Chemotactic responses play a significant role during Leishmania (V.) braziliensis differentiation... more Chemotactic responses play a significant role during Leishmania (V.) braziliensis differentiation through its life cycle and during infection. The aim of this description has been to portray the modified “two-chamber capillary chemotaxis assay” as a technique useful for quantitative in vitro evaluation of Leishmania chemotaxis after reviewing the methods described until now to assess chemotaxis in vitro in Leishmania sp. This valued simple and reproducible method convenient for parasite migration determination, was tested by the use of controlled changes in monosaccharide (D-glucose and D-fructose) concentrations as referent ligands. The validation of the method demonstrates that this technique is useful to evaluate the relationship existing between parasite migration towards the monosaccharides and sugar concentration. This means that within specific ranges, parasites attracted by the monosaccharide migrate towards more concentrated solutions and accumulate (higher number of parasites) at that spot. Interestingly, both the time course of the experiment and the osmolality of the solution influence parasite migration capacity. Our validation suggests that this improved methodology quantitatively evaluates taxis of Leishmania towards/against different substances. On the basis of our herein presented data, we conclude that this technique is a novel, rapid and reliable screening method to evaluate chemotaxis in Leishmania.• The two-chamber capillary chemotaxis assay was standardized for Leishmania.• The technique is useful to quantitatively evaluate in vitro chemotaxis in Leishmania.• Parasite migration was characterized by monosaccharide chemical gradients.• This assay is a novel, rapid and reliable screening method to evaluate chemotaxis. Contain between 1 and 3 bullet points highlighting the customization rather than the steps of the procedure.
By means of copper(I)-and ruthenium(II)-catalyzed click reactions of quinine-and quinidinederived... more By means of copper(I)-and ruthenium(II)-catalyzed click reactions of quinine-and quinidinederived alkynes with azide-substituted chalcones a systematic series of novel cinchona-chalcone hybrid compounds, containing 1,4-disubstituted-and 1,5-disubstituted 1,2,3-triazole linkers, were synthesized and evaluated for their cytotoxic activity on four human malignant cell lines (PANC-1, COLO-205, A2058 and EBC-1). In most cases, the cyclization reactions were accompanied by the transition-metal-catalyzed epimerization of the C9-stereogenic centre in the cinchona fragment. The results of the in vitro assays disclosed that all the prepared hybrids exhibit marked cytotoxicity in concentrations of low micromolar range, while the C9-epimerized model comprising quinidineand (E)-1-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl) fragments, connected by 1,5-disubstituted 1,2,3-triazole linker, and can be regarded as the most potent lead of which activity is probably associated with a limited conformational space allowing for the adoption of a relatively rigid well-defined conformation identified by DFT modelling. The mechanism of action of this hybrid along with that of a model with markedly decreased activity were approached by comparative cell-cycle analyses in PANC-1 cells. These studies disclosed that the hybrid of enhanced antiproliferative activity exerts significantly more extensive inhibitory effects in subG1, S and G2/M phases than does the less cytotoxic counterpart.
One of the most focused-on point in biomedical and biotechnological research is the fabrication o... more One of the most focused-on point in biomedical and biotechnological research is the fabrication of complex tissue engineering scaffolds, with the ultimate objective being a functional, biocompatible and biodegradable implant that could facilitate and enhance tissue regeneration. Creating such implants is a highly challenging task. Physical and chemical characteristics have to be optimized and the balance between biodegradability, mechanical strength and overall practicality cannot be easily obtained. In this regard, composite materials have been regularly used in numerous areas of science and engineering as they incorporate advantages from two or more component materials. Our objective was to fabricate a composite, fibrous mesh composed of both degradable and non-degradable elements, that could be applicable as an implant with reliable mechanical properties without hindering in vivo tissue integration. In the manuscript, we present the fabrication, chemical, physical, mechanical and cytotoxic evaluation of co-electrospun polysuccinimide/poly(vinyl alcohol) (PSI/PVA) meshes. Results confirmed the presence and random distribution of both PSI and PVA fibres in the fabricated meshes. Mechanical studies indicate that meshes are competent for implantation while cell viability study revealed no cytotoxic effects.
