فهرست مطالب ahmad zavaran hosseini

Pathogen recognition receptors (PRRs), which play a crucial role in responding to pathogens, affect the function of mesenchymal stem cells (MSCs). One important group of PRRs is the toll-like receptors (TLRs). When PRRs are activated, they can alter the expression of specific surface markers, the ability of MSCs to differentiate, and the types of substances they secrete. These modifications in MSC function may have unexpected consequences for patients. In this study, we examined how Leishmania major (L. major) promastigotes affect the properties of MSCs. MSCs were isolated from adipose tissue and categorized into two groups: one group left untreated and the other group exposed to L. major. Giemsa staining was employed to accurately quantify the number of parasites that entered the cells. After 72 hours, real-time polymerase chain reaction was utilized to assess the expression of TLRs. Additionally, the flow cytometry technique was used to evaluate the expression of surface markers on the MSCs. Our results showed that MSCs can engulf parasites and increase the expression of TLR4 and TLR6. The pro-inflammatory cytokine increased, and the transforming growth factor-β decreased significantly. The parasite exposure increased reactive oxygen species production. Additionally, the percentage of cluster differentiation (CD) 73 decreased, and the mean fluorescent index of CD29 and CD73 was down-regulated by L. major. Exposure to parasites diminishes the immunomodulatory capacity of MSCs. This discovery holds significance for the application of MSCs in addressing parasite infections and underscores the need for additional research to enhance their therapeutic effectiveness.

Induction of a protective immune response against Leishmania major requires the activation of both TH1 and CD8+ T lymphocytes. Because L. major is an intra-phagosomal parasite, its antigens do not have access to MHC-I. The present study aimed to evaluate the effect of cysteine peptidase A (CPA)/cysteine peptidase B (CPB) conjugated to α-AL2O3 on autophagy induction in L. major infected macrophages and subsequent activation of cytotoxic CD8+ T lymphocytes.

Recombinant CPA and CPB of L. major were produced in expression vectors and purified. Aldehyde functionalized α-AL2O3 were conjugated to hydrazine-modified CPA/CPB by a chemical bond was confirmed by Fourier-transform infrared spectroscopy (FTIR). The High efficient internalization of α-AL2O3 conjugated CPA/CPB to macrophages was confirmed using a fluorescence microscope and flowcytometry. Induction of the acidic autophagosome and LC3 conversion in macrophages was determined by acridine orange (AO) staining and western blot. Autophagy-activated macrophages were used for CD8+ T cell priming. Cytotoxic activity of the primed CD8+ T cell against L. major infected macrophages was measured using apoptosis assay.

α-AL2O3 conjugated CPA/CPB enhances macrophages antigen uptake and increases acidic vacuole formation and LC-3I to LC-3II conversion. Co-culture of autophagy-activated macrophages with CD8+ T cells augmented CD8+ T cells priming and proliferation more than in other study groups. These primed CD8+ T cells induce significant apoptotic death of L. major infected macrophages compared with non-primed CD8+ T cells.

α-AL2O3 nanoparticles enhance the cross-presentation of L. major antigens to CD8+ T cells by inducing autophagy. This finding supports the positive role of autophagy and encourages the use of α-AL2O3 in vaccine design.

Fibroblast-like synoviocytes (FLSs) play a major role in the pathogenesis of rheumatoid arthritis (RA). Endoplasmic reticulum (ER) stress and dysregulation of unfolded protein response are involved in the resistance to apoptosis of FLSs in RA (RA-FLSs). MicroRNA (MiR)-211 plays an important role in controlling ER stress and apoptotic genes in a PKR-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-dependent manner. We investigated the effect of miR-211-5p overexpression on ER stress and apoptotic genes in RA-FLSs. FLSs were isolated from synovial tissues of trauma (n=10) and RA (n=10) patients. MiR-211-5p and mRNA expression of the selected genes involved in the PERK pathway and apoptosis regulation were measured in RA, trauma, and thapsigargin (Tg)-treated RA-FLSs. Afterward, Tg-treated RA-FLSs following miR-211-5p overexpression were evaluated for miR-211-5p and mRNA levels of the study genes. The expression of miR-211-5p, PERK, BAX, and BCL2 showed no differences between RA and trauma. However, the expression of ATF4 and BCL-XL showed a significant increase in trauma. In addition, the levels of C/EBP homologous protein (CHOP) and MCL1 indicated a significant increase in RA-FLSs. Tg treatment significantly increased the expression of PERK, ATF4, and CHOP in RA-FLSs with no effect on miR-211-5p, BAX, BCL2, BCL-XL, and MCL1. Furthermore, Tg treatment following miR-211-5p overexpression in RA-FLSs showed a significant increase in levels of miR-211-5p with no changes in apoptotic genes. MiR-211-5p overexpression in stimulated RA-FLSs did not alter the levels of selected genes involved in apoptosis regulation. However, more investigations are necessary to determine the ER stress role in apoptosis regulation in RA-FLSs.

