ABSTRACT
CD19 is a pan B cell marker that is recognized as an attractive target for antibody-based therapy of B-cell disorders including autoimmune disease and hematological malignancies. The object of this study was to stably express the human CD19 antigen in the murine NIH-3T3 cell line aimed to be used as an immunogen in our future study. Total RNA was extracted from Raji cells in which high expression of CD19 was confirmed by flow cytometry. Synthesized cDNA was used for CD19 gene amplification by conventional PCR method using Pfu DNA polymerase. PCR product was ligated to pGEM-T Easy vector and ligation mixture was transformed to DH5 alpha competent bacteria. After blue/white selection, one positive white colony was subjected to plasmid extraction and direct sequencing. Then, CD19 cDNA was sub-cloned into pCMV6-Neo expression vector by double digestion using Kpnl and HindIII enzymes. NIH-3T3 mouse fibroblast cell line was subsequently transfected by the construct using Jet-PEI transfection reagent. After 48 hours, surface expression of CD19 was confirmed by flow cytometry and stably transfected cells were selected by G418 antibiotic. Amplification of CD19 cDNA gave rise to 1701 bp amplicon confirmed by alignment to reference sequence in NCBI database. Flow cytometric analysis showed successful transient and stable expression of CD19 on NIH-3T3 cells [29 and 93%, respectively]. Stable cell surface expression of human CD19 antigen in a murine NIH-3T3 cell line may develop a proper immunogene which raises specific anti-CD19 antibody production in the mice immunized sera
Subject(s)
NIH 3T3 Cells , Cell Line , B-Lymphocytes , Cloning, Organism , Gene Expression , Immunogenetics , MiceABSTRACT
The peroxisome proliferator-activated receptors [PPARs] are a group of nuclear receptor proteins whose functions as transcription factors regulate gene expressions. PPARs play essential roles in the regulation of cellular differentiation, development, and metabolism [carbohydrate, lipid, protein], and tumorigenesis of higher organisms. This study attempts to determine the effect of baicalin, a PPAR? activator, on erythroid differentiation of cluster of differentiation 133+ [CD133+] cord blood hematopoietic stem cells [HSCs]. In this experimental study, in order to investigate the effects of the PPAR? agonists baicalin and troglitazone on erythropoiesis, we isolated CD133+ cells from human umbilical cord blood using the MACS method. Isolated cells were cultured in erythroid-inducing medium with or without various amounts of the two PPAR? activators [baicalin and troglitazone]. Erythroid differentiation of CD133+ cord blood HSCs were assessed using microscopic morphology analysis, flow cytometric analysis of erythroid surface markers transferrin receptor [TfR] and glycophorin A [GPA] and bycolony forming assay. Microscopic and flow cytometric analysis revealed the erythroid differentiation of CD133+ cord blood HSCs under applied erythroid inducing conditions. Our flow cytometric data showed that the TfR and GPA positive cell population diminished significantly in the presence of either troglitazone or baicalin. The suppression of erythroid differentiation in response to PPAR? agonists was dose-dependent. Erythroid colony-forming ability of HSC decreased significantly after treatment with both PPAR? agonists but troglitazone had a markedly greater effect. Our results have demonstrated that PPAR? agonists modulate erythroid differentiation of CD133+ HSCs, and therefore play an important role in regulation of normal erythropoiesis under physiologic conditions. Thus, considering the availability and application of this herbal remedy for treatment of a wide range of diseases, the inhibitory effect of baicalin on erythropoiesis should be noted
ABSTRACT
