CD3 receptor modulation in Jurkat leukemic cell line.

CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low) (217.6), CD3+(217.9) or CD3(low) (217.7). The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones

Wpływ zmian ekspresji genów kontrolujących cykl komórkowy na zaburzenia proliferacji limfocytów T u osób chorych na reumatoidalne zapalenie stawów

Praca wykonana w: Katedra i Zakład Fizjopatologii

The in vitro modulatory effect of TNFα on the mRNA expression and protein levels of zinc finger protein ZNF334 in CD4+ lymphocytes of healthy people

We have shown before that the expression of ZNF334 gene, coding for a newly described zinc finger protein of as yet unknown function, is extremely reduced in CD4+ lymphocytes of rheumatoid arthritis (RA) patients regardless of their age, and thus can be considered a new molecular marker of the disease. Based on the promoter sequence of the gene we speculated that it might be regulated by TNFα. Here we have tested that hypothesis, studying the in vitro influence of TNFα on the ZNF334 gene expression and protein levels in resting and stimulated CD4+ cells of healthy volunteers. We have confirmed that treatment with TNFα modifies the levels of ZNF334 expression in the CD4+ cells ex vivo; however, the effect varied for different individuals and reduction of expression was seen only for those cell samples that initially exhibited high transcriptional activity of the gene, while for those exhibiting initially very low expression, some increase in the transcriptional activity was observed. Incubation with TNFα significantly reduced the amounts of two isoforms of ZNF334 protein (initially high in all subjects) in parallel to the reduced transcription. Finally, the expression of ZNF334 in CD4+ lymphocytes isolated after various periods of anti-CD3 stimulation generally increased with longer culture times, and the effect of TNFα treatment was negligible. Concluding, our results obtained in vitro for helper lymphocytes of healthy individuals seem to mimic the regulatory effect of TNFα on the expression of ZNF334 in the cells of RA patients

CD3 receptor modulation in Jurkat leukemic cell line.

CD3 antigen is a crucial molecule in T cell signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3(low) (217.6), CD3+(217.9) or CD3(low) (217.7). The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor--chelerythrine or calcineurin blocker--cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones

Determinants Related to Oxidative Stress Parameters in Pediatric Patients with Type 1 Diabetes Mellitus

Adequate glycemic management is one of the main goals in treating type 1 diabetes mellitus (T1DM) and preventing the early onset of diabetic complications. Improperly controlled diabetes mellitus (DM) will result in oxidative stress (OS) and lead to further related health issues. Therefore, the aim of this study was to evaluate the body’s ability to defend against OS depending on the duration of T1DM, metabolic management, antioxidant intake and modern glycemic monitoring systems (GMS). The study included 103 adolescents with T1DM aged 10–17 years. The control group consisted of 65 healthy peers. The patients’ blood was assayed for antioxidant enzymes, minerals and toxic elements. In addition, their dietary intake of antioxidant components was assessed. The T1DM group had higher total oxidant status, oxidative stress index and Cu/Zn ratio values, higher concentrations of malondialdehyde and lower total antioxidant status (TAS) and chromium, zinc, superoxide dismutase and catalase levels than their healthy peers. The comparison between GMS types revealed favorable changes in OS parameters for the flash and continuous systems. Furthermore, an effect of vitamin A and C dietary intake on serum TAS concentrations was detected. More than 82% of the patients with high TAS fulfilled the estimated average requirement norm for vitamin A, and more than 60% fulfilled the vitamin C requirement. In youths with T1DM, it is advisable to observe the antioxidant activity of the body to prevent the accelerated development of diabetic complications

Serum Levels of α-Klotho, Inflammation-Related Cytokines, and Mortality in Hemodialysis Patients

It has been hypothesized that α-Klotho deficiency might contribute to chronic inflammation in patients with end-stage renal disease (ESRD), especially those on hemodialysis (HD). Serum Klotho levels by some authors are considered a potential predictor of cerebrovascular events. Therefore, we analyzed serum levels of α-Klotho with ELISA and inflammation-related cytokines in HD patients. Sixty-seven HD patients and 19 healthy people were recruited between November 2017 and June 2021. A Cytometric Bead Array (CBA) was used to determine the level of different cytokines: IL-12p70, TNF, IL-10, IL-6, IL-1β, and IL-8. A human Klotho ELISA kit was used to determine the level of α-Klotho in the plasma samples of HD patients. There was no difference in serum levels of α-Klotho between HD patients and healthy people. Patients had increased serum IL-6 and IL-8. Significant positive correlations existed between the concentration of α-Klotho and the serum concentrations of IL-12p70, IL-10, and IL-1β. However, in a multivariable linear regression analysis, only patients’ age was associated independently with α-Klotho level. Serum α-Klotho was not associated with higher mortality risk in HD patients. While these results draw attention to potential relationships between α-Klotho proteins and inflammatory markers in HD patients, our cross-sectional study could not confirm the pathogenic link between α-Klotho, inflammation, and cardiovascular mortality

Concentration of Zinc, Copper, Selenium, Manganese, and Cu/Zn Ratio in Hair of Children and Adolescents with Myopia

The aim of this work was to determine the concentration of trace elements, zinc, copper, selenium, manganese, and Cu/Zn ratio, in scalp hair samples of children and adolescents with myopia. The study included 92 children (mean age 14.5 ± 2.5 years) with myopia and 43 healthy persons (mean age 11.8 ± 4.7 years). Each patient had a complete eye examination. Trace element concentrations in hair were determined by atomic absorption spectrometry. Cu/Zn ratio was also calculated. The zinc level in the hair of myopic patients was significantly higher (260 µg/g) in comparison to the control group (130 µg/g). There was a significantly lower Cu/Zn ratio in myopic patients (0.045) compared with controls (0.07). An insignificant difference was observed in the hair level of copper, selenium, and manganese between patients and controls. The results show that trace elements may play a significant role in the pathogenesis of myopia. Further studies should pay more attention to determine the effect of trace element on children myopia

Gluten-Free Cereals and Pseudocereals as a Potential Source of Exposure to Toxic Elements among Polish Residents

Background: Nutritional food quality plays a crucial role in maintaining human health. However, food and drinking water, along with occupational exposure, are the main routes of exposure to toxic elements for humans. The main aim of this study was to determine the content of As, Cd, Pb and Hg in naturally gluten-free grains and products made from buckwheat, millet, maize, quinoa and oat. The safety of consumption of the products tested was also assessed. Methods: The contents of As, Cd and Pb were determined using inductively coupled plasma mass spectrometry (ICP-MS). To measure Hg, an atomic absorption spectrometry method (AAS) with the amalgamation technique was applied. To assess the level of consumption of the tested products, an online survey was conducted. To estimate health risk, three indicators were used: the target hazard quotient (THQ), cancer risk (CR) and hazard index (HI). The research material obtained 242 different samples without replications. Results: The highest average content of As, Cd, Pb and Hg were observed for the following groups of products: oat (10.19 µg/kg), buckwheat (48.35 µg/kg), millet (74.52 µg/kg) and buckwheat (1.37 µg/kg), respectively. For six samples, exceedance of established limits was found—three in the case of Cd and three of Pb. Due to the lack of established limits, As and Hg content of the tested products was not compared. Generally, no increased health risks were identified. Conclusions: Based on the obtained results, the consumption of gluten-free cereals and pseudocereals available on the Polish market seems to be safe. However, there is a great need to establish maximum levels of the toxic elements, especially As and Hg in cereal products in European legislation