T-lymphocytes have critical functions in the immune responses against viral and intracellular bacterial infections as well as cancers. Antigen (Ag)-specific T-lymphocyte clones enriched and expanded in vitro are valuable tools in the study of immune responses in animal models and adoptive T-cell therapy of patients with cancer or infection. We described a method for inducing, enriching, and replicating Ag-specific poly-clonal T-cells from BALB/c mice infected with live Bacillus Calmette Guérin (BCG) bacterium. During a 7-8 days procedure, T-lymphocytes were purified from immune cells of lymph nodes stimulated with immunodominant Ag of BCG, TB10.4, and expanded by interleukin -2 cytokine. We evaluated the effect of Ag doses (1, 10, and 100 μg/mL) and exposure method of Ag presenting cells (APCs) to T-cells, on T-cells’ proliferation, viability, and Interferon-gamma (IFN-γ) secretion at 2, 5, and 7 days after Ag stimulation. Increasing Ag concentration increased the average cell division, but at the highest dose of Ag (100 μg/mL), T-cell viability is decreased. Only clones induced by 10 μg/mL Ag produced a desirable amount of IFN-γ. Incubation of Ag and APCs, 24 h before T-lymphocytes addition, increased the proliferation and viability of cells. T cells are in a more favorable condition around day 5 of Ag stimulation in terms of proliferation and survival, and it is the desired time for T cell restimulation. For optimal preparation of specific T-cells for adoptive cell transfer, optimization of Ag dose, the order of APCs and T-cells exposure with Ag, and the duration of initial Ag stimulation, as well as the time for restimulation, is essential.

Exosomes are extracellular vesicles that are involved in intracellular communication and different biological processes. Recently, the importance of microRNAs (miRNAs) in exosomes has been considered as biomarkers in asthma diagnosis. This study aimed to determine the expression of selective miRNAs from plasma-derived exosomes in moderate and severe asthmatic patients compared with healthy controls. Forty-six subjects including 22 patients with severe and mild to moderate allergic asthma and 24 healthy controls have entered this study. MiRNAs were extracted from the plasma exosomes and selective miRNAs (miR-21, miR-16, miR-Let7, miR-148a, miR-155, miR-125, miR-150, miR-146a, miR-223, miR-126) expressions levels were determined; using quantitative polymerase chain reaction (qPCR). In this study, we found a significant up-regulation of miR-223 and miR-21 in moderate asthmatic patients compared to the healthy controls (p=0.002, p=0.006). MiR-223 and miR-21 had the probability of 83% and 76% diagnosis estimation in moderate asthmatic patients respectively. Therefore, they could be used as biomarkers in these patients.  No expression of miR-125, miR-126, and miR-155 was found in plasma exosomes by qPCR in this study. The other miRNAs had no significant expression between different groups. Based on our findings,miR-223 and miR-21 may be considered biomarkers or used for targeted immunotherapies in asthma.

Sepsis is a systemic inflammatory disease in response to the pathogens that leads to vital organ failures the failure of vital organs. Appropriate animal models should be developed to measure the effectiveness of therapeutic methods. Cecal Ligation and Puncture (CLP) is the most widely used methods of creating the sepsis model. Some variables interfere in the creation of the CLP model which terminated to result in an unrepeatable dynamic of the inflammatory responses. The current research, suggests presents the simultaneous study of inflammatory responses in serum and liver as a criterion for determining the inflammatory status of the CLP model. CLP model was induced in 15 female C57bl/6 mice. IL-6, TNF-α, IL-10, and TGF-β1 cytokines levels were measured at 24, 48, and 72 hours after CLP induction in both serum and liver tissue by ELISA method. Serum levels of liver enzymes were analyzed by the clinical chemistry analyzer. All studies were performed in healthy mice as well. The results were reported as Mean±SD. The levels of IL-10 and TGF- β1 in the liver is were significantly (P≤0.05) higher than serum. The production of IL-10 and TGF- β1 in the serum and liver reaches its maximum at peaked 24 and 72 hours after CLP induction. The level of TNF-α in the liver is was significantly (P≤0.05) higher than serum with a maximum production 24 hours after CLP induction. Serum is not a good representative of the inflammatory condition in sepsis. Therefore, it is suggested that local inflammatory responses be considered in evaluating the model, and the determination of drug efficacy.

