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Regulatory T (Treg) cells take part in immune homeostasis and play a pivotal role in maintaining peripheral tolerance. The aim of this study was to evaluate the frequency and function of Treg cells in active and untreated ulcerative colitis (UC) patients. Thirty-two subjects with newly diagnosed UC and 31 age-matched healthy controls were included in this survey. The frequency of Tregs was analyzed with flow cytometry using CD4, CD25, CD127 and FoxP3 markers. We used surface expression of CD4+, CD25+and CD127lowmarkers for isolation of a relatively pure Treg population. Suppressive activity of Tregs was determined by measuring their ability to inhibit the proliferation of T responder cells. UC patients had a lower frequency of CD4+CD25+CD127lowFoxP3+Treg cells. Additionally, Treg cell-mediated suppression was lower in UC patients compared to controls. The frequency and suppressive capacity of Tregs and MFI of FoxP3 were inversely correlated with disease activity. These results suggest that CD4+CD25+CD127lowFoxP3+Treg cells may contribute to immunopathogenesis of UC, and assessment of Treg cell frequency and function may have clinical value. [ABSTRACT FROM PUBLISHER]

Soluble forms of nonclassical human leukocyte antigen (HLA)-G have recently been suggested as immunomodulatory factors in multiple sclerosis (MS). HLA-G inhibits the effecter function of T cells and natural killer (NK) cells. Also regulatory T cells (Treg) are considered as pivotal players in MS pathogenesis. Thus, we aimed to evaluate the presence of HLA-G molecules and Treg cells in Relapsing-Remitting Multiple Sclerosis (RRMS) patients and compare it to healthy controls. Patients with RRMS (n=205, mean age=31.32±8.53) and healthy subjects (n=205, mean age=32.2±7.48) were studied. The patients subgrouped to untreated and treated with Interferon beta. Then sHLA-G levels (sHLA-G1 and sHLA-G5) were measured using ELISA method. Treg (CD4+ CD25+ Foxp3+) cells in patients who had sHLA-G>10 U/ml were characterized by using flow cytometry. Our data showed that there was no significant differences between RRMS patients and healthy controls in sHLA-G concentration (p>0.05). Treg cell frequencies were higher in the patients who had sHLA-G >10 U/ml compared to healthy subjects (p<0.05). Collectively, there was significant correlation between sHLA-G and frequency of Treg cells in treated RRMS patients and healthy individuals. It seems that high level sHLA-G has been instrumental in raising frequency of Treg cells in treated patients and could be associated with remission of MS disease. [ABSTRACT FROM AUTHOR]

Abstract: Background: Development of agents that specifically kill cancer cells and simultaneously elicit antitumor immune response is a step forward in cancer therapy. In the present study, we investigated whether the administration of artemether contributes to the augmentation of antitumor immunity and the regression of tumor tissues in a mouse model of breast cancer. Methods: An optimal immunostimulatory dose of artemether (ART) was defined by DTH reaction and antibody production in sRBC-challenged mice. Subsequent experiments were carried out on tumor-bearing BALB/c mice. In the first group of tumor-bearing mice, the dose of 10mg/kg/day of artemether were intraperitoneally administered to each animal for six times. The second group was treated with 20mg/kg/day of cyclophosphamide as a positive control, and the last group (negative control) received the ART diluents. Tumor size was measured during the 10-day experiment; on the last day, mice were sacrificed and their splenocytes and tumor infiltrating lymphocytes were harvested. The concentration of IL-4 and IFN-γ cytokines (using ELISA assay) and the percentage of splenic and tumor Treg cells (using Flowcytometry analysis) were measured. Results: Artemether could increase both DTH reaction and the production of hemagglutinating antibody in normal mice. Administration of ART profoundly suppressed the progression of tumor tissues. As well, it was significantly effective in the depletion of splenic CD4+ CD25+ Foxp3+ Treg cells (p-value>0.05). ART also increased the production of IL-4 (p-value<0.05) and IFN-γ (p-value>0.05). As a conclusion, the cytotoxic and immunomodulatory properties of artemether were acknowledged in vivo [Copyright &y& Elsevier]

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.

Introduction: 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., Methods: 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., Results: 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., Conclusion: 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., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s).)

Objective: 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., Materials and Methods: 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., Results: 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., Conclusion: 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., Competing Interests: There is no conflict of interest in this study., (Copyright© by Royan Institute. All rights reserved.)

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.

Objective: 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., Materials and Methods: 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., Results: 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., Conclusion: 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.

Background: 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., Objective: To investigate the cytotoxic and immunomodulatory properties of Arteether in-vivo and in-vitro., Methods: 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 T-reg cells were measured., Results: 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., Conclusion: These results underscore antitumor properties of Arteether that may aid in development of more effective antitumor agents.

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.

