Culture density of menstrual blood-derived stromal/stem cells determines the quality of T cell responses: An experimental study

Background: Menstrual blood-derived stromal/stem cells (MenSCs) are a new population of refreshing and highly proliferative stem cells. Immunomodulatory effects of MenSCs profoundly depend on their relative density. Objective: To find whether MenSCs cultured at varying numbers would differentially affect the allogenic peripheral blood mononuclear cells (PBMCs) key features. Materials and Methods: PBMCs were co-cultured with various MenSCs numbers. PBMCs proliferation was investigated via 3H-thymidine incorporation. Flow cytometry was used to assess human leukocyte antigen (HLA)-DR, HLA-ABC, HLA-G, and costimulatory markers on MenSCs and the percentage of regulatory T cells (Tregs) among PBMCs. The concentration of cytokines was determined in supernatant of co-cultures. Results: The support of PBMCs proliferation at low MenSCs densities correlated with higher levels of pro-inflammatory interferon gamma (IFN-γ) in MenSCs/PBMCs co-culture and increased expression of HLA-DR by MenSCs. On the other hand, the suppressive property of MenSCs at higher densities was independent of Treg frequency, but correlated with a high concentration of Interleukin (IL)-6 and IL-10 in the co-cultures. Conclusion: Totally, at different seeding densities, MenSCs could differentially interact with PBMCs leading to significant changes in the level of anti- and/or pro-inflammatory factors. These preliminary in vitro results are suggested to be taken into consideration in experimental models of MenSC-based immunomodulation. Nonetheless, for efficient utilization of MenSCs anti-inflammatory features in pre-clinical disease models, we still need to broaden our knowledge on MenSC-immune system cross-talk; this could play a part in designing more optimized MenSCs injection modalities in the case of future pre-clinical and subsequently clinical settings. Key words: Menstrual, Stromal cells, T cell response, Interferon

Affinity Determination of Monoclonal Antibodies (mAbs) Using Enzyme- Linked Immunosorbent Assay (ELISA); A Protocol

Monoclonal antibodies (mAbs) have changed diagnostics and therapy due to their high specificity and affinity to the target antigens (Ags). Accurately measuring the affinity of mAbs is critical to understanding their binding properties. It represents the strength of binding between an antibody (Ab) and its target Ag and enables decision making in the development and optimization of these antibodies to improve their efficacy in diagnostics and therapy. Various methods such as the equilibrium dissociation constant, ELISA, surface plasmon resonance (SPR), and microarray-based platforms can be used to determine mAb affinity. The non-competitive ELISA is simple and available method for many laboratories, based on the law of mass action that compares the OD50 of three sigmoidal curves of serial Ab dilutions on plates coated with different Ag concentrations to determine the binding strength between a mAb and its Ag. This protocol provides a step-by-step guide to determining mAb affinity using modified ELISA and enables researchers to make informed decisions about the development and application of mAbs in their respective fields

Immunophenotypic characterization of the leukemic B-cells from Iranian patients with chronic lymphocytic leukemia : association between CD38 expression and disease progression

Background: Patients with B-cell chronic lymphocytic leukemia (B-CLL) have hetero-geneous clinical courses, thus several biological parameters need to be added to the cur-rent clinical staging systems to predict disease outcome. Recent immunophenotypic stud-ies performed mainly in Western populations have demonstrated the prognostic value of CD38 and ZAP-70 expression in B-CLL. Objectives: To investigate the expression pat-tern of a variety of membrane antigens on leukemic cells from Iranian patients with CLL and to find out if there are any differences in the expression of these markers between in-dolent and progressive groups. Methods: In the present study, peripheral blood samples from 87 Iranian patients with B-CLL were analysed by flow cytometry. Results: In all cases, the neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/sIg+). The vast ma-jority of the cases expressed CD23, but failed to stain for CD3 or CD14. The leukemic cells of most patients expressed CD27 (84/87, 95.4%) and CD45RO (74/87, 83.9%) molecules, suggesting a memory B-cell phenotype. Comparison between the indolent (n=42) and progressive (n=37) patients revealed significantly higher frequency and inten-sity of CD38 expression in progressive group (40.5%) compared to indolent (11.9%) pa-tients (p<0.05). None of the other membrane antigens were differentially expressed in these two groups of patients. Conclusion: Our results obtained in an Asian ethnic popula-tion confirm and extend previous findings obtained from Western populations regarding the association of CD38 expression and disease progression in B-CLL.Tehran University of Medical Sciences and the Ministry of Health and Medical Education of Iran.Publishe

