiting time, as well as false-negative results are daunting challenges facing us. In this study, we aimed to report the diagnostic value of laboratory findings in COVID-19 patients, with an extensive focus on the differences between PCR-positive and PCR-negative cases. PATIENTS AND METHODS: We did a retrospective single-centre study on a large cohort of 1546 COVID-19 patients in Tehran, Iran. Based on clinical symptoms, chest CTs were performed for all the patients. Also, molecular testing of swab specimens was also performed for 1450 cases. RESULTS: All the data on laboratory results were retrospectively extracted from medical records. Of the 1546 patients, 1040 (67.5%) were male and 506 (32.5%) were female with the mean age of 55.67. On admission, 31.4% of the whole study population displayed lymphopenia and 38.9% showed neutrophilia. Decreased hemoglobin and mild thrombocytopenia were also found in 40% and 18.6% of cases, respectively. Elevated lactate dehydrogenase in nearly 75% of COVID-19 cases was the most common alteration amongst biochemical parameters which together with increased ESR and CRP could serve as diagnostic markers in SARS-CoV-2 infection. Of the 1450 patients with a PCR result, 439 (28.3%) were PCR-negative and 1011 (65.3%) were PCR-positive. Notably, lymphopenia and increased AST were higher in the PCR-positive group than their negative counterparts. Albeit being in the normal range, a significant decrease in the number of monocytes was also evident in the PCR-positive cases. CONCLUSIONS: As far we are aware, this is the first time that we reported a comprehensive exploration of laboratory characteristics of a large cohort of hospitalized COVID-19 patients from Iran, hoping that these data will cast more light on the diagnostic significance of these parameters.

An evaluation of the effects of Pistacia atlantica gum hydro-alcoholic extract on the phagocytosis ability of macrophages and atherosclerosis development in hypercholesteremic rats.

BACKGROUND: Atherosclerosis is an inflammatory disease that various factors affect the onset and its progression, including free radicals, hypertension, diabetes, genetic changes, hypercholesterolemia, and even some microorganisms such as herpes viruses and chlamydia. Therefore, compounds that can be effective in any of the above cases may be considered as a useful therapeutic agent in the process of atherosclerosis. The aim of the present study was to evaluate the effects of Pistacia atlantica gum hydro-alcoholic extract on macrophage phagocytosis ability and development of atherosclerosis in hypercholesterolemic rats. METHODS: The statistical population of the present study consisted of 25 rats that were randomly divided into 5 groups (one control group under standard diet, 4 treatment groups under high-fat diet). After consumption of high-fat food for 45 days, the treatment groups orally received 100, 200, and 400 mg/kg of Pistacia atlantica gum hydro-alcoholic extract for 30 days. Then, peritoneal macrophages were isolated and blood samples were collected to measure the level of nitroblue tetrazolium (NBT), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG). P ˂ 0.05 was considered significant in all evaluations. RESULTS: The level of cholesterol (503.66 ± 17.15), TG (436.66 ± 16.80), LDL-C (343.66 ± 11.59), HDL-C (54.33 ± 7.02), and NBT (0.64 ± 0.02) decreased in the treatment groups. Besides, exactly in a concentration-dependent manner, plant extract significantly reduced the level of respiratory potential level in macrophages. CONCLUSION: Hydro-alcoholic extract of Pistacia atlantica gum could effectively decrease hypercholesterolemia and increase phagocytic ability of macrophages. Therefore, it can be suggested for more investigation as a blockage of atherosclerosis.

Biological properties the novel application of N-trimethyl chitosan nanospheres as a stabilizer and preservative in tetanus vaccine.

PURPOSE: Chitosan is a natural polymer that has excellent properties include biocompatibility, biodegradability, no cytotoxicity, high charge density, low cost, mucoadhesive, permeation enhancing (ability to cross tight junction), and immunomodulating ability that makes the spectrum of its applicability much broader. This study was conducted to investigate the stabilizing, preservative and immunogenicity properties of N-trimethyl chitosan nanospheres (N-TMCNS). MATERIALS AND METHODS: The tetanus toxoid (TT) was encapsulated into N-TMCNS and then characterized by scanning electron microscope, atomic force microscope, and dynamic light scattering. For stabilizer assay of N-TMCNS after storage of TT-N-TMCNS at different temperatures for 3 weeks, they were used for immunization of mice and different temperatures groups' anti-TT-N-TMCNS production compared with other groups. Finally, the immunized mice were challenged with tetanus toxin. The preservation activity of TT-N-TMCNS against Escherichia coli was compared with thimerosal formulated TT. RESULTS: Our results revealed that heat-treated TT-N-TMCNS could induce higher titer of neutralizing immunoglobulin G in compared to TT vaccine and was able to protect the mice better than TT vaccine in challenge test. Furthermore, N-TMCNS as a preservative inhibited the growth of E. coli more effective than thimerosal. CONCLUSION: Overall, the obtained results indicated that the N-TMCNS is one of the best stabilizer and preservative agent that can be used in the formulation of TT vaccine.

