Csaba Orbán

Prevalence of Regulatory T-Cell Subtypes in Preeclampsia.

The prevalence of regulatory T cells (Tregs) is lower in preeclampsia (PE) compared with healthy pregnancy (HP). However, the proportion of recently described Treg subtypes has not been investigated.

Different calcium influx characteristics upon Kv1.3 and IKCa1 potassium channel inhibition in T helper subsets

Functional imbalance between T helper subsets plays important role in the pathogenesis of autoimmune disorders. Transient increase of cytoplasmic calcium level, and sustention of negative membrane potential by voltage sensitive Kv1.3 and calcium‐dependent IKCa1 potassium channels are essential for short‐term lymphocyte activation, thus present possible target for selective immunomodulation. We aimed to investigate calcium influx sensitivity to the inhibition of potassium channels in the main T helper subsets. Peripheral blood from 11 healthy individuals was drawn and calcium influx kinetics following activation with phytohemagglutinin in Th1, Th2, Th17, and Treg cells were evaluated. Alteration of calcium influx induced by specific inhibitors of Kv1.3 and IKCa1 potassium channels, and the expression of Kv1.3 channels were also assessed. Highest cytoplasmic calcium concentration was observed in stimulated Th1 cells, while the lowest level was measured in Treg cells. In Th1 and Th17 cells, inhibition of both investigated potassium channels decreased calcium influx. In Th2 cells only the inhibitor of Kv1.3 channels, while in Treg cells none of the inhibitors had significant effect. Upon the inhibition of IKCa1 channels, short‐term activation of proinflammatory cells was specifically decreased without affecting anti‐inflammatory subsets, indicating that selective immunomodulation is possible in healthy individuals.

Modulation of T Lymphocyte Calcium Influx Patterns Via the Inhibition of Kv1.3 and Ikca1 Potassium Channels in Autoimmune Disorders

Currently available immunotherapies have improved the treatment of autoimmune diseases; however, these therapies are known to have considerable side-effects, such as increasing the susceptibility to infections. Therefore, there is an unmet need for novel immunosuppressive strategies with different mechanisms of action and higher specificity for disease-causing autoreactive T lymphocytes from existing immunomodulators. The increase of the cytoplasmic calcium concentration from intra- and extracellular sources (i.e., the endoplasmic reticulum and store-operated calcium entry through the plasma membrane) is the cornerstone of T lymphocyte activation and functionality. In the course of lymphocyte activation, potassium channels maintain the driving force for sustained calcium influx from the extracellular milieu as they grant the efflux of potassium from the cytoplasm, thus conserving an electrochemical potential gradient between the intra- and extracellular spaces. There are two major types of potassium channels in T cells: the voltage-gated Kv1.3 and the calcium-activated IKCa1 channels. The relation between the calcium currents through calcium release-activated calcium (CRAC) channels and the efflux of potassium makes the proliferation and activation of lymphocytes sensitive to pharmacological modulation of Kv1.3 and IKCa1 channels, and provides an opportunity for targeted intervention. Specific inhibition of these channels results in a diminished calcium influx in lymphocytes and a lower level of lymphocyte activation. Previous data suggest that selective modulation of lymphocyte activation through specific inhibition of potassium channels may be a possible therapeutic approach for the treatment of autoimmune disease (1–,6). Beeton et al. showed that terminally differentiated effector memory T (TEM) cells play a pivotal role in the pathogenesis of autoimmunity (4). Wulff et al. described that the characteristic potassium channel phenotype of TEM cells in multiple sclerosis (MS) is Kv1.3high IKCa1low, contrasting naive, and central memory T (TCM) cells, which exhibit a Kv1.3low IKCa1high channel phenotype (1). Therefore the therapeutic relevance of specific Kv1.3 channel inhibitors is of outstanding interest, as they may offer the possibility for selective modulation of pathogenic TEM cells, while naive and TCM cells (needed for physiological immune responses) would escape the inhibition through upregulation of IKCa1 channel expression. Beeton et al. demonstrated that the symptoms of experimental autoimmune encephalitis, a murine model of MS, significantly improved after treatment with selective Kv1.3 inhibitors (5). Although results from animal models are promising, limited data is available on the effects of potassium channel inhibition on T cell function in humans. Furthermore, besides naive and memory T cells, alterations in the activation pattern of effector (CD4+ helper and CD8+ cytotoxic) T lymphocytes have not been described upon Kv1.3 and IKCa1 inhibition. Although these cells might have a less-specific role in the maintenance of autoreactivity compared to TEM cells, their inhibition have important consequences on the overall immune response. Therefore, over the recent years, we have investigated calcium influx characteristics in effector T cell subsets in a number of autoimmune disorders. We isolated peripheral blood mononuclear cells from MS, rheumatoid arthritis (RA) and type 1 diabetes mellitus (T1DM) patients and applied a novel flow cytometry-based approach for the detection of calcium influx (7–10). Until the recent past, single-cell techniques were used for the investigation of calcium influx during lymphocyte activation. There has been no high-throughput method available to study the kinetics of lymphocyte activation in more subsets at the same time. Single-cell techniques are restricted by not being capable of characterizing this process in complex cellular systems, thus ignoring the interaction between the different lymphocyte subsets that may modulate the course of their activation. Therefore, over the recent years we have developed a novel algorithm that allows simultaneous monitoring of calcium influx in several lymphocyte subsets. Our software (FacsKin) fits functions to median values of the data of interest and calculates relevant parameters describing each function. By selecting the best fitting function, this approach provides an opportunity for the mathematical analysis and statistical comparison of kinetic flow cytometry measurements of distinct samples (more details available at www.facskin.com). Our findings indicate important differences in calcium influx kinetics in the studied autoimmune disorders compared to healthy controls.