Immunophenotypic characterization of the leukemic B-cells from Iranian patients with chronic lymphocytic leukemia

Background: Patients with B-cell chronic lymphocytic leukemia (B-CLL) have heterogeneous clinical courses, thus several biological parameters need to be added to the current clinical staging systems to predict disease outcome. Recent immunophenotypic studies performed mainly in Western populations have demonstrated the prognostic value of CD38 and ZAP-70 expression in B-CLL. Objectives: To investigate the expression pattern of a variety of membrane antigens on leukemic cells from Iranian patients with CLL and to find out if there are any differences in the expression of these markers between indolent and progressive groups. Methods: In the present study, peripheral blood samples from 87 Iranian patients with B-CLL were analysed by flow cytometry. Results: In all cases, the neoplastic cells displayed B-CLL phenotype (CD5+/CD19+/sIg+). The vast majority of the cases expressed CD23, but failed to stain for CD3 or CD14. The leukemic cells of most patients expressed CD27 (84/87, 95.4%) and CD45RO (74/87, 83.9%) molecules, suggesting a memory B-cell phenotype. Comparison between the indolent (n=42) and progressive (n=37) patients revealed significantly higher frequency and intensity of CD38 expression in progressive group (40.5%) compared to indolent (11.9%) patients (p<0.05). None of the other membrane antigens were differentially expressed in these two groups of patients. Conclusion: Our results obtained in an Asian ethnic population confirm and extend previous findings obtained from Western populations regarding the association of CD38 expression and disease progression in B-CLL.Tehran University of Medical sciences, Tehran, IranPublishe

Construction and characterization of a new chimeric antibody against HER2

Aim: Immunotherapy with anti-HER2 antibodies has shown promising results in patients with HER2-positive breast cancer. We have recently reported characterization of a mouse monoclonal antibody (mAb) against HER2, which binds to an epitope different from that recognized by trastuzumab and specifically inhibits proliferation of tumor cells overexpressing HER2. In the present study we report chimerization of this antibody. Materials & methods: The immunoglobulin variable region heavy and light chain genes of 1T0 hybridoma cells were amplified and ligated to human -1 and constant region genes using splice overlap extension PCR. The chimeric antibody was subsequently expressed and characterized by ELISA, western blot and flow cytometry. Results: The purified chimeric antibody specifically binds to recombinant HER2 and HER2-overexpressing tumor cells and inhibits proliferation of these cells. The binding affinity of the chimeric mAb was comparable with the parental mouse mAb. Conclusion: This chimeric anti-HER2 mAb is a potentially valuable tool for targeted immunotherapy.noneManuscrip

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

Ligation of human Fc receptor like-2 (FCRL2) by monoclonal antibodies downregulates B cell receptor mediated signaling

B cell antigen receptor (BCR) signaling and its regulation through negative and positive regulators are critical for balancing B cell response and function. Human Fc receptor like-2 (FCRL2), a member of the newly identified FCRL family, could influence B cell signaling due to possession of both immunoreceptor tyrosine-based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified yet, we generated FCRL2-specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signaling in a FCRL2-expressing B cell line. Two anti-FCRL2 mAb-producing hybridoma clones (5A7-E7 and 3D8-G8) were selected. None of the mAbs displayed any cross-reactivity with the other members of the FCRL family including recombinant FCRL1, 3, 4 and 5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signaling mediators such as calcium mobilization and phosphorylation of the MAP kinases Erk, p38 and Jnk MAP. These findings indicate that the FCRL2 ITIM motifs are functional and the anti-FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells.Food and Drug Administration of the Ministry of Health, Treatment and Medical Education of Iran (grant number S87P/3/414). MS was also supported by EFIS-IL fellowship grant.Accepte

Conjugation of R-Phycoerythrin to a Polyclonal Antibody and F (ab&apos;)2 Fragment of a Polyclonal Antibody by Two Different Methods

Abstract R-Phycoerythrin (R-PE), a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F(ab&apos;)2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry (ICC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity