Production of hemo- and immunoregulatory cytokines by erythroblast antigen(+ )and glycophorin A(+ )cells from human bone marrow

BACKGROUND: Erythroid nuclear cells (ENC) of the bone marrow (BM) have not previously been considered as important producers of wide spectrum of haemo- and immunoregulatory cytokines. The aim of the current work was to confirm the production of the main hemo- and immunoregulatory cytokines in human ENC from BM. RESULTS: We used native human BM ENC in our experiments. We for the first time have shown, that the unstimulated erythroblasts (Gl A(+ )or AG-EB(+)) produced a wide spectrum of immunoregulatory cytokines. Human BM ENC produce cytokines such as interleukn (IL)-1β, IL-2, IL-4, IL-6, interferon (IFN)-γ, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α and IL-10. They can be sub-divided into glycophorin A positive (Gl A(+)) and erythroblast antigen positive (AG-EB(+)) cells. To study potential differences in cytokine expression between these subsets, ENC were isolated and purified using specific antibodies to Gl A and AG-EB and the separated cells were cultivated for 24 hours. The cytokine contents of the supernatant were measured by electrochemiluminescence immunoassay. Quantitative differences in TGF-β1 and TNF-α production were found between Gl A(+ )and AG-EB(+ )BM ENC. Furthermore, in vitro addition of erythropoietin (EPO) reduced IFN-γ and IL-2 production specifically by the AG-EB(+ )ENC. Thus, Gl A(+ )and AG-EB(+ )ENC produce IL-1β, IL-2, IL-4, IL-6, IFN-γ, TGF-β1 and TNF-α. Gl A(+ )ENC also produce IL-10. CONCLUSION: Cytokine production by erythroid nuclear cells suggests that these cells might be involved in regulating the proliferation and differentiation of hematopoietic and immunocompetent cells in human BM

EFFECT OF DNA CONSTRUCTIONS ELECTROPORATION ON DENDRITIC CELLS

Today transfection of mammalian cell with DNA or RNA construction is the only method for delivering programmed information into the cell nucleus. Electroporation is most commonly used method of transfection in experiments with dendritic cell. The aim of electroporation is to permeabilize the membrane by passing electric impulse through the cell. Due to the increase permeability of the membrane chance DNA or RNA construction getting inside into the cell is increased, wherein survival of the cells is decreased.In the study male mice C57Bl/6 line 2-4 months old were used. From femur bones was isolated 20 × 106 bone marrow cells, which were cultured in 20 mL of complete RPMI-1640 for 7 days. To generate dendritic cells from BM cells, 100 ng/mL of rmFlt3-L was added to culture media at day 0. After 7 days of cultivation, the cell cultures were electroporated with control noncoding plasmids p5 (EP P5) or pmaxCCR9 encoding mouse chemokine receptor CCR9 (EP CCR9). The controls were cell cultures electroporated without any plasmids (mock EP) and cell cultures without electroporation (none EP). 5 × 105 cells were electroporated and resting for 10 minutes. After 10 minutes, cells were harvested and seeded into 24-well plates in 1 mL of culture medium and conditioning medium (1:1). Then, 50 ng/mL of Flt3-L was added to each well. The next day, transfected cells were collected and used for flow cytometry, qRT-PCR analysis.It was found that after electroporation in the groups mock EP, EP P5, EP CCR9 relative amount of live CD11c+ dendritic cells was significantly less than in the non EP group. Moreover, in the EP P5 and EP CCR9 groups the frequency of live CD11c+ dendritic cells was significantly less than in the mock EP group. Expression of the CD86 marker in the EP P5 and EP CCR9 groups was significantly higher than in the non EP and mock EP groups. Expression of the I-AB(MHCII) in the EP CCR9 group on cDC2s was significantly higher than in the non EP group. On plasmacytoid DCs (pDCs) and conventional type 2 DCs (cDC2s) in the EP CCR9 group expression of CCR9 was significantly higher than in the non EP group. Therefore, in this study, we demonstrated the effectiveness of electroporation, accompanied by the decrease in the survival rate and maturation of DCs

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 −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 −3609C/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 TNF receptors and TNF -mediated signaling