Chronic myeloid leukemia [CML] is a myeloproliferative disease. The cytogenetic hallmark of CML is Philadelphia [Ph] chromosome. This study aimed to diagnose suspected CML patients, to monitor CML patients under therapy using cytogenetic and fluorescence in situ hybridization [FISH] techniques to analyze their bone marrow [BM] and peripheral blood [PB] samples, and finally to compare their obtained results for both specimens. This study was conducted during one-year period [2012-2013]. The participants were recruited from the Hematology and Oncology Clinic of Shahid Gazi [Emam Reza] Hospital of Tabriz University of Medical Sciences, Tabriz, East Azerbaijan Province, Iran. We analyzed 90 samples from 60 suspected CML patients [30 BM and 60 PB samples]. All samples were analyzed using G-banding, 5 samples using dual fusion FISH [DF-FISH] probes, as well as 30 samples using both FISH and G-banding. Among the 90 analyzed samples of 60 patients, 25 [41.66%] were Ph+ using karyotyping, whereas five cases were not analyzable, so FISH was applied and the results confirmed that only two individuals were BCR-ABL+. In the comparison between 25 BM and 25 PB samples using karyotyping, 15 [60%] and 10 [40%] were ph+, respectively. The comparison of FISH and karyotyping on 30 samples showed that 9 [30%] and 8 [26.66%] were Ph+, respectively, and only 18.18% of Ph+ patients showed atypical patterns. In the comparison between BM-cytogenetic and PB-interphase-FISH [I-FISH], BM-cytogenetic was more reliable than PB-I-FISH in detecting Ph. Our data demonstrate that FISH analysis is a rapid, reliable and sensitive technique. The comparison between BM and PB showed that PB can not be replaced by BM, even in detecting by FISH
ABSTRACT
Evidence from several lines of investigations suggests that Toll-like receptor 4 [TLR4] is involved in atherosclerosis as a bridge between innate and acquired immunity. Percutaneous coronary intervention [PCI] can trigger inflammation through activation of human TLR4 [hTLR4] on monocytes. Hydrocortisone as an anti-inflammatory and immuno-suppressant agent has multiple mechanisms of action. In this study, we aimed at assessing the effects of hydrocortisone on monocyte expression and activity of hTLR4 in patients underwent PCI. Blood samples were taken from a total of 71 patients with chronic stable angina who were scheduled for a PCI, before the intervention. Thirty patients received 100 mg hydrocortisone prior to the procedure. Control group was composed of 41 patients underwent PCI without receiving hydrocortisone. Blood collection was repeated 2 and 4 h after PCI. The expression of hTLR4 on the surface of CD14[+] monocytes and the serum levels of TNF- alpha and IL-1 beta were measured using flowcytometry and Sandwich ELISA. Compared with controls, hydrocortisone significantly reduced monocyte expression of hTLR4 in test group [P<0.01]. In addition, it had a significant effect on reduction of serum concentrations of TNF- alpha and IL-1 beta in test group in a time-dependent manner [P<0.01]. In this study, hydrocortisone was able to reduce the hTLR4/CD14 positive monocytes and its related pro-inflammatory cytokines, thus it can decrease inflammatory responses following PCI
ABSTRACT
The amount of literature concerning the implication of the red cell distribution width [RDW] in the assessment of ulcerative colitis [UC] activity is rather limited. The aim of this study is to investigate the potential role of RDW in the evaluation of UC disease activity. A total of 96 patients with UC and 51 age and sex-matched healthy volunteers were included in a cross-sectional study. Clinical disease activity was defined using the numerical Disease Activity Index [DAI]. In addition to RDW, serum C-reactive protein [CRP] levels, erythrocyte sedimentation rates [ESR], and platelet counts [PLT] were measured. AThere were 47 [about 49%] patients with that had active UC. The RDW was significantly higher in patients with UC than in controls [p=0.001] and active versus patients in remission [p<0.001]. RDW was significantly correlated with DAI scores, ESR, CRP and PLT in active patients. There was a significant correlation between RDW with DAI scores and CRP levels in patients who were in remission. RDW was elevated in UC patients in comparison with healthy controls and increased markedly in active disease. It was also strongly correlated with clinical disease activity scores and inflammatory parameters such as ESR and CRP. RDW, as a cost-effective tool, may be an additional parameter to assess disease activity in UC.