Leishmaniasis is of public health problems, especially in endemic areas. The activation of macrophages, as the main host of leishmania and promotion of the TH1 immune responses, are the main goal of im-munotherapy methods. Recently, the immunomodulatory role of mesenchymal stem cells (MSCs) in infectious disease has been considered. Different in vitro studies demonstrated the immunostimulatory effect of MSCs on macrophages in response to L.major. In this study, the effect of MSCs on cutaneous leishmaniasis in BALB/c mice was assessed.

To do this experimental research, BALB/c mice infected with L. major that was followed by multiple subcutaneous injections of MSCs at infection site at different intervals. Footpad thickness, spleen parasite burden, lymph node, and spleen cytokine production were measured to determine the efficacy of cell therapy.

Significant (P<0.05) reduction in footpad thickness and delayed wound formation was observed in MSCs treated group. The spleen of the MSCs-treated group indicated a two-fold reduction in parasite burden compared with non-treated infected mice. In addition, nitric oxide (NO), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α) production of lymph node isolated cells and splenocytes changed to the benefit of macrophage activation in response to L. major in MSCs treated group. A two-fold increase in interferon-gamma (IFN-γ) production in the lymph node was determined in the MSCs-treated group.

Although MSCs therapy could not clear the parasite, the results confirm the ability of MSCs to enhance immune responses against leishmania by induction of inflammatory responses and slowing down the spread of parasites. However, further studies needed to improve the efficacy of this method and provide a therapeutic protocol.

Mesenchymal stem cells (MSCs)-derived exosomes can function similar to MSCs in repairing damaged tissues and modulating immune responses. They are considered as an effective tool in regenerative medicine as well as autoimmune diseases and cancer.

MSCs were isolated from adipose tissue of mice, and characterized. Cell supernatant was used for extraction of exosomes using ultracentrifugation with 110000 g and polyethylene glycol (PEG). Dynamic light scattering (DLS) technique, transmission electron microscopy, and Bradford assay were used to evaluate the accuracy of the isolated exosomes.

The results of isolation of exosomes derived from MSCs using PEG showed that most of the extracted spherical exosomes were in the range of 50-300 nm; but the results of isolation of exosomes derived from MSCs using ultracentrifuge showed the range of 30-100 nm. Using Bradford test, the concentration of exosomes extracted was recorded as 1296.7 mg/ml by ultracentrifugation and 1322.4 mg/ml by PEG.

Comparison of two methods of separation of exosomes showed that the extracted exosomes were more purified by ultracentrifugation; but in PEG method, the exosomes were larger and less purified due to PEG deposition on the particles. However, because of less access to ultracentrifuge, PEG separation is also applicable.

Leishmania major (L.major) is a causative agent of leishmaniasis. The disease has now become a public health problem. Modulation of the immune responses to inhibit the infection or the proliferation of the parasite is an effective way to overcome the leishmaniosis. Leishmania parasite secretory-excretory antigens are responsible for immune system modulation; therefore, they are used in vaccine design and immunotherapy. Nanoparticles have been considered in recent years due to the features that stimulate the immune system. The purpose of the present study was to produce a combination of nanoparticles and secretory-excretory antigens to induce immune responses. L.major secretory-excretory antigens were isolated from supernatant of parasite culture using ultracentrifugation and protein molecular weight and concentration were determined using SDS-PAGE and Bradford methods. The chitosan nanoparticle were loaded with antigens which confirmed by FTIR. MTT test was performed to determine the cytotoxic effect of nanoparticles on macrophages. Five groups of female BALB/c mice were sensitized with nanoparticles and nanoparticles loaded with the protein on days 0.10 and 21. The macrophages were isolated from the mice and infected with L.major for one hour. After 72 hours of incubation, NO production and inhibition of parasite growth were measured in response to L.major. In the present study, the excretory-secretory antigens of the L.major were in the range of 75-110 kDa, and loaded with chitosan nanoparticle by %76 efficacy. The results showed that the excretory-secretory antigens of the L.major parasites significantly increased the production of nitric oxide in the macrophages and the presence of chitosan induced this increase (P<0.01). In addition, the group that received chitosan loaded with the excretory secretory antigens of the L.major showed the least proliferation of the parasites (P<0.05). If chitosan nanoparticles loaded with L.major excretory-secretory antigens can increase the activity of macrophage killing function by increasing nitric oxide production; therefore, they can be nominated as candidates for vaccine development and disease improvement.