SERUM LEVEL AND PRODUCTION OF CYTOKINES BY PBMC IN PATIENTS WITH ATOPIC DERMATITIS

The рарег presents the results of measurements of cytokine levels in serum and conditioned medium of PBMC cultures from the patients with atopic dermatitis with the exacerbation and remission in comparison with the healthy donors. We have shown that the serum levels of the key cytokines IL-5 and IL-13, proinflammatory cytokines IL-1β and IL-6 and the main Th1 cytokine — IFNγ — were higher compared to healthy donors. In the conditioned media of peripheral blood mononuclear cells in contrast, we have found a significant decrease of the spontaneous secretion of key cytokines IL-10 and IL-17. We have shown that the stimulated secretion of IL-5 and IL-13, IL-12 and INF-γ is significantly reduced in comparison with the control level. Only IL-1β revealed a statistically significant higher level of stimulated secretion without exacerbation of atopic dermatitis. The contemporary therapy has no effect on cytokine production

PENTOXIFYLLINE ENHANCES IN VITRO T-CELL ANTITUMOR RESPONSE IN BREAST CANCER PATIENTS

The NF-κB transcription factor controls the expression of genes responsible for cell cycle, apoptosis, and other immunoregulatory functions. Some nonspecific NF-κB inhibitors were found after discovering the possibility of blocking tumor growth through suppression of NF-κB activity, but their use was complicated by multiple side effects, such as interleukin-1β-related systemic inflammation or non-immunerelated complications, which may be due to inhibition of the p65 NF-κB subunit that plays a central role in organogenesis and inflammation. Inhibition of the c-Rel subunit leads to tumor growth restriction by modulating the T-regulatory cell activity.In 2017, Grinberg-Bleyer and co-authors checked the hypothesis that selective inhibition of the c-Rel subunit can be performed using pentoxifylline and will effectively regulate Treg activity during tumor growth. The authors showed that pentoxphylline, an FDA-approved drug, could indeed induce selective degradation of c-Rel without affecting p65, and suggested that such an effect could be effective in suppressing tumor growth. In this regard, we aimed to investigate in vitro how pentoxifylline affect the functional activity and antitumor cytotoxic potential of T-cells in cancer patients.The objects of the study were peripheral blood mononuclear cells from 25 patients with primary breast cancer (no metastases), 15 patients with metastatic breast cancer, and 25 healthy women without breast pathology. Informed consent was obtained from all donors and patients. The study was approved by the local ethics committee.Here we showed that pentoxifylline treatment in vitro enhances the pro-apoptotic and cytotoxic antitumor response via increasing the expression of TRAIL on T-lymphocytes, mainly in healthy donors and patients with metastatic breast cancer, both on intact T-cells and in response to the cells of the tumor line of human breast carcinoma ZR-75-1. In healthy donors, in the presence of pentoxifylline, a population of highly expressing TRAIL CD4 and CD8 T-cells appears

CO-EXPRESSION OF MEMBRANE-BOUND TUMOR NECROSIS FACTOR-α RECEPTORS IN MAJOR SUBPOPULATIONS OF IMMUNOCOMPETENT CELLS IN HEALTHY INDIVIDUALS AND PATIENTS WITH RHEUMATOID ARTHRITIS AS WELL AS BRONCHIAL ASTHMA

A pleiotropic cytokine TNFα is an important inflammatory mediator of a number of diseases; its biological functions are fulfilled through two different receptors, TNFR1 and TNFR2. Changes in the ratio between these types of receptors shifting the balance between the pro-apoptotic and proliferation signaling pathways play a crucial role in eliciting the cell response to TNFα. The pathological processes in the body can alter the levels of TNFR1 and TNFR2 expression on the cells involved in disease development. Therefore, this study was aimed at investigating the level of co-expression of type 1 and 2 TNFα receptors in the major subpopulations of peripheral blood cells in patients with rheumatoid arthritis (RA) and bronchial asthma (BA). The greatest changes in the percentage of cells expressing TNFR1 and TNFR2 were revealed for the B-lymphocyte subpopulation. For the T-lymphocyte subpopulation, there were some differences in the percentage of cells expressing exclusively TNFR1 in RA and BA patients compared with those in healthy subjects, as well as between the RA and BA groups. A higher percentage of double-negative monocytes was observed in patients with BA and RA compared to healthy subjects. These findings indicate that the coexpression profile of TNFR1 and TNFR2 receptors in patients with RA and BA differ within these groups as well as compared to that in healthy subjects. These immune cell populations are actively involved in the pathogenesis of both rheumatoid arthritis and bronchial asthma, so the results may indicate that these cells might show different responses to TNFα as the percentage and the number of receptors on their surface vary

