Evaluation of mRNA Expressions of TOX and NR4As in CD8+ T cells in Acute Leukemia

Background: Thymocyte selection-associated high mobility group box protein (TOX) and members of the nuclear receptor 4A (NR4A) are known as transcription factors involved in T cell exhaustion.Objective: To evaluate the mRNA expression of TOX and NR4A1-3 in CD8+ T cells in acute leukemia.Methods: Blood samples were obtained from 21 ALL and 6 AML patients as well as 20 control subjects. CD8+ T cells were isolated using MACS. Relative gene expression of TOX and NR4A1-3 was then evaluated using qRT-PCR.Results: Comparison of mRNA expression of TOX in CD8+ T cells showed no significant difference among the study groups (p>0.05), while the expression of NR4A1 was significantly lower in AML patients than in the control group (p=0.0006). Also, the expression of NR4A2 and NR4A3 was significantly lower in both ALL (p=0.0049 and p=0.0005, respectively) and AML (p=0.0019 and p=0.0055, respectively) patients.Conclusion: NR4As expressions were found to be lower in CD8+ T cells from patients with AML and ALL compared to controls, whereas the mRNA expression of TOX showed no significant difference. Although TOX and NR4As are associated with CD8+ T cell exhaustion in solid tumors, they might play different roles in acute leukemia, which requires further investigation

Comparative expression profile of orphan receptor tyrosine kinase ROR1 in Iranian patients with lymphoid and myeloid leukemias

It has recently been shown that ROR1, a member of the receptor tyrosine kinase family, is overexpressed in leukemic B cells of Chronic Lymphocytic Leukemia (CLL) and a subset of Acute Lymphoblastic Leukemia (ALL). In this comparative study the expression profile of ROR1 mRNA was investigated in Iranian patients with CLL and Acute Myelogenous Leukemia (AML) and the results were compared with those previously reported in our Iranian ALL patients. RT-PCR was performed on bone marrow and/or peripheral blood samples of 84 CLL and 12 AML patients. CLL samples were classified into immunoglobulin heavy chain variable region (IGHV) gene mutated (n = 55) and unmutated (n = 29) and also indolent (n = 42) and progressive (n = 39) subtypes. ROR1 expression was identified in 94% of our CLL patients, but none of the AML patients expressed ROR1. No significant differences were observed between different CLL subtypes for ROR1 expression. Taken together the present data and our previous results on ROR1 expression in ALL, our findings propose ROR1 as a tumor-associated antigen overexpressed in a large proportion of lymphoid (CLL and ALL), but not myeloid (AML) leukemias. Expression of ROR1 seems to be associated to lineage and differentiation stages of leukemic cells with a potential implication for immunotherapy.Tehran University of Medical SciencesPublishe

Comparative expression profile of orphan receptor tyrosine kinase ror1 in iranian patients with lymphoid and myeloid leukemias

It has recently been shown that ROR1, a member of the receptor tyrosine kinase family, is overexpressed in leukemic B cells of Chronic Lymphocytic Leukemia (CLL) and a subset of Acute Lymphoblastic Leukemia (ALL). In this comparative study the expression profile of ROR1 mRNA was investigated in Iranian patients with CLL and Acute Myelogenous Leukemia (AML) and the results were compared with those previously reported in our Iranian ALL patients. RT-PCR was performed on bone marrow and/or peripheral blood samples of 84 CLL and 12 AML patients. CLL samples were classified into immunoglobulin heavy chain variable region (IGHV) gene mutated (n=55) and unmutated (n=29) and also indolent (n=42) and progressive (n=39) subtypes. ROR1 expression was identified in 94% of our CLL patients, but none of the AML patients expressed ROR1. No significant differences were observed between different CLL subtypes for ROR1 expression. Taken together the present data and our previous results on ROR1 expression in ALL, our findings propose ROR1 as a tumor-associated antigen overexpressed in a large proportion of lymphoid (CLL and ALL), but not myeloid (AML) leukemias. Expression of ROR1 seems to be associated to lineage and differentiation stages of leukemic cells with a potential implication for immunotherapy.Tehran University of Medical Sciences and the Ministry of Health and Medical Education of Iran.Publishe

Enhancement of immune responses by vaccine potential of three antigens, including ROP18, MIC4, and SAG1 against acute toxoplasmosis in mice