Coherences of Instrumental and Sensory Characteristics: Case Study on Cherry Tomatoes

The aim of this study was to investigate 6 cherry tomato varieties in terms of morphological, instrumental, and sensory attributes. Hungarian cherry tomato landraces have not been investigated in comparison with new commercial varieties for these traits. Parameters investigated were water-soluble antioxidant capacity (FRAP, DPPH, and TEAC), and total polyphenol, vitamin C, β-carotene, lycopene, total soluble solids, and acid contents. Colorimetric measurements as well as sensory analyses were conducted. It was concluded that varied antioxidant assays should be used in parallel to overcome the selectivity of any 1 method. Total phenolic content significantly contributed to results of antioxidant assays for the investigated varieties. The sensory profiles of the 6 cherry tomato varieties have been created. The differences between the products based on the 18 attributes were analyzed by Tukey post hoc test. The biplot of the principal component analysis showed that the sensory panel could discriminate the samples along the principal components. No correlation was found between colorimetric data a* and b* measured from pulp and lycopene, but a negative connection of β-carotene and hue was noted. Total polyphenol content showed correlations with colorimetric results, except for b*. The influence of tomato skin color on color perception is significant as in the present study instrumental data measured from pulp did not match that of the panelists evaluating intact fruit. Instrumental results of sugar content were supported by the ratings of the sensory panel.

Calcium influx kinetics, and the features of potassium channels of peripheral lymphocytes in primary Sjögren’s syndrome

OBJECTIVE The transient increase of the cytoplasmic free calcium level plays a key role in the process of lymphocyte activation. Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx and present a possible target for selective immunomodulation. DESIGN Case-control study. SUBJECTS AND METHODS We took peripheral blood samples from 8 healthy individuals and 15 primary Sjögrens syndrome (pSS) patients. We evaluated calcium influx kinetics following activation in peripheral T lymphocytes. We also assessed the sensitivity of T lymphocytes to specific inhibition of the Kv1.3 and IKCa1 potassium channels, and the Kv1.3 channel expression. RESULTS The basal cytoplasmatic calcium levels were lower in both Th1 and Th2 lymphocytes in pSS compared to controls. The peak of calcium influx in lymphocytes isolated from pSS patients is reached later, indicating that they respond more slowly to stimulation compared to controls. In healthy individuals, the inhibition of the IKCa1 channel decreased calcium influx in Th2 and CD4 cells to a lower extent than in Th1 and CD8 cells. On the contrary, the inhibition of Kv1.3 channels resulted in a larger decrease of calcium entry in Th2 and CD4 than in Th1 and CD8 cells. In the pSS group, neither of the inhibitors induced alteration in calcium influx. Expression of Kv1.3 channels on CD4, Th2 and CD8 lymphocytes in pSS was significantly higher compared to controls. CONCLUSION The altered expression and specific inhibition of potassium channels seem to be related to altered calcium influx kinetics in pSS which distinguish pSS either from healthy controls or other systemic autoimmune diseases.

Altered calcium influx of peripheral Th2 cells in pediatric Crohn’s disease: infliximab may normalize activation patterns

Objective Crohns disease is a chronic inflammation of the gastrointestinal tract with an abnormal immune phenotype. We investigated how intracellular calcium kinetics of Th1 and Th2 lymphocytes alter upon specific inhibition of Kv1.3 and IKCa1 channels in pediatric Crohns disease. Study design Blood was taken from 12 healthy and 29 Crohns disease children. Of those, 6 were switched to infliximab and re-sampled after the 4th infliximab treatment. Intracellular calcium levels were monitored using flow cytometry in the presence or absence of specific inhibitors of Kv1.3 and IKCa1 potassium channels. Results In Crohns disease treated with standard therapy, calcium response during activation was higher than normal in Th2 cells. This was normalized in vitro by inhibition of Kv1.3 or IKCa1 potassium channels. After the switch to infliximab, potassium channel function and expression in Th2 lymphocytes were comparable to those in Th1 cells. Conclusion These results may indicate that potassium channels are potential immune modulatory targets in Crohns disease.

Real time kinetic flow cytometry measurements of cellular parameter changes evoked by nanosecond pulsed electric field.