A growing body of evidence suggests the existence of abnormalities in the immune system of schizophrenic patients. The current study examined serum levels of interleukin (IL) -1β, IL-6, IL-2,interferon(IFN) -γ, and tumor necrosis factor(TNF)-α in schizophrenic patients before and after treatment with risperidone and correlated levels of these cytokines with symptomatology. The study group consisted of 24 schizophrenic patients as defined by Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-IV) criteria and 24 healthy controls. Serum cytokine levels were examined using enzyme-linked immunosorbent assay (ELISA). Schizophrenic symptomatology was assessed with the Positive and Negative Syndrome Scale (PANSS) questionnaire. The serum levels of TNF-α, IL-1β and IL-6 were significantly higher in participants before treatment compared with the healthy controls and after treatment (p<0.001). IFN-γ and IL-2 levels were significantly lower in participants after treatment compared with before treatment and the healthy controls (p<0.001). Except for IL-6 (p<0.05), there was no significant difference in the levels of TNF-α and IL-1β between the patients receiving treatment and the healthy subjects. Moreover, there was no significant difference in levels of IFN-γ and IL-2 between patients before treatment and the healthy subjects. There were no significant correlations between the concentration of cytokines studied and the PANSS. Positive intercorrelations between the production of IFN-γ and IL-2 were detected for sums of all groups(r=0.33, p=0.005). Clinical improvement of treated patients was associated with a reduction in the studied cytokines. It seems that changes in the cytokines level may play a significant role in the psychopathology of these patients.

Leishmaniasis is a public health problem caused by different types of Leishmania. The induction of apoptosis in infected macrophages is one of the main mechanisms for the escape of the parasite from the immune system. Preventing apoptosis and enhancing the ability to kill macrophages can be effective in treating or controlling leishmaniasis.  In this study, the effect of chitosan nanoparticle loaded with Leishmania major secretory and excretory antigens on the number of apoptotic macrophages on exposure to Leishmania was investigated.   Secretory and excretory proteins were isolated from the supernatant of the Leishmania major. The protein molecular weight range and its concentration were determined by SDS-PAGE and Bradford method. Proteins were coupled with chitosan nanoparticles. The coupled proteins and nanoparticles were confirmed by FTIR. The nanoparticle toxicity on macrophages was determined by MTT assay. Rats were treated intraperitoneally with protein, nanoparticle, protein coupled with nanoparticles on days 0, 10, and 21. After 28 days, the macrophages were isolated and their apoptosis percentage in presence and absence of parasites was investigated by flow cytometry.   The maximum production of Leishmania major secretory and excretory proteins was 72 hours after parasite culture. Proteins were in the range of 35-110 kDa. At a concentration of 250 μg/ml, the highest percentage of nanoparticles was 76%. The results of the MTT confirmed non-toxicity of all chitosan concentrations coupled with Leishmania major secretory and excretory proteins. The results of apoptotic measurements by Annexin showed that apoptosis in macrophages treated with chitosan and chitosan coupled with Leishmania major secretory and excretory proteins was significantly (p<0.05) less than untreated macrophages.   Chitosan coupled with Leishmania major secretory and excretory proteins can increase the ability of infected macrophages to remove parasites by reducing apoptosis.