Toxoplasma gondii (T. gondii) causes considerable financial losses in the livestock industry and can present serious threats to pregnant women, as well as immunocompromised patients. Therefore, it is required to design and produce an efficient vaccine for controlling toxoplasmosis. The present study aimed to evaluate the protective immunity induced by RMS protein (ROP18, MIC4, and SAG1) with Freund adjuvant, calcium phosphate nanoparticles (CaPNs), and chitosan nanoparticles (CNs) in BALB/c mice. The RMS protein was expressed in Escherichia coli (E. coli) and purified using a HisTrap HP column. Thereafter, cellular and humoral immunity was assessed by injecting RMS protein on days 0, 21, and 35 into four groups [RMS, RMS-chitosan nanoparticles (RMS-CNs), RMS-calcium phosphate nanoparticles (RMS-CaPNs), and RMS-Freund]. Phosphate buffered saline (PBS), CNs, CaPNs, and Freund served as the four control groups. The results displayed that vaccination with RMS protein and adjuvants significantly elicited the levels of specific IgG antibodies and cytokines against toxoplasmosis. There were high levels of total IgG, IgG2a, and IFN-γ in vaccinated mice, compared to those in the control groups, especially in the RMS-Freund, indicating a Th-1 type response. The vaccinated and control mice were challenged intraperitoneally with 1 × 103 tachyzoites of the T. gondii RH strain four weeks after the last injection, and in RMS-Freund and RMS-CaPNs groups, the highest increase in survival time was observed (15 days). The RMS can significantly increase Th1 and Th2 responses; moreover, multi-epitope vaccines with adjuvants can be a promising strategy for the production of a vaccine against toxoplasmosis

Galectin-9 Expression Profile in Patients with Gastric Cancer and Peptic Ulcer Disease

Background: Chronic inflammation and dys-regulation of the immune system mechanisms are now well established as primary triggers of gastric cancer and peptic ulcer disease. Galectin-9 (Gal-9) is a member of the galectin family and known as an inducer of cell aggregation, adhesion and apoptosis. Gal-9 interaction with its main ligand T-cell immunoglobulin and mucin domain protein-3 (Tim-3) leads to the apoptosis of T cells and dys-regulation of the immune responses in different tumors. In the current study, the mRNA expression pattern of Gal-9 was evaluated in gastric biopsies of patients with gastric cancer and peptic ulcer disease. Methods: In this case control study, gastric biopsies were obtained from 46 patients with gastric cancer, 44 patients with peptic ulcer disease and 41 cases with non-ulcer dyspepsia served as controls who underwent endoscopy for evaluation of their gastric problems. Infection with Helicobacter pylori was determined by rapid urease test for all participants and H&E staining for GC patients. Total RNA was extracted from all gastric tissues and used for cDNA synthesis. Relative expression of Gal-9 mRNA was determined by Real-Time PCR using β-actin as a housekeeping gene. Results: Gal-9 was similarly expressed in all three studied groups. No statistical difference was found for Gal-9 expression between gastric cancer patients and control group (2-ΔCt =0.022 vs 0.0144, p=0.30) and also between peptic ulcer and control groups (2-ΔCt =0.088 vs 0.144, p=0.16). No correlation was found for Gal-9 expression and infection with Helicobacter pylori (2-ΔCt = 0.1 vs 0.129, p=0.51). Conclusion: Similar expression of Gal-9 in gastric tissues from patients with gastric cancer, peptic ulcer and also non-ulcer dyspepsia individuals suggest no possible role of this molecule on tumorigenesis and immunoregulatory mechanisms of these gastric disorders

Designing an ELISA Method for Measurement of Human IgG and IgM Antibodies against SARS-CoV-2

Background and purpose: Coronavirus disease 2019 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, an indirect ELISA method was designed to measure the human IgM and IgG antibodies against SARS-CoV-2. Materials and methods: Protein sequence of nucleocapsid antigen from SARS-CoV-2 was expressed in E. coli BL21 and then was purified by chromatography. The purified protein was confirmed by SDS-PAGE and Western blotting. An indirect ELISA method was designed to measure the specific IgG and IgM antibodies against SARS-CoV-2 using recombinant N protein. The optimized ELISA method was then applied to measure the IgG and IgM antibodies in 61 infected or recovered COVID-19 patients and in 31 healthy controls. Finally, data obtained from the designed ELISA method were compared with those of a commercially approved ELISA kit. Results: The recombinant nucleocapsid protein was successfully expressed and purified which was confirmed by SDS-PAGE and Western blotting. The amount of optical densities obtained from the designed ELISA method was similar to those of the commercial kit in 61 patients and 31 controls. The sensitivity and specificity of the designed ELISA method for IgG were 100% compared with the commercial ELISA kit, while the sensitivity and specificity for IgM were 96.72 and 96.77, respectively. Conclusion: Serological tests alone are not suitable for diagnosis; however, their combination with molecular tests increases the accuracy and sensitivity of the COVID-19 diagnosis. These tests are also vauable for epidemiological studies

