16 research outputs found
Polymorphisms in the Tumor Necrosis Factor Receptor Genes Affect the Expression Levels of Membrane-Bound Type I and Type II Receptors
The level of TNF receptors on various cells of immune system and its association with the gene polymorphism were investigated. Determining the levels of membrane-bound TNF receptors on peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry using BD QuantiBRITE calibration particles. Soluble TNF receptor (sTNFRs) levels were determined by ELISA and genotyping was determined by PCR-RFLP. Homozygous TT individuals at SNP β609G/T TNFRI (rs4149570) showed lower levels of sTNFRI compared to GG genotype carriers. Homozygous carriers of CC genotype at SNP β1207G/C TNFRI (rs4149569) had lower expression densities of membrane-bound TNFRI on intact CD14 + monocytes compared to individuals with the GC genotype. The frequency differences in the CD3 + and CD19 + cells expressing TNFRII in relation to SNP β1709A/T TNFRII (rs652625) in healthy individuals were also determined. The genotype CC in SNP β3609C/T TNFRII (rs590368) was associated with a lower percentage of CD14 + cells expressing TNFRII compared to individuals with the CT genotype. Patients with rheumatoid arthritis had no significant changes in the frequencies of genotypes. Reduced frequency was identified for the combination TNFRI β609GT + TNFRII β3609CC only. The polymorphisms in genes represent one of cell type-specific mechanisms affecting the expression levels of membrane-bound TNF receptors and TNF -mediated signaling
Generation of populations of antigen-specific cytotoxic T cells using DCs transfected with DNA construct encoding HER2/neu tumor antigen epitopes
Abstract Background Recent fundamental and clinical studies have confirmed the effectiveness of utilizing the potential of the immune system to remove tumor cells disseminated in a patientβs body. Cytotoxic T lymphocytes (CTLs) are considered the main effectors in cell-mediated antitumor immunity. Approaches based on antigen presentation to CTLs by dendritic cells (DCs) are currently being intensively studied, because DCs are more efficient in tumor antigen presentation to T cells through their initiation of strong specific antitumor immune responses than other types of antigen-presenting cells. Today, it has become possible to isolate CTLs specific for certain antigenic determinants from heterogeneous populations of mononuclear cells. This enables direct and specific cell-mediated immune responses against cells carrying certain antigens. The aim of the present study was to develop an optimized protocol for generating CTL populations specific for epitopes of tumor-associated antigen HER2/neu, and to assess their cytotoxic effects against the HER2/neu-expressing MCF-7 tumor cell line. Methods The developed protocol included sequential stages of obtaining mature DCs from PBMCs from HLA A*02-positive healthy donors, magnet-assisted transfection of mature DCs with the pMax plasmid encoding immunogenic peptides