Ficolins are proteins that bind to carbohydrates, act as opsonins and play an important role in innate immunity. Polymorphism in ficolin-3 gene (FCN3) can lead to complement deficiency and increase the risk of some disorders such as diabetes. The aim of this study was to investigate the frequency of FCN3+1637delC as a single nucleotide polymorphism in this gene in healthy and diabetic subjects of Iran. Blood was taken from 36 diabetics and 37 healthy subjects who had referred to the Iranian Blood Transfusion Organization. Blood sugar was analyzed using a calorimetric method. After DNA extraction using salting out method, polymerase chain reaction (PCR) was carried out and the restriction fragment length polymorphism (RFLP) method was accomplished using ApaI restriction enzyme. Consequently, the resulted fragments were evaluated using electrophoresis on 2% L-agarose gel. Evaluation of the results indicated that the heterozygote form of single-nucleotide polymorphism FCN3+1637delC was seen in three samples (8.1%) of the studied healthy subjects and in two samples (5.6%) of the diabetic individuals. Besides, the homozygous form of the mutation was not seen in the studied healthy and diabetic subjects. Results of this study showed that FCN3 variant of single-nucleotide polymorphism FCN3+1637delC was not considered as a risk factor for type 2 diabetes mellitus in Iranian subjects.

CD19 is a transmembrane glycoprotein of immunoglobulin superfamily. In order to treat lymphoma, monoclonal antibodies (mAb) can target different antigens, including CD19, CD20 and CD22 on the surface of B-cells. Along with biotechnology progress, a new generation of antibodies is introduced, with the purpose of eliminating the defects of the previous generation. Among the most developed one are nanobodies (Nb). Nbs are a unique kind of camelid single domain antibody fragments with a broad range of medical applications. Unique physicochemical properties of Nbs have made them ideal candidates for therapeutic and diagnostic applications.
An immune gene library was created, and several CD19 specific Nbs were selected through antigen panning process, and their molecular properties as well as specificity, sensitivity, affinity and immunoreactivity against CD19 positive and negative cells were evaluated.
The Nb library was prepared with 7.2 x107 members. We managed to isolate a panel of CD19-specific Nbs after the last round of selection with the affinity of isolated Nbs being estimated at the standard range of 15-35 nM. Sequence analysis of positive clones was indicative of the fact that 12 variable sequences were confirmed. Of all these 12 clones, 2 clones with the greatest level signal in ELISA underwent subsequent analysis. Our sequencing results indicated high sequence homology (approximately 90%) between the Nb and Homa variable immunoglobulin domains.
Specific Nbs possess the potential to be used as novel therapeutic approaches in order to treat autoimmune diseases and B-cell lymphoma.

Colorectal cancer (CRC) is one of the most prevalent cancers in the world. Cancer stem cells (CSC) have been reported in many human tumors and are thought to be responsible for tumor initiation, therapy resistance, progression, relapse, and metastasis. Recent studies revealed that microRNAs (miRNA) play important roles in maintaining stemness of embryonic stem cells and CSCs.

With regard to the crucial role of cancer stem cells in colon cancer initiation and maintenance, the miRNA expression profile in primary and cell line cancer stem cells compared to non-stem cells cancer cells were evaluated.

In this study, a population of colon CSCs from primary colon cancer and human HT-29 colonic adenocarcinoma cell line was isolated from serum free medium and their microRNA profiles were evaluated using miRNA PCR array.

The isolated cells with high expression of EPCAMﰠ markers showed greater colony-forming efficiency and higher tumorigenic potential. Furthermore, expression of ‘‘stemness’’ genes including C-myc, Klf4, Nanog, Sox2 and Oct4 was higher in isolated cancer stem cells. Moreover, miRNA expression profile of colon CSCs was performed using miRNA PCR array. There are 39 differentially expressed miRNAs, 24 of which had lower mean expression in the cancer stem cells samples. By contrast, 15 miRNAs had higher expression levels in CSCs samples. Of these, miR-495, miR-125, and miR-199a were the most significantly up-regulated miRNAs.

Our results suggest that miRNAs might play important roles in maintenance and regulation of colon CSCs and specific miRNA expression signatures may contribute to cancer initiation and expansion.