The solvation and dissociation of 4-benzylaniline hydrochloride in chlorobenzene

A reaction scheme is proposed to account for the liberation of 4-benzylaniline from 4-benzylaniline hydrochloride, using chlorobenzene as a solvent at a temperature of 373 K. Two operational regimes are explored: “closed” reaction conditions correspond to the retention of evolved hydrogen chloride gas within the reaction medium, whereas an “open” system permits gaseous hydrogen chloride to be released from the reaction medium. The solution phase chemistry is analyzed by 1H NMR spectroscopy. Complete liberation of solvated 4-benzylaniline from solid 4-benzylaniline hydrochloride is possible under “open” conditions, with the entropically favored conversion of solvated hydrogen chloride to the gaseous phase thought to be the thermodynamic driver that effectively controls a series of interconnecting equilibria. A kinetic model is proposed to account for the observations of the open system

Production of immunoregulatory molecules by induced erythroblasts at various stages of cell differentiation

Introduction. Bone marrow erythroblasts produce a wide range of cytokines with opposite biological effects. This may be due to a change in the spectrum of production of immunoregulatory mediators during differentiation and small qualitative and quantitative differences in the spectrum of cytokines produced at each stage of differentiation, which may be important for the regulation of hemo- and immunopoiesis.   The aim. To study the spectrum of production of mediators by erythroblasts at different stages of differentiation.   Methods. Erythroblasts were obtained from CD34+ bone marrow cells of healthy donors in the presence of recombinant cytokines. Phenotype assessment was performed using flow cytometry for erythroid (CD45, CD71, CD235a, CD44) and lymphoid markers (CD3, CD4, CD8, CD16, CD19). Blocking of erythroblast differentiation at different stages was carried out using specific blocking monoclonal antibodies to melanocortin receptors (MCR) of types 1, 2 and 5. Cytokine analysis in conditioned erythroblast media was performed using the Bio-Plex Pro Human Cytokine 48-Plex Screening Panel (Bio-Rad Laboratories, USA). Cytokine production was analyzed using the CytokineExplore online tool.   Results. The resulting erythroblasts are divided into positive and negative populations according to the CD45 marker, carry markers of erythroid cells CD71, CD235a and do not express linear markers of lymphoid cells. In type 1 MCR blockage, polychromatophilic erythroblasts predominate, in type 2 MCR blockage, basophilic erythroblasts predominate, and in type 5 MCR blockage, orthochromatophilic erythroblasts accumulate. According to the production of cytokines, it was shown that when using any of the blocking antibodies, we obtain cells that differ qualitatively and quantitatively in a number of mediators from the initial population of induced erythroblasts.   Conclusion. Thus, we have shown qualitative and quantitative differences in the production of mediators by erythroblasts depending on the stage of differentiation, which can lead to different regulatory effects

THE FREQUENCY OF ALLELIC VARIANTS OF GENES TNFRI AT POSITIONS -609 AND -1207 AND TNFRII AT POSITIONS -1709 AND -3609 IN HEALTHY DONORS AND PATIENTS WITH RHEUMATOID ARTHRITIS

In this paper we studied the frequency of allelic variants of genes TNFRI at positions -609 and -1207 and TNFRII at positions -1709 and -3609 in patients with rheumatoid arthritis and in population control group. It was shown that the resulting frequency distribution of alleles and genotypes of TNFRI gene promoter in a group of population control is characteristic for Asian populations, for TNFRII gene polymorphisms and the frequency distribution differed from the European and Asian populations. Comparative analysis of allele and genotype frequencies of TNFRI gene promoter at positions -609 and -1207 and TNFRII and -3609 and positions -1709 in patients with rheumatoid arthritis and in population control group showed no statistically significant differences. However in the analysis of combinations has been shown that in patients with rheumatoid arthritis compared with the control population was significantly less common combination of genotypes TNFRI-609GT + TNFRII-3609CC