HER2 p369β377 (E75 peptide) and HER2 p689β697 (E88 peptide), coculture of antigen-activated DCs with autologous lymphocytes, magnetic-activated sorting of CTLs specific to HER2 epitopes, and stimulation of isolated CTLs with cytokines (IL-2, IL-7, and IL-15). Results The resulting CTL populations were characterized by high contents of CD8+ cells (71.5% in cultures of E88-specific T cells and 90.2% in cultures of E75-specific T cells) and displayed strong cytotoxic effects against the MCF-7 cell line (percentages of damaged tumor cells in samples under investigation were 60.2 and 65.7% for E88- and E75-specific T cells, respectively; level of spontaneous death of target cells was 17.9%). Conclusions The developed protocol improves the efficiency of obtaining HER2/neu-specific CTLs and can be further used to obtain cell-based vaccines for eradicating targeted tumor cells to prevent tumor recurrence after the major tumor burden has been eliminated and preventing metastasis in patients with HER2-overexpressing tumors
Polymorphisms in the Tumor Necrosis Factor Receptor Genes Affect the Expression Levels of Membrane-Bound Type I and Type II Receptors
The level of TNF receptors on various cells of immune system and its association with the gene polymorphism were investigated. Determining the levels of membrane-bound TNFΞ± receptors on peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry using BD QuantiBRITE calibration particles. Soluble TNFΞ± receptor (sTNFRs) levels were determined by ELISA and genotyping was determined by PCR-RFLP. Homozygous TT individuals at SNP -609G/T TNFRI (rs4149570) showed lower levels of sTNFRI compared to GG genotype carriers. Homozygous carriers of CC genotype at SNP -1207G/C TNFRI (rs4149569) had lower expression densities of membrane-bound TNFRI on intact CD14+ monocytes compared to individuals with the GC genotype. The frequency differences in the CD3+ and CD19+ cells expressing TNFRII in relation to SNP -1709A/T TNFRII (rs652625) in healthy individuals were also determined. The genotype CC in SNP β3609C/T TNFRII (rs590368) was associated with a lower percentage of CD14+ cells expressing TNFRII compared to individuals with the CT genotype. Patients with rheumatoid arthritis had no significant changes in the frequencies of genotypes. Reduced frequency was identified for the combination TNFRI -609GT + TNFRII -3609CC only. The polymorphisms in genes represent one of cell type-specific mechanisms affecting the expression levels of membrane-bound TNFΞ± receptors and TNFΞ±-mediated signaling
Expression Density of receptors to immunoregulatory mediators as a modulating component of biological effects of mediators on cell. Part 2
Π ΠΎΠ±Π·ΠΎΡΠ΅ ΠΎΠ±ΠΎΠ±ΡΠ΅Π½Ρ ΠΏΠΎΡΠ»Π΅Π΄Π½ΠΈΠ΅ ΠΌΠΈΡΠΎΠ²ΡΠ΅ Π½Π°ΡΡΠ½ΡΠ΅ Π΄Π°Π½Π½ΡΠ΅ ΠΎ ΡΠΎΠ»ΠΈ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠΎΠ² ΠΊ ΠΈΠΌΠΌΡΠ½ΠΎΠΌΠ΅Π΄ΠΈΠ°ΡΠΎΡΠ°ΠΌ Π² ΡΠ΅Π³ΡΠ»ΡΡΠΈΠΈ Π±ΠΈΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΡΡΠ΅ΠΊΡΠΎΠ², ΠΎΠΊΠ°Π·ΡΠ²Π°Π΅ΠΌΡΡ
Π½Π° ΠΊΠ»Π΅ΡΠΊΠΈ. ΠΠ»Ρ ΠΎΡΠ½ΠΎΠ²Π½ΡΡ