Autophagy as a cellular pathway facilitates several immune responses against infection. It also eliminates invading pathogens through transferring content between the cytosol and the lysosomal vesicles and contributes to the cross-presentation of exogenous antigens to T lymphocytes via MHC class I pathway. Autophagy induction is one of the main targets for new drugs and future vaccine formulations. Nanoparticles are one of the candidates for autophagy induction. Cysteine Peptidase A (CPA) and Cysteine Peptidase B (CPB) are two members of papain family (Clan CA, family C1) enzyme that have been considered as a virulence factor of Leishmania (L.) major, making them suitable vaccine candidates. In this research, Leishmania major cysteine peptidase A and B (CPA and CPB) conjugation to alpha alumina nanoparticle was the main focus and their entrance efficacy to macrophages was assessed.
For this purpose, CPA and CPB genes were cloned in expression vectors. Related proteins were extracted from transformed E. coli and purified using Ni affinity column. Alpha alumina nanoparticles were conjugated to CPA/CPB proteins using Aldehyde/Hydrazine Reaction. Autophagy induction in macrophages was assessed using acridine orange staining.
CPA/CPB protein loading to nanoparticles was confirmed by Fourier Transform Infrared Spectroscopy. α-alumina conjugated CPA/CPB antigen uptake by macrophages at different concentrations was confirmed using fluorescence microscope and flowcytometry. Highly efficient CPA/CPB protein loading to α-alumina nanoparticles and rapid internalization to macrophages introduced these nanocarriers as a delivery tool. Acridine orange staining demonstrated higher autophagy induction in CPA/CPB protein conjugated with α-alumina nanoparticles.
α-alumina nanoparticles may be a promising adjuvant in the development of therapeutic leishmania vaccines through antigen delivery to intracellular compartments, induction of autophagy and cross presentation to CD8 lymphocytes.

Lymph node stromal fibroblasts are interconnected with TCD4 cells and affect their phenotype and function. Understanding the nature of these interactions under unusual conditions like infections will help to their application in control and regulation of immune responses.
Lymph node fibroblasts were affected in BCG primed immune environment by both in-vitro and in vivo method. Fibroblasts isolated through enzymatic digestion and surface markers and co-cultured with spleen isolated naive T cells for 72-hours in a cell-to-cell contact manner. Then naive T cells were removed and cultured 72-hours more and assessed for regulatory T cell marker expression and IL-4, TGF-β1, IL-10 and IFN-γ cytokine production.
Results showed that in in-vivo stimulation method, more regulatory T cells was induced by BCG stimulated fibroblasts compared to non-stimulated fibroblasts. In this method, IFN-γ cytokine production was reduced and IL-10 and TGF-β1 regulatory cytokine production was increased. In contrast, in in-vitro stimulation method, regulatory T cell induction by BCG stimulated fibroblasts was significantly less than non-stimulated ones. In addition, both IL-10 and IFN-γ production was increased that indicate induction of IFN-γ producing T-cells.
Present study shows that fibroblasts affect regulatory T-cell induction and T-cell function. The result of this effect is dependent to environment. In this study, fibroblasts were isolated from two different BCG stimulated immune environment. They induced more regulatory responses in in-vivo condition and reduced regulatory responses in in-vitro environment. This result demonstrates different fibroblast behavior at different phases of infection.

Recent evidence has suggested that epithelial cancers including colorectal cancer (CRC) have driven by a small population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which could be responsible for recurrence of cancer. Aldehyde dehydrogenase 1 (ALDH1) activity has used as a functional stem cell biomarker to isolate CSCs in different cancers such as colorectal cancer. The main aim of this research was to determine the utility of ALDH1 activity along with CD44 and EPCAM in identifying stem cell-like cells in human HT-29 colonic adenocarcinoma cell line. In this experimental study, colon CSCs biomarkers including CD44, EPCAM and ALDH1 in colonospheres and parent cells have analyzed by flow cytometry. The expression levels of stemness genes in spheroid and parental cells have investigated using SYBR Green real-time PCR. In addition, in vivo xenografts assay has performed to determine tumorigenic potential of tumor spheroid cells in nude mice. According to results, over 92% of spheroids were CD44+/EpCAM+, while parent cells only have expressed 38% of CD44/EpCAM biomarkers (P < 0.001). Controversially, ALDH activity was about 2-fold higher in the parent cells than spheroid cells (P < 0.05). In comparison with the parental cells, expression levels of ‘‘stemness’’ genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 have significantly increased in colonosphere cells (P < 0.05). Further, administration of 2500 spheroids could be sufficient to initiate tumor growth in nude mice, while 1x106 of parental cells has needed to form tumor. For the first time, we have shown that colonospheres with low ALDH1 activity has indicated increased tumorigenic potential and stemness properties. So, it hasn’t seemed that ALDH1 could become a useful biomarker to identify CSCs population in HT-29 cell line.