Inflammation, a Key Factor in Cancer Ambush

Inflammatory condition is the consequence of defensive mechanism of immune system against viral and bacterial infection, tissue injury, UV radiation, stress and etc. Persistently acute inflammation leads to chronic phase which is characterized by production of pro-inflammatory mediators from T cells. These molecules (e.g. IL-6, TNF-&alpha, IL-1&beta and IL-17) are mostly pleiotropic cytokines involved in multiple signaling cascades. NF-&kappaB, STAT3, and HIF-1&alpha are the major engaged pathways directing to several downstream targets associating with tumorigenesis and inflammation. Carcinogenesis processes such as DNA mutation/damage, proliferation, angiogenesis, apoptosis, and invasion are implicated to inflammation. Clearly there is a closely association between cancer and inflammation reported as “Seven Hallmark of Cancer”. The elucidation of relationship between inflammation and cancer and their interaction may result in effective therapy and prevention. Gastric cancer is one of the main cancer involved in complex correlation of inflammation and cancer. Inflammation in gastric epithelium could trigger cellular transformation and promote invasion by inducing immune responses and utilizing signaling cascades. Gastric tumor microenvironment has inverse association by providing cytokines and inflammatory mediators. This closely relationship facilitates gastric tumor development and the induction of chronic inflammation in tumor microenvironment. The current review will focus on describing the possible and critical ways in which inflammation and cancer are linked together with specific view to gastric cancer and inflammation. Finally, it introduces some putative treatment generally used in this way in order to direct more attention for further exploration

Immunophenotypic Characterization of Patients with Chronic Lymphocytic Leukemia by Flow Cytometry; Association with Disease Prognosis

Background and purpose: Chronic lymphocytic leukemia (CLL) is characterized by clonal proliferation and accumulation of long-lived B lymphocytes within the peripheral blood, bone marrow, and secondary lymphoid organs. In this study, immunophenotypic characteristics of leukemic cells and their association with disease prognosis were investigated in CLL patients. Materials and methods: A case-control study was carried out in 25 CLL patients and 15 healthy individuals. Complete blood count was performed for all subjects. CLL patients were classified into different clinical stages based on the Rai staging system. For immunophenotypic characterization of leukemic cells, peripheral blood mononuclear cells (PBMCs) were isolated from CLL patients and stained using specific monoclonal antibodies. They were then analyzed by flow cytometry. Results: The CD5, CD19, CD20, and CD23 were expressed significantly more in advanced clinical stages of CLL compared to those in primary stages (P= 0.03, 0.01, 0.02, and 0.02, respectively). The mean fluorescence intensity (MFI) of CD38 in advanced progressive stages of CLL patients was higher than that of primary non-progressive stages (P=0.02). Conclusion: Our results indicated significant immunophenotypic profile and higher CD38 expression in CLL patients with advanced clinical stages. These immunophenotypic characteristics are useful biomarkers for the disease monitoring and therapy

Blimp-1 Expression as an Exhaustion Transcription Factor in Chronic Lymphocytic Leukemia

Background: PPreviously, it was shown that exhausted CD4+ and CD8+ T cells in chronic lymphocytic leukemia (CLL) co-express the two immune-inhibitory receptors, Tim-3 and PD-1. Present study investigated the expression of Blimp-1, a transcription factor involved in T cell exhaustion, in patients with CLL. Materials and Methods: Peripheral blood mononuclear cells were collected from 25 untreated CLL patients and 15 sex- and age-matched normal subjects. CLL patients were clinically classified according to the Rai staging system. The relative expression of Blimp-1 mRNA was determined by quantitative Real Time Polymerase Chain Reaction (qRT-PCR) after normalization with β-actin. Results: Expression of Blimp-1 mRNA was much higher in CLL patients than in normal controls (p=0.001). Moreover, Blimp-1 was more expressed in patients with advanced clinical stages of CLL compared to those with early stages of the disease (p=0.01). Interestingly, the Blimp-1 expression was correlated with the frequencies of exhausted Tim-3+/PD-1+/CD4+ and Tim-3+/PD-1+/CD8+ T cells in CLL patients. Conclusion: Our results highlight the role of Blimp-1 transcription factor in T cell exhaustion of CLL. &nbsp

Effect of Intralipid Infusion on Pregnancy Outcome in Infertile Women with History of Implantation Failure: A Single Blind Randomized Clinical Trial

Background and purpose: Recurrent implantation failure is one of the problems associated with in vitro fertilization cycles. The aim of this study was to evaluate the results of pregnancy using intralipid infusion in infertile women with history of two implantation failures. Materials and methods: A clinical trial was conducted in 80 infertile women in the infertility center in Sari Imam Khomeini Hospital, Iran 2020. In all samples, the level of peripheral blood natural killer (NK) cells was measured two weeks before the intervention using flow cytometry. The participants were divided into an intervention group (n=40) and a control group (n=40) by SAS statistical software. Two days before embryo transfer, patients in intervention group underwent intra lipid infusion (2 ml of 20% solution in 250 ml sterile saline) for two hours and the control group received normal saline (250 ml) infusion. Clinical pregnancy (detection of fetal heart rate by ultrasound) rate was compared between the two groups. Data were analyzed using Chi-square, Mann-whitney, and t-test. Results: Clinical pregnancy rate was higher in intervention group after intralipid infusion (30%) compared with the control group (10%) (P=0.025). There were no significant differences between the two groups in the level of NK cells and peripheral blood lymphocytes (P<0.05). Conclusion: Intralipid infusion improved clinical pregnancy rate in women with history of recurrent implantation failure. However, further studies are required on the possible mechanism of immunological effects of interlaipid in NK cells. (Clinical Trials Registry Number: IRCT20140305016858N5