Unfavorable effect of levetiracetam on cultured hippocampal neurons after hyperthermic injury.

BACKGROUND: The aim of this study was to examine the viability of neurons and the putative neuroprotective effects of second-generation antiepileptic drug, levetiracetam (LEV), on cultured hippocampal neurons injured by hyperthermia. METHODS: Primary cultures of rat's hippocampal neurons at 7day in vitro (DIV) were incubated in the presence or absence of LEV in varied concentrations under hyperthermic conditions. Cultures were heated in a temperature of 40°C for 24h or in a temperature of 41°C for 6h. Flow cytometry with Annexin V/PI staining as well as fluorescent microscopy assay were used for counting and establishing neurons as viable, necrotic or apoptotic. Additionally, the release of lactate dehydrogenase (LDH) to the culture medium, as a marker of cell death, was evaluated. Assessment was performed after 9DIV and 10 DIV. RESULTS: Incubation of hippocampal cultures in hyperthermic conditions resulted in statistically significant increase in the number of injured neurons when compared with non-heated control cultures. Intensity of neuronal destruction was dependent on temperature-value. When incubation temperature 40°C was used, over 80% of the population of neurons remained viable after 10 DIV. Under higher temperature 41°C, only less than 60% of neurons were viable after 10 DIV. Both apoptotic and necrotic pathways of neuronal death induced by hyperthermia were confirmed by Annexin V/PI staining. CONCLUSIONS: LEV showed no neuroprotective effects in the current model of hyperthermia in vitro. Moreover, drug, especially when used in higher concentrations, exerted unfavorable intensification of aponecrosis of cultured hippocampal neurons.

Decreased Frequencies of Peripheral Blood CD4+CD25+CD127-Foxp3+ in Patients with Graves' Disease and Graves' Orbitopathy: Enhancing Effect of Insulin Growth Factor-1 on Treg Cells.

Graves' orbitopathy (GO) is characterized by orbital T cell infiltration. We evaluated the regulatory T (Treg) cell fractions induced with IGF-1 in Graves' disease (GD) with and without GO. Peripheral blood mononuclear cells (PBMCs) were obtained from 13 patients with GD without eye manifestations; 10 patients with active GO; and 12 patients with nodular goiter (NG). All the patients from GD, GO, and NG were subclinical hyperthyroid. We analyzed the expression of Treg cell markers (CD4, CD25, CD127-, Foxp3) on T cells and their ability to respond to IGF-1 stimulation. In patients with GD without GO, we found lowered percentages of CD4+ Foxp3+ cells, as compared to nodular goiter 1.77 vs. 5.42% (p=0.0276). Similarly, significantly reduced frequencies of CD4+CD25+CD127-Foxp3+ and CD4+CD25+CD127- cells were observed in GD patients as compared to nodular goiter patients with hyperthyreosis, (0.7 vs. 1.48%) (p=0.0071) and (14.5 vs. 37.2%) (p=0.0051), respectively. In GO with active GO, only the percentage of CD4+CD25+CD127- cells was found to be decreased versus nodular goiter (9.35 vs. 37.2) (p=0.0275). Stimulation of PBMC derived from GO patients with IGF-1 resulted in significant increase of frequency of both CD4+ Foxp3+ and CD4+CD25+CD127- Foxp3 cells. Decreased frequencies of peripheral blood CD4+CD25+CD127-Foxp3+ in patients with GD and GO could be an useful marker of autoimmune process and perhaps a possible target for future therapies. This is the first study demonstrating Treg-enhancing effects of IGF-1. Thus IGF-1 can be accounted for modulating Treg cell-related action in GO.

Decrease of interleukin (IL)17A gene expression in leucocytes and in the amount of IL-17A protein in CD4+ T cells in children with Down Syndrome.

BACKGROUND: Down Syndrome is by far the most common and best known chromosomal disorder in humans. It expresses multiple systemic complications with both structural and functional defects as part of the clinical manifestation. The mechanisms of immune changes occurring in Down Syndrome are complex and include an extra gene copy of chromosome 21 and secondary dysregulation of numerous intercellular interactions. Recent studies suggest a role of interleukin 17A (IL-17A), a pro-inflammatory cytokine located on 6p12 chromosome, in the pathogenesis of inflammatory and autoimmune diseases. We aimed to analyze IL17A gene expression in peripheral white cells and IL-17A intracellular expression on CD4+ T-cells. METHODS: The research was carried out on a group of 58 children aged 6-12 years including a group of 30 children with Down Syndrome (simple trisomy of chromosome 21 only) and a reference group of 28 healthy children. We evaluated gene IL17A expression using real-time PCR and intracellular IL-17A analyzed by flow cytometry. RESULTS: We found significantly decreased gene expression in white cells and significantly decreased expression of IL-17A levels on CD4+ T-cells in Down Syndrome. CONCLUSIONS: Our data indicate that decreased IL-17A expression may play a significant role in the etiology of infections in Down Syndrome. Moreover, we demonstrated that in Down Syndrome the other gene located outside the extra chromosome 21 is also affected.

Fibroblast-Like Synovial Cells in Rheumatoid Arthritis--the Impact of Infliximab on Hexosaminidase Activity.

BACKGROUND: The effect of multiple infusions of infliximab (INF), a chimeric anti-tumor necrosis factor alpha antibody, on the concentration of hexosaminidase (HEX) activity in a synovial cell culture derived from human synovial inflamed fluid obtained from patients suffering from rheumatoid arthritis (RA) has been evaluated. OBJECTIVES: The aim of this study was to prove INF efficacy in RA. MATERIAL AND METHODS: Inflamed synovial fluid was taken from RA patients (a study group) and patients who had undergone knee trauma within 7 days (a control group). The following solutions of infliximab were used: 40, 60 and 140 µg/mL. Determination of the concentration of HEX activity in cell cultures was performed after 24, 48, 72 and 96 h of infliximab administration. To identify synoviocytes in cell culture immunohistochemical staining with vimentin and pancytokeratin was performed. RESULTS: A predominance of fibroblast-like synovial cells has been observed in the study group. In the control group the concentration of HEX activity without adding infliximab to the cell culture was 283.00 nkat/mL. After 96 h of incubation with infliximab, the concentrations of HEX activity in cultured synoviocytes according to infliximab doses of 40, 60 and 140 µg/mL were respectively: 280.00, 271.50 and 293.50 nkat/mL. In the study group, the concentration of HEX activity without adding infliximab to the cell culture was 542.27 nkat/mL. The final concentrations of HEX activity of cultured fibroblast-like synovial cells measured after 96 h of incubation with infliximab were: 471.72, 498.27 and 556.72 nkat/mL, according to infliximab doses of 40, 60 and 140 µg/mL. In all groups (besides the infliximab concentration of 140 µg/mL after 96 h of incubation), the level of concentration of HEX activity was significantly higher in the study group compared to the control group, irrespective of infliximab concentration and time of infliximab incubation. CONCLUSIONS: Infliximab changes the concentration of HEX activity depending on the drug dose and time of administration.