ΠΊΠ»Π°ΡΡΠΎΠ² ΡΠ΅Π³ΡΠ»ΡΡΠΎΡΠΎΠ² ΠΈΠΌΠΌΡΠ½Π½ΠΎΠΉ ΡΠΈΡΡΠ΅ΠΌΡ (ΠΈΠ½ΡΠ΅ΡΠ»Π΅ΠΉΠΊΠΈΠ½ΠΎΠ², ΠΈΠ½ΡΠ΅ΡΡΠ΅ΡΠΎΠ½ΠΎΠ², ΡΠ°ΠΊΡΠΎΡΠΎΠ² ΡΠΎΡΡΠ° ΠΈ ΡΠ°ΠΊΡΠΎΡΠΎΠ² Π½Π΅ΠΊΡΠΎΠ·Π° ΠΎΠΏΡΡ
ΠΎΠ»ΠΈ) ΠΏΡΠ΅Π΄ΡΡΠ°Π²Π»Π΅Π½Ρ Π²Π°ΡΠΈΠ°Π½ΡΡ ΡΡΠ°ΡΡΠΈΡ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠΎΠ² ΠΊΠ°ΠΊ ΠΊΠΎΠΌΠΏΠΎΠ½Π΅Π½ΡΠΎΠ² Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ ΡΠΈΡΠΎΠΊΠΈΠ½/ΠΊΠ»Π΅ΡΠΊΠ°, Π½Π° ΠΏΡΠΈΠΌΠ΅ΡΠ΅ invitro ΠΈ invivo ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΠΉ. ΠΠΎΠΊΠ°Π·Π°Π½Π° ΡΠΏΠΎΡΠΎΠ±Π½ΠΎΡΡΡ ΠΏΠΎΠΊΠ°Π·Π°ΡΠ΅Π»Π΅ΠΉ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΠΈ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠΎΠ² ΠΌΠ΅Π½ΡΡΡ Ρ
Π°ΡΠ°ΠΊΡΠ΅Ρ ΠΈ ΡΠΈΠΏ Π΄Π°Π½Π½ΠΎΠ³ΠΎ Π²Π·Π°ΠΈΠΌΠΎΠ΄Π΅ΠΉΡΡΠ²ΠΈΡ. ΠΡΠΎΠ°Π½Π°Π»ΠΈΠ·ΠΈΡΠΎΠ²Π°Π½Ρ Π΄Π°Π½Π½ΡΠ΅ ΠΎΠ± ΡΡΠ°ΡΡΠΈΠΈ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠΎΠ² ΠΊ ΡΠ΅Π³ΡΠ»ΡΡΠΎΡΠ½ΡΠΌ ΠΌΠΎΠ»Π΅ΠΊΡΠ»Π°ΠΌ Π² ΡΠ°Π·Π²ΠΈΡΠΈΠΈ ΠΈΠΌΠΌΡΠ½ΠΎΠΎΠΏΠΎΡΡΠ΅Π΄ΠΎΠ²Π°Π½Π½ΡΡ
Π·Π°Π±ΠΎΠ»Π΅Π²Π°Π½ΠΈΡΡ
ΡΠ°Π·Π»ΠΈΡΠ½ΠΎΠ³ΠΎ Π³Π΅Π½Π΅Π·Π°. ΠΡΠΎΠ΄Π΅ΠΌΠΎΠ½ΡΡΡΠΈΡΠΎΠ²Π°Π½ΠΎ, ΡΡΠΎ ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΠ΅ ΡΡΠΎΠ²Π½Ρ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΠΈ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠΎΠ² ΠΈΠΌΠ΅Π΅Ρ Π±ΠΎΠ»ΡΡΠΎΠ΅ Π·Π½Π°ΡΠ΅Π½ΠΈΠ΅ Π² ΠΎΡΠ΅Π½ΠΊΠ΅ ΡΡΠ½ΠΊΡΠΈΠΎΠ½Π°Π»ΡΠ½ΠΎΠ³ΠΎ ΠΎΡΠ²Π΅ΡΠ° ΠΊΠ»Π΅ΡΠΊΠΈ Π½Π° ΠΌΠ΅Π΄ΠΈΠ°ΡΠΎΡ ΠΈ Π² ΡΠ°Π·Π²ΠΈΡΠΈΠΈ ΠΏΠ°ΡΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΡ
ΡΠΎΡΡΠΎΡΠ½ΠΈΠΉ. Π ΠΈΡΡΠ»Π΅Π΄ΠΎΠ²Π°Π½ΠΈΡΡ
ΠΏΠΎΠ΄ΡΠ²Π΅ΡΠΆΠ΄Π΅Π½Ρ Π΄Π°Π½Π½ΡΠ΅ ΠΎ Π²Π»ΠΈΡΠ½ΠΈΠΈ ΠΏΠ»ΠΎΡΠ½ΠΎΡΡΠΈ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠΎΠ² Π½Π° ΠΏΡΠΎΡΠ΅ΡΡΡ ΠΏΡΠΎΠ»ΠΈΡΠ΅ΡΠ°ΡΠΈΠΈ ΠΈ Π°ΠΏΠΎΠΏΡΠΎΠ·Π°, Π° ΡΠ°ΠΊ ΠΆΠ΅ ΠΎΠ±ΠΌΠ΅Π½Π½ΡΠ΅ ΠΏΡΠΎΡΠ΅ΡΡΡ, ΡΡΠΎ ΡΠ²Π»ΡΠ΅ΡΡΡ ΡΡΠΈΠ³Π³Π΅ΡΠΎΠΌ Π² ΡΠ°Π·Π²ΠΈΡΠΈΠΈ Π°ΡΡΠΎΠΈΠΌΠΌΡΠ½Π½ΡΡ
, ΠΎΠ½ΠΊΠΎΠ»ΠΎΠ³ΠΈΡΠ΅ΡΠΊΠΈΡ
ΠΈ Π΄ΠΈΡΡΡΠΎΡΠΈΡΠ΅ΡΠΊΠΈΡ
Π·Π°Π±ΠΎΠ»Π΅Π²Π°Π½ΠΈΠΉ. ΠΠ»Ρ Π²ΡΠ΅Ρ
ΡΠ°ΡΡΠΌΠΎΡΡΠ΅Π½Π½ΡΡ
ΠΊΠ»Π°ΡΡΠΎΠ² ΡΠ΅Π³ΡΠ»ΡΡΠΎΡΠ½ΡΡ
ΠΌΠΎΠ»Π΅ΠΊΡΠ» Ρ
Π°ΡΠ°ΠΊΡΠ΅ΡΠ½ΡΠΌ ΡΠ²Π»ΡΠ΅ΡΡΡ ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΠ΅ ΠΏΠ»ΠΎΡΠ½ΠΎΡΡΠΈ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΠΈ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠΎΠ² ΠΊΠ°ΠΊ ΠΎΠ΄Π½ΠΎΠ³ΠΎ ΠΈΠ· ΠΊΠ»ΡΡΠ΅Π²ΡΡ
ΠΌΠΎΠΌΠ΅Π½ΡΠΎΠ² ΡΠ΅Π³ΡΠ»ΠΈΡΠΎΠ²Π°Π½ΠΈΡ ΡΡΠ½ΠΊΡΠΈΠΎΠ½Π°Π»ΡΠ½ΠΎΠΉ Π°ΠΊΡΠΈΠ²Π½ΠΎΡΡΠΈ ΠΊΠ»Π΅ΡΠΎΠΊ. Π’Π°ΠΊΠΈΠΌ ΠΎΠ±ΡΠ°Π·ΠΎΠΌ, ΠΈΠ·ΡΡΠ΅Π½ΠΈΠ΅ ΡΡΠΎΠ²Π½Ρ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΠΈ ΡΠ΅ΡΠ΅ΠΏΡΠΎΡΠΎΠ² Π½Π° ΠΌΠ΅ΠΌΠ±ΡΠ°Π½Π΅ ΠΊΠ»Π΅ΡΠΎΠΊ ΡΠ²Π»ΡΠ΅ΡΡΡ Π²Π°ΠΆΠ½ΡΠΌ Π² ΠΏΠΎΠ½ΠΈΠΌΠ°Π½ΠΈΠΈ ΠΏΠ°ΡΠΎΠ³Π΅Π½Π΅Π·Π°, Π° ΠΈΠ·ΠΌΠ΅Π½Π΅Π½ΠΈΠ΅ ΡΡΠΎΠ²Π½Ρ ΡΠΊΡΠΏΡΠ΅ΡΡΠΈΠΈ ΠΌΠΎΠΆΠ΅Ρ ΡΠ°ΡΡΠΌΠ°ΡΡΠΈΠ²Π°ΡΡΡΡ ΠΊΠ°ΠΊ ΡΠ΅ΡΠ°ΠΏΠ΅Π²ΡΠΈΡΠ΅ΡΠΊΠ°Ρ ΠΌΠΈΡΠ΅Π½Ρ Π² Π»Π΅ΡΠ΅Π½ΠΈΠΈ ΡΠ°Π·Π»ΠΈΡΠ½ΡΡ
Π·Π°Π±ΠΎΠ»Π΅Π²Π°Π½ΠΈΠΉ
Redistribution of TNF Receptor 1 and 2 Expression on Immune Cells in Patients with Bronchial Asthma