Proliferation and expansion of cancer stem cells as spheroids were proved in previous studies. But, capability of primary tumor-derived stem cells to keep their unique properties in vitro is still disputed. So, the goal of this study was to isolate, expand and characterize of colon cancer-derived stem cells.In the present work, colon cancer stem cells markers including CD44 and EPCAM in spheroid and parental cells were analyzed by flow cytometry. The expression levels of stemness genes in both spheroid and parental cells were investigated using real-time PCR. Tumorigenic potential of spheroid cells was evaluated and used implantation of tumor xenografts into nude mice. Our data shows 79% of spheroids were CD44+/EpCAM+, while parental cells only expressed 20% of CD44/EpCAM markers (p< 0.01). In compared with the parental cells, the expression levels of ‘‘stemness’’ genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 were significantly increased in spheroid cells (p< 0.05). Furthermore, as little as 1000 spheroid cells were sufficient to obtain tumor growth in nude mice, while 1x106 of parental cells was needed to generate tumor.Sphere formation assay is a useful method to enrich cancer stem cells. Spheroid cells showed increasing expression of stemness genes and tumorigenic activity in nude mice.

Cancer stem cells are subpopulation of cancer cells that show self-renewal potential and the capacity to differentiate into diverse populations comprising a tumor. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free media. The aim of this study was isolation of colon cancer stem cells by sphere formation assay and characterization of them in human colonic adenocarcinoma HT-29. In this experimental study, colon CSCs markers including CD44 and EPCAM in spheroid and HT-29 cells were analyzed by flow cytometry. The expression levels of stemness genes in both spheroid and HT-29 cells were investigated using real-time PCR. Tumorigenic potential of spheroid cells was evaluated using in vivo xenografts assay. Our data showed over 92% of spheroids were CD44+/EpCAM+, while HT-29 cells only have expressed 37% of CD44/EpCAM markers. In compared with the HT-29 cells, expression levels of ‘‘stemness’’ genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 were significantly increased in spheroid cells (p< 0.05). Further, As little as 2500 spheroid cells were sufficient to obtain tumor growth in nude mice, while 1x106 of HT-29 cells was needed to form tumor. Our data suggest that spheroid formed by colon cancer cell lines highly enriched in CSCs and showed increasing expression of stemness genes and tumorigenic in nude mice.

Immunotoxins (ITs) have been developed for the treatment of cancer, and comprise of antibodies linked to toxins. Also vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis, and the blockade of VEGF receptor- 2 (VEGFR2) inhibits angiogenesis and tumor growth. The aim of this study was to produce anti-VEGFR2/rPE (Pseudomonas exotoxin) 38 IT to test its cytotoxic activity and mechanism of action. In this basic research and experimental study, at first, DNA that encodes recombinant PE38 protein was inductively expressed in Escherichia coli (E.coli) and purified by nickel-sepharose chromatography and further analyzed by western blot. Then, for production of IT, rPE38 was chemically conjugated to anti- VEGFR2. The cytotoxicity response of IT treatment was evaluated by 3-(4,5 Dimethylthiazol- 2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) test in Human Umbilical Vein Endothelial Cell (HUVEC) and Michigan Cancer Foundation-7 (MCF-7) (VEGFR2+) cell lines. The mechanism of IT cytotoxicity was observed by Annexin V staining and flow cytometry. Continuous variables were compared with the analysis of variance (ANOVA; for all groups). P values less than 0.05 were considered statistically significant. SDS-PAGE showed 98% purity of rPE38 and IT. In vitro dose-dependent cytotoxicity assay demonstrated that anti-VEGFR2/PE38 is toxic to VEGFR2-positive cells. IT treatment significantly inhibited proliferation of HUVEC and MCF-7 in a VEGFR2- specific manner as compared with the control groups (p<0.05). Flow cytometry showed that the mechanism of IT induced cell death is mediated by apoptosis. IT treatment also caused remarkable synergistic cytotoxicity characterized by decreased cell viability, and an increased apoptotic index by both anti-VEGFR2 and PE38. Thus these results raise the possibility of using anti-VEGFR2/PE38 IT for cancer therapy because nearly all tumors induce local angiogenesis with high VEGFR expression.