Objective: Macrophages influence their environment and surrounding immune cells as soon as stimulators affect them. Different sources of macrophages induce different reactions in their neighboring immune cells,which result in non-uniform immunologic outcomes. In this experimental research, we compare the behavior of peritoneal macrophages to lipopolysaccharide (LPS) stimulation from BALB/cmice as an indicator of a type 2 immune response and from C57BL/6 mice as an indicator of a type 1 immune response., Materials and Methods: In this experimental study, peritoneal macrophages prepared from thioglycolate stimulated BALB/c and C57BL/6 micewere treated with 1µg/ml LPS. At different time points after LPS treatment, nitric oxide (NO), interferon gamma (IFN-λ), interleukin 4 (IL-4),transforming growth factor β1(TGF-β1), interleukin 17 (IL-17), and interleukin 10(IL-10) production were measured in the supernatants of all macrophage cultures. Indoleamine 2, 3 dioxygenase (IDO) and phagocytic activitywere analyzed in the different experimental groups. The supernatant effects of LPS-treated macrophages on splenocyte proliferation was assessed by the colorimetric method using a 3-(4,5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reagent., Results: According to cytokine analysis, different mouse strains show different cytokine patterns in response to LPS. C57BL/6 macrophages produced more IL-17, IL-10, and IFN-λ, while BALB/c macrophages produced more TGF-β1 and IL-4. There was no significant difference in IDO activity between strains (p≤0.05). BALB/c mice produced more NO inthe first 24 hours after LPS treatment,but C57BL/6 produced more NO at 72 hours post-LPS treatment. Macrophages from both strains hada suppressor effect on splenocyte proliferation, but this effect was stronger in BALB/c mice., Conclusion: The results show that macrophages from different genetic backgrounds respond differently to the same stimulus in aspects of type, intensity, and time of response. The consideration of these aspects will enableresearchers to use correct treatment programs for immune-regulation or immunotherapy.

Background: 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., Objective: The aim of current investigation was to study the M. tuberculosis lipid antigens in tuberculosis pathogenesis., Methods: In the present study M. tuberculosis lipid antigens were extracted. Monocytes and macrophages from multidrug-resistant tuberculosis (MDR-TB), TB patients, asymptomatic healthy individuals with positive tuberculin skin test positive and healthy individuals with negative tuberculin skin 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 measured by enzyme-linked immunosorbent assay., Results: The IL-12 production by monocytes in response to M. tuberculosis total sonicate antigens in the MDR-TB patients did not show a considerable difference with the PPD positive healthy subjects, whereas in the active TB patients, IL-12 levels significantly decreased (p<0.05). IL-10 production by monocytes in TB patients in response to total lipid antigens showed a significant increase in comparison to MDR-TB patients and healthy individuals., Conclusion: 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.

Background: HLA-G gene contains 15 alleles including a null allele, HLA-G*0105N. Previous studies have shown that HLA-G*0105N does not encode the complete HLA-G1 or HLA-G5 isoforms but encodes a functional HLA-G protein with the ability to inhibit NK cell cytolysis. Thus, although the biological functions of HLA-G1 and HLA-G5 proteins are abrogated, other isoforms such as HLA-G2 can replace their roles. Studies on the null allele of HLA-G gene could be useful in understanding the genetic variants of HLA-G alleles in ethnic groups., Objective: The goal of this research was to determine the frequency of HLA-G*0105N null allele in Iranian healthy subjects., Methods: The frequency of HLA-G*0105N null allele was evaluated in Iranian healthy subjects by PCR-RFLP method. Genomic DNA was isolated from the whole blood of 100 randomly selected, healthy, unrelated Iranian individuals using salting-out technique followed by PCR amplification of the exon 3 of HLA-G gene. PCR products were digested with PpUM-1 and the resulted fragments were analyzed using gel electrophoresis., Results: In this study the restriction enzyme digestion confirmed homozygous HLA-G*0105N null allele for 9 % of the population. Furthermore obtained results indicated that the total frequency of HLA-G*0105N null allele was 20 % in the studied population of Iran., Conclusion: The final data analysis showed that the total frequency of this allele in Iranian people was higher than other ethnic groups that have been studied so far.

Studies on HLA-G, a nonpolymorphic antigen of non-classical HLA class I, is of basic and clinical significance. In the present study, the expression of HLA-G proteins in the human skin tissue sections of normal and autoimmune pemphigus vulgaris (PV) individuals were investigated using monoclonal antibodies. The antibodies recognized both membrane-bound and soluble isoforms of HLA-G. RT-PCR was performed to assess the patterns of HLA-G mRNA transcripts in the epidermal cells of PV and normal subjects. HLA-G expression could only be detected at transcriptional level in normal skin tissues. However cells derived from PV subjects expressed detectable HLA-G molecules at both transcriptional and translational levels. In addition, the RT-PCR patterns of HLA-G amplification revealed a reduction in HLA-G2 and an increase in HLA-G1 transcripts in epidermal cells of PV patients as compared to normal cells. These observations further support suggestions in the literature regarding the role of HLA-G in induction of tolerance in autoimmune individuals.

Nitric oxide (NO) derived from activated macrophages has been shown to be crucial for the host's leishmanicidal activities. Excess NO, however, can induce apoptosis in some cell types, including macrophages. In the present investigation, we studied the role of NO in inducing apoptosis of BALB/c mice macrophages infected with Leishmania major in vitro. The peritoneal macrophages were harvested and cultured with or without L.major in the presence of a donating reagent (s-Nitroso-N-Acetylpenicillamine (SNAP)) or an inhibitor of NO synthase (NG -Methyl-L-Arginine (NMMA)). The concentration of NO in culture supernatants was measured after 18 hours incubation. Simultaneously, macrophages undergoing apoptosis were identified by fluorescence and electron microscopy. The results showed an increase in apoptosis rate in parallel to nitrite production in macrophages cultured in the presence of SNAP. Although macrophages infected with L.major had no significant increase in NO production, they showed a significant increase in apoptosis rate. Besides, macrophages cultured with NMMA, had a decreased NO production but the apoptosis rate increased. Therefore, mechanisms involved in apoptosis induction in the last two groups may be different from NO overproduction.