Background: The co-expression patterns of type 1 and 2 tumor necrosis factor (TNF)-α membrane receptors (TNFR1/TNFR2) are associated with the presence, stage, and activity of allergic diseases. The aim of this study was to assess the expression levels and dynamics of TNFRs on immune cells and to assess associations between their expression and severity of bronchial asthma (BA). Methods: Patients with severe (n = 8), moderate (n = 10), and mild (n = 4) BA were enrolled. As a comparison group, data from 46 healthy volunteers (HV) were accessed. Co-expression of TNFR1/2 was evaluated as a percentage of cells and the number of receptors of each type per cell. Multivariate logistic regression analysis was used to identify diagnostic biomarkers of BA. Results: More than 90% of the monocytes in patients with mild BA were TNFR1+TNFR2+ but had significantly lower TNFR1 expression density compared with HV (7.82- to 14.08-fold, depending on disease severity). Lower percentages of the TNFR+ B-lymphocytes were observed in combination with significantly lower receptors density in BA compared with HV (2.59- to 11.64-fold for TNFR1 and 1.72- to 3.4-fold for TNFR2, depending on disease severity). The final multivariate model for predicting the presence of BA included the percentage of double-positive CD5+ B-lymphocytes and average number of TNFR1 molecules expressed on cytotoxic naive T-lymphocytes and T-helper cells (R2 = 0.87). Conclusions: The co-expression patterns of TNFRs on immune cells in BA differed significantly compared with HV. The expression differences were associated with disease severity. TNFR1 expression changes were key parameters that discriminated patients with BA from those with HV
Co-Expression Profile of TNF Membrane-Bound Receptors Type 1 and 2 in Rheumatoid Arthritis on Immunocompetent Cells Subsets
Introduction: Tumor necrosis factor (TNFα) is an important proinflammatory cytokine in rheumatoid arthritis (RA) immune processes. However, TNFα activity and functions may be regulated by soluble receptors, which act as decoys, and by number, density, and co-expression of its membrane-bound receptors type 1 and 2 (TNFR1 and TNFR2). The aim of this study was to reveal associations between TNFR1/2 co-expression profile parameters and RA disease activity indicators. Methods: PBMC were analyzed from 46 healthy donors and 64 patients with RA using flow cytometry. Patients were divided according to the disease activity score (DAS) 28 index into groups with high (n = 22, 34.4%), moderate (n = 30, 46.9%), and low (n = 12, 18.8%) disease activity. Co-expression of TNFR1 and TNFR2 was studied by evaluating the percentage of cells, with different receptors, and by counting the number of receptors of each type per cell, using QuantiBritePE beads. Associations between disease severity and activity indicators and parameters of TNFα receptor expression in subpopulations of immune cells were studied. Results: T cell subsets from RA patients were characterized by co-expression of TNFR1 and TNFR2, and were found to differ significantly compared with healthy donors. Memory cells both among T helper cells and cytotoxic T cells demonstrated the most significant differences in TNFR-expression profile. Multivariable logistic regression revealed model to identified