Chemo-immunotherapy is one of the new achievements for treatment of cancer, by which the success of anti-cancer therapy can be increased. In vitro studies have been shown that Arteether (ARE) induces apoptosis in tumor cells, but not in normal cells. To investigate the cytotoxic and immunomodulatory properties of Arteether invivo and in-vitro. In this study, we used MTT assay for evaluation of cytotoxicity of Arteether on tumor cell line and Peripheral Blood Mononuclear Cells (PBMCs) from healthy individuals. Balb/c mice were subcutaneously transplanted with tumor tissue taken from Spontaneous Mouse Mammary Tumor (SMMT) bearing female mice. Arteether was administered to breast tumor-bearing Balb/c mice at a dose of 6mg/kg/day intraperitoneally. Tumor sizes, lymphocyte proliferation, cytokines production, and the percentage of splenic Treg cells were measured. We observed that ARE could reduce the cell growth of 4T1 cell line in a dose-dependent manner but it had no cytotoxic effect on the growth of peripheral blood lymphocytes. ARE administered intraperitoneally to tumor-bearing Balb/c mice could reduce the tumor growth rate and splenic T-reg cells. No difference in the IFN-γ, IL-10 and IL-4 production was observed between tumor antigen-stimulated splenocytes of mice treated with ARE and control mice. These results underscore antitumor properties of Arteether that may aid in development of more effective antitumor agents.

Helicobacter pylori is the causative agent of peptic ulcer disease and a co-factor in development of gastric malignancies. LPS are among toxic substances produced by H. pylori exhibiting low endotoxic activity compared to typical bacterial LPS. The aim of this study was to investigate bioactivity of LPS produced by different serotypes of Helicobacter pylori compared to Escherichia coli and Brucella abortus LPS. Bacterial LPS was extracted by the hot phenol-water method. Biological activities of LPS were determined via the limulus lysate assay, pyrogenic assay, and blood pressure and PBMC induction test in rabbits. Biological activity of O2 serotype LPS of H. pylori was less than the biological activity of other H. pylori serotypes. Our data supported the hypothesis that the unique bacterial LPS of the O2 serotype must be included in the formulation of a multivalent H. pylori vaccine.

Recent studies have indicated the profound anti-tumor activity of artemisinin's compounds, among which; arteether is an oil-soluble derivative of artemisinin with an endoperoxide bridge that can induce apoptosis in tumor cells but not in the normal cells. An experiment was carried out on tumor-bearing Balb/c mice to estimate the effects of Arteether on tumor growth and antitumor immune responses. Briefly, 6mg/kg/day of Arteether and diluents were administered to two groups of mice. Tumor sizes were measured using digital verniercallipers. Mice were sacrificed and splenocytes were harvested for lymphocyte proliferation assay, the level of IL-4 and IFN-γ cytokines, and the percentage of splenic T regulatory cells were measured. According to the findings, there were no statistical differences between the groups with respect to the level of IFN-γ, IL-4 and proliferation assay; while our results showed that Arteether is effective in the reduction of tumor growth rate. In general, intra-tumoral injection of Arteether as an oil-soluble derivative of artemisinin brings to light some antitumor properties that may aid in development of more effective antitumor agents.

Various prokaryotic and eukaryotic expression systems havebeen developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum (L.infantum). The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 (rLPG3) to further research in vaccine designing against leishmaniasis.

Mycobacterium tuberculosis lipid antigens take part in pathogenicity of the bacterium but the response of monocytes/macrophages to these antigens in tuberculosis is not well known. The aim of current investigation was to study the M. tuberculosis lipid antigens in tuberculosis pathogenesis. In the present studyM. tuberculosis lipid antigens were extracted. Monocytes and macrophages from multidrug- resistant tuberculosis (MDR-TB), TB patients, asymptomatic healthy individualswith positive tuberculin skin test positive and healthy individuals with negative tuberculinskin test were collected using magnetic cell sorting. The cells were stimulated by M.tuberculosis total lipid antigens and IL-12 and IL-10 in their supernatants were measuredby enzyme-linked immunosorbent assay. The IL-12 production by monocytesin response to M. tuberculosis total sonicate antigens in the MDR-TB patients didnot show a considerable difference with the PPD positive healthy subjects, whereas inthe active TB patients, IL-12 levels significantly decreased (p<0.05). IL-10 productionby monocytes in TB patients in response to total lipid antigens showed a significant increase in comparison to MDR-TB patients and healthy individuals. In the MDR-TB patients, IL-10 and IL-12 production by monocytes in response to M. tuberculosis lipid antigens are similar to the healthy subjects.