Nanosecond pulsed electric field (nsPEF) is a novel method to increase cell proliferation rate. The phenomenon is based on the microporation of cellular organelles and membranes. However, we have limited information on the effects of nsPEF on cell physiology. Several studies have attempted to describe the effects of this process, however no real time measurements have been conducted to date. In this study we designed a model system which allows the measurement of cellular processes before, during and after nsPEF treatment in real time. The system employs a Vabrema MitoplicatorTM nsPEF field generating instrument connected to a BD Accuri C6 cytometer with a silicon tube led through a peristaltic pump. This model system was applied to observe the effects of nsPEF in mammalian C6 glioblastoma (C6 glioma) and HEK‐293 cell lines. Viability (using DRAQ7 dye), intracellular calcium levels (using Fluo‐4 dye) and scatter characteristics were measured in a kinetic manner. Data were analyzed using the FACSKin software. The viability and morphology of the investigated cells was not altered upon nsPEF treatment. The response of HEK‐293 cells to ionomycin as positive control was significantly lower in the nsPEF treated samples compared to non‐treated cells. This difference was not observed in C6 cells. FSC and SSC values were not altered significantly by the nsPEF treatment. Our results indicate that this model system is capable of reliably investigating the effects of nsPEF on cellular processes in real time.

Kv1.3 lymphocyte potassium channel inhibition as a potential novel therapeutic target in acute ischemic stroke

Stroke-induced immunosuppression (SIIS) leads to severe complications in stroke patients, including an increased risk of infections. However, functional alterations of T lymphocytes during SIIS are poorly described in acute ischemic stroke (AIS). We aimed to characterize Ca(2+) influx kinetics in major lymphocyte subsets (CD4, Th1, Th2, CD8) in AIS patients without infection 6 hours and one week after the CNS insult. We also assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 lymphocyte K(+) channels. We took peripheral blood samples from 12 non-stroke individuals and 12 AIS patients. We used an innovative flow cytometry approach to determine Ca(2+) influx kinetics and the surface expression of Kv1.3 channels. Our results indicate that Ca(2+) influx kinetics is altered in the Th2 and CD8 subsets in AIS which may play a role in the development of SIIS. Specific inhibition of Kv1.3 channels selectively decreased Ca(2+) influx in the CD8 and Th2 subsets of AIS patients. The surface expression of Kv1.3 channels is also altered compared to non-stroke individuals. Kv1.3 channel inhibition might have beneficial therapeutic consequences in AIS, selectively targeting two distinct T cell subsets at two different time points following the CNS insult. Within hours after the insult, it might prevent excessive tissue injury through the inhibition of CD8 cells, while at one week after the insult, it may improve the inflammatory response through the inhibition of Th2 cells, thus reducing the unwanted clinical consequences of SIIS.

Effects of caffeine and phosphodiesterase inhibitors on activation of neonatal T lymphocytes

Caffeine and selective PDE inhibitors are widely used in clinical management of preterm and term neonates. However, little is known about how these compounds interact with the neonatal adaptive immune system. We aimed to describe the effects of caffeine, milrinone and sildenafil on the activation and cytokine production of T cells from umbilical cord blood (UCB) compared to adult peripheral blood (APB). We isolated mononuclear cells from 10 APB and 6 UCB samples. We assessed intracellular cytokine production (IFN-γ, IL-2, IL-4, IL-6, IL-17) of stimulated CD4 cells and parameters of calcium influx and ROS production following treatment with caffeine, milrinone, sildenafil, dbcAMP or a specific A2A receptor antagonist, ZM241385 using flow cytometry. In ABP, only ZM241385 caused a 1.14-fold increase in calcium influx, while all compounds increased calcium influx in UCB. This effect was more pronounced in case of caffeine (1.41-fold) and dbcAMP (1.3-fold) compared to milrinone (1.22-fold), sildenafil (1.23-fold) or ZM241385 (1.23-fold). Intracellular levels of the studied cytokines were unaffected by the applied compounds in both APB and UCB samples. Caffeine increases calcium influx upon activation in neonatal T lymphocytes to a larger extent than milrinone or sildenafil. This effect appears to be mediated primarily via increased cAMP levels rather than A2A receptor inhibition. Overall, the application of caffeine, sildenafil or milrinone does not appear to have immunosuppressive effects on neonatal T cells.

Altered activation of peripheral CD8+ T cells in pediatric Crohn's disease

AIM Although Crohns disease (CD) is an extensively investigated autoimmune condition, knowledge on early phase activation of lymphocytes, especially CD8+ Tc cells is scarce. Our aim was to investigate the calcium influx characteristics of CD8+ cells upon activation as well as the expression and function of Kv1.3 and IKCa1 lymphocyte potassium channels. METHODS We took peripheral blood from 12 healthy controls, 23 CD children on conventional therapy and 6 severe CD children before and after infliximab therapy. Intracellular calcium levels were monitored in CD8+ lymphocytes using flow cytometry. RESULTS In CD treated with standard therapy calcium response during activation was elevated. This was not affected by the inhibition of Kv1.3 or IKCa1 potassium channels. After the switch to infliximab potassium channel function and expression of CD8+ lymphocytes were comparable to healthy controls in severe CD. CONCLUSION Calcium handling of CD8+ lymphocytes is altered in pediatric CD, which is normalized by infliximab therapy.