RA patients from healthy individual based on the TNFR1/2 co-expression parameters. Conclusion: The profile of TNFR12 co-expression differs in RA comparing with health. Proportion of TNFR1+TNFR2- cells increased significantly among memory T helper cells and activated cytotoxic T cells, and decreased significantly among naïve cytotoxic T cells and T regulatory cells as compared with health. The parameters of TNFR12 co-expression in RA are associated with clinical and laboratory indicators of disease activity
Additional file 2: of Generation of populations of antigen-specific cytotoxic T cells using DCs transfected with DNA construct encoding HER2/neu tumor antigen epitopes
Typical scatter plots of gates used in DCs phenotyping analysis. DCs were analyzed by flow cytometry in the region of large granular leukocytes. A β Events corresponding to large granular leukocytes in terms of their phenotypic parameters (forward and side light scattering) are gated. B β Events corresponding CD14-FITC-labeled cells are gated. C β Events corresponding CD83-APC-labeled cells are gated. D β Events corresponding CD86-PE-Cy7-labeled cells are gated. E β Events corresponding double-positive HLA-DR-PerCP-Cy5 and CD11c-PE-labeled cells are gated. (DOCX 785 kb
The Influence of Severity and Disease Duration on TNF Receptorsβ Redistribution in Asthma and Rheumatoid Arthritis
One of the mechanisms of cellular dysfunction during the chronization of immune-system-mediated inflammatory diseases is a change in the profile of expression and co-expression of receptors on cells. The aim of this study was to compare patterns of redistribution of TNF receptors (TNFRs) among patients with different durations of rheumatoid arthritis (RA) or asthma. Subgroup analysis was performed on RA (n = 41) and asthma (n = 22) patients with disease duration10 years and on 30 comparable healthy individuals. The co-expression profile of TNFR1 and TNFR2 was assessed in T cells, B cells, monocytes, regulatory T cells, T-helper subsets, and cytotoxic T-lymphocyte subsets. Percentages of cells with different co-expression combinations and receptor density per cell were estimated. Longer disease duration was significantly associated with a redistribution of receptors in immunocompetent cell subsets with an increase in the expression of TNFR1 in asthma but did not correlate with significant unidirectional changes in receptor expression in RA. In asthma, a higher proportion of cells with a certain type of TNF receptor (as compared with the healthy group) was correlated with a simultaneous greater density of this receptor type. In RA, an inverse correlation was observed (compensatory lower receptor density). Mechanisms of long-term changes in the expression of TNF receptors differ significantly between the diseases of autoimmune and allergic etiology. The formation of irreversible morphostructural alterations was strongly correlated with changes in the expression of TNFR1 in asthma and with changes in the expression of TNFR2 in RA