Dynamic changes of epigenetic signatures during chondrogenic and adipogenic differentiation of mesenchymal stem cells.

Extensive studies have been performed to clarify the processes during which mesenchymal stem cells (MSCs) differentiate into their lineage fates. In vitro differentiation of MSCs into distinct lineages have attracted the focus of a large number of clinical investigations. Although the gene expression profiling during differentiation of MSC toward bone, cartilage, and adipocytes is well established, the master regulators by which MSC fate can be controlled are not entirely determined. During differentiation of MSCs into a special cell fate, epigenetic mechanisms considered as the primary mediators that suppress the irrelevant genes and activate the genes required for a specific cell lineage. This review dedicated to addressing the changes of various epigenetic mechanisms, including DNA methylation, histone modifications, and micro-RNAs during chondrogenic and adipogenic differentiation of MSC.

Development of T follicular helper cells and their role in disease and immune system.

The T follicular helper cells (TFH) are a subset of CD4+ T cells specialized to regulate antibody responses. The production of these cells is associated with the dendritic cells (DCs) and B cells. TFH cells help B cells form germinal centers (GC) differentiate into memory and plasma cells (antibody-secreting cells) as humoral responses. In addition, there is strong evidence that TFH cells play a pivotal role in the development of long-lived humoral immunity. Molecular factors such as transcription factors, surface receptors, cytokine and micro RNAs are involved in the formation of TFH cells. Such TFH cells are diagnosed by transcription factor (BCL-6), surface marker expression (including CXCR5, PD-1, ICOS and CD40L) and a unique cytokine production pattern (such as IL-21 and IL-6). Memory TFH cells, accompanied by memory B cells, are known to be formed during antibody responses. It is now clear that the precise control of TFH cells is critically important for both inducing the optimal affinity maturation of antibody responses and preventing self-reactivity. Exclusive controls of TFH cell function and production are essential for human health. However, it is important to note that excessive activities may lead to autoimmune diseases, while reduced activity often results in immunodeficiency. It has also been shown that TFH cells are associated with cancers such as angioimmunoblastic T-cell lymphoma (AITL), follicular T-cell lymphoma (FTCL) and nonspecific Peripheral T-cell lymphomas (PTCLs). The biology of TFH cells, including their differentiation and transcriptional regulation will be described in the present review. Some of The developments of these cells in immunodeficiency diseases, autoimmunity and cancer will also be taken into account.

Synergistic Effect of Expressed miR-128 and Puma Protein on Targeted Induction of Tumor Cell Apoptosis.

BACKGROUND: Puma is a highly robust pro-apoptotic protein. The protein becomes activated by p53 ensuing beyond-repair DNA damage. Downregulation of SIRT 1, by miR-128, elevates activated p53 that foment Puma indirectly. OBJECTIVES: In the present study, we used two-expression Adeno-Associated Virus (AAV) system for co-expression of miR-128 and Puma in order to evaluate apoptotic response; both in the tumor and normal cells, respectively. MATERIALS AND METHODS: Three recombinant AAVs constructs were generated. The First rAAV bearing Puma under the control of hTERT (p-AAV), the second construct designed such that to carry miR-128 downstream of CMV (mi-AAV), and the last construct comprises of the both CMV-miR-128 and hTERT- Puma. Real-Time PCR and western blotting were used to evaluate expression levels of the transduced genes. RESULTS: MTT assay and DAPI staining shown suicidal effect of each recombinant AAV vectors. p-AAV cytotoxicity was recorded for 62% of the tumor cells, while for normal cells it was only 20% cytotoxic. The second construct, mi-AAV, was not as potent and selective as p-AAV. This construct was shown to be 27% and 16% cytotoxic for BT-474 and HEK-293 cells, respectively. Co-expression of Puma and miR-128 (p-mi-AAV) was accomplished with a selective cytotoxicity toward BT-474. This construct was 85% toxic for tumor cells, although it was only 25% toxic for the normal cell line (HEK-293). CONCLUSIONS: In this study, we have shown that not only Puma is able to instigate apoptotic response but also its co-expression along with miR-128 could significantly enhance apoptosis in a synergistic manner.