PECAM-1 Is Down-Regulated in γδT Cells during Remission, but Up-Regulated in Relapse of Multiple Sclerosis.

Introduction. PECAM-1 and NKRP1A are both involved in the vascular transmigration of T lymphocytes. Vascular transmigration is a crucial process in multiple sclerosis pathogenesis. Methods and aim. The current paper presents an analysis of PECAM-1 and NKRP1A expression on γδ T cells. Expression of PECAM-1 and NKRP1A on subsets of γδ T cells was performed with flow cytometry. Results. Based on the flow cytometry data, PECAM1 was slightly differentially modulated on γδ T cells-it was up-regulated during relapse, but down-regulated during remission. Moreover, a significant downregulation of CD3 expression was noted on γδ T cells from MS patients, most notably during relapse. Conclusions. This may be a sign of the overall activation of γδ T cells in the course of multiple sclerosis.

IL-15 Is Overexpressed in γδ T Cells and Correlates with Disease Severity in Relapsing-Remitting Multiple Sclerosis.

Interleukin 15 (IL-15) is known to be involved in the pathogenesis of multiple sclerosis (MS). An animal study revealed a distinct subset of IL-15-producing γδ T cells that correlate with disease severity. The aim of the current study was to test whether such a subset is also present in humans and its importance for the pathogenesis of MS. The peripheral blood from 29 patients with relapsing-remitting MS (including 6 relapses) and 22 controls was stained with monoclonal antibodies and analyzed with flow cytometry. The existence of IL-15+ γδ T cells was confirmed. Moreover, the percentage of IL-15+ γδ T is significantly increased in MS patients and correlates with disease severity. Nevertheless, additional functional studies are needed to fully understand the importance of those cells in multiple sclerosis pathogenesis.

RORγT is overexpressed in iNKT and γδ T cells during relapse in relapsing-remitting multiple sclerosis.

The aim of the current study is to evaluate IL-17 production and RORγT, and IL-23R expression by iNKT, Th17 and γδ T cells in the peripheral blood of relapsing-remitting multiple sclerosis patients. Samples of peripheral blood from 21 relapse patients and 12 remission patients, and 15 healthy volunteers were stained with monoclonal antibodies for flow cytometry analysis. No significant differences in iNKT, γδ T and Th17 percentages were noted. The significant overexpression of RORγT was observed in all three subpopulations - therefore, iNKT, γδ T and Th cells may be an important source of IL-17 shortly prior to the relapse.

Decrease in Lipid Droplets in Adrenal Cortex of Male Wistar Rats after Chronic Exposure to Energy Drinks.

Background and objectives: Energy drinks are popular non-alcoholic beverages. They are consumed in large amounts, mainly by active, young people. Although they are easily accessible and marketed as safe, numerous cases of adverse effects have been published, including cardiac arrest, arrythmias, acute hepatitis, and renal failure. The aim of the current study is the assessment of energy drink influence on the histological structure of adrenal cortex in rats. Material and Methods: 15 male young Wistar rats were equally divided into three groups: control (C), experimental (E) and reversibility control (RC). C group received water and standard rodent food ad libitum while both E and RC groups had additionally unlimited access to energy drinks. C and E groups were decapitated after 8 weeks and RC was given another 8 weeks without energy drinks. Adrenal glands were embedded in paraffin blocks and 5 µm slides were prepared and stained according to standard H&E and Masson's trichrome protocols. Additionally, immunohistochemical stainings against Ki-67, p53, CTGF and caspase-3 were prepared. Results: Decreased vacuolization and numerous pyknotic nuclei were noted in E and RC groups. Overexpression of caspase-3 was noted both subcapsular in zona glomerulosa and along sinusoids in zona fasciculata. Increased collagen deposition in zona glomerulosa and zona fasciculata of E and RC was observed. Insular and irregular overexpression of CTGF was noted. The overall picture of CTGF expression matched the Masson's trichrome. No significant difference was observed in Ki-67 expression. Conclusions: The results of the current study suggest that the stimulation is so intense that it causes significant damage to adrenal cortical cells, resulting in their apoptosis. It seems, however, that the observed effects are at least partially reversible.

Determination of affinity and efficacy of acetylcholinesterase inhibitors using isothermal titration calorimetry.

BACKGROUND: Acetylcholinesterase (AChE), an enzyme rapidly terminating nerve signals at synapses of cholinergic neurons is an important drug target in treatment of Alzheimer's disease and related memory loss conditions. Here we present comprehensive use of isothermal titration calorimetry (ITC) for investigation of AChE kinetics and AChE-inhibitor interactions. METHODS: Acetylcholinesterase (AChE, EC 3.1.1.7) from Electrophorus electricus was assayed for interactions with five well known AChE inhibitors, galanthamine, tacrine, donepezil, edrophonium and ambenonium. In ITC experiments the inhibitors were injected to the enzyme solution solely (for thermodynamic characterization of binding) or in presence of the substrate, acetylcholine (for determination of inhibitors potency). RESULTS: Detailed description of various experimental protocols is presented, allowing evaluation of inhibitors potency (in terms of IC50 and Ki) and thermodynamic parameters of the binding. The potency of tested inhibitors was in nano to micromolar range which corresponded to activities determined in conventional method. Binding of all inhibitors showed to be enthalpy driven and obtained Ka values demonstrated good correlation with the data from standard Ellman's assay. CONCLUSIONS: Obtained results confirmed the usability of the ITC technique for comprehensive characterization of AChE-inhibitor interactions and AChE kinetics. The method reduced the complexity of reaction mixture and interference problems with the advantage of using natural substrates. GENERAL SIGNIFICANCE: The work reports complete thermodynamic characteristics of the AChE - inhibitor complexes. Due to the universal character of ITC measurements, described protocols can be easily adapted to other enzymatic systems.