Tetrahymena pyriformis is one of the most frequently used unicellular, eukaryotic models in molec... more Tetrahymena pyriformis is one of the most frequently used unicellular, eukaryotic models in molecular and cell biology. The suitability of the model is supported by several molecular level homologies to higher ranked vertebrates, like (i) identical and inducible receptor pools of the surface membrane (e.g., insulin receptor) and cytoplasm (e.g., steroids); (ii) similar elements of signaling pathways (e.g., cyclic nucleotide phosphates, Ca 2+ -calmodulin system, phosphatidylinositol metabolism); (iii) homologous cell physiological responsiveness (e.g., chemotaxis, phagocytosis, proliferation, and metabolic processes) induced by natural ligands (e.g., peptide hormones, chemokines, drugs, and artificial signals); (iv) high-level sensitivity and molecular level distinctiveness are shown by diverse activity or structurally closely related molecules (e.g., bradykinins, crystalline, and amorphous insulins). Practical properties like the size of the cells (20 × 50 μm), short generation time (about 150 min), chemically defined media, fast and easy handling as well as the potential of accurate application of test substances all support using Tetrahymena cells not only as an advantageous model in biology and medicine but also as a good candidate of microbiorobotics too. Motion itself and the wide range of random and vectorial forms of locomotion represent one of the most essential and physiological responses of T. pyriformis . However, these cells – as regular ciliates – show no cell adhesion. The axis of “cell adhesion – chemotaxis – phagocytosis” is still observed as creeping on solid surfaces represent a functionally analogous process to adhesion, which provides the possibility of reorientation followed by chemotaxis which directs cells to nourishment and finished by phagocytosis, the target reaction of chemotaxis. The structural backgrounds of motion in Tetrahymena are well detectable both on microscopic and molecular biological levels. In small scale, about 600 cilia beating on the surface of the cells cover the force generation requirements, while coordinated interaction of several families of cytoskeletal proteins (e.g., tubulins, nexins, dyneins, etc.) support the structural basis of migration. The changes in swimming behavior of our model provide a good index to be analyzed. Computer-based path evaluation of consecutive swimming patterns (run, rest, and turn) and mathematical simulations of chemoattractant induced, concentration dependent migration (e.g., time-delayed model) are sensible and high-tech investigations of the classic phenomena of motion (chemotaxis, chemokinesis, and necrotaxis), while the model is also a proper one to evaluate a list of other migratory responses elicited by transient or definitive shifts of environmental stimuli (e.g., phototaxis, magnetotaxis, galvanotaxis). It is obvious that majority of the above listed migratory responses are elicited via specific signaling pathways. Investigation of these processes was more detailed in the case of chemotaxis, where groups of professional signal molecules were identified as strong chemoattractants (e.g., fMLF and IL-8) or chemorepellents (e.g., serotonin); however, several other non-professional chemotaxis inducer molecules are also capable of eliciting strong migratory responses (e.g., vasoactive peptides, melatonin). It is worth mentioning that classes of membrane-receptors identical to vertebrate ones were demonstrated by tools of molecular genetics as significant ones in evolving chemotactic responses of Tetrahymena and more intracellular signaling pathways (e.g., PI3K pathway) were also described as triggered ones. On the basis of processes described above, more migration-specific phenomena like (i) chemotactic range fitting, (ii) chemotactic selection, and (iii) chemotactic drug-targeting were also reported using Tetrahymena chemotaxis as a referent one. In each case, the increased ability for molecular (membrane) level discrimination of the individual cells provides the possibility to gain distinct groups of cells with increased responsiveness to a specific concentration of chemicals. However, the possibility to form long-term sub-populations of cells by the help of their migratory responses shows that genetic/nuclear level mechanisms have also significant role in development of migratory responsiveness of cells as individual ones and in populations, too. A theoretically underlined aspect of experiments on Tetrahymena motion is that, due to its basic cell physiological properties, investigations of chemotaxis provide data significant in respect of phylogenetics. Evaluation of chemotactic properties of amino acids and comparison of these data with consensus sequences describing order of appearance of amino acids in the primordial soup show that the first ones in molecular phylogeny (e.g., G, E, P) are chemoattractant while the latest ones (e.g., W, F, Y) are chemorepellent.
The effects of mammalian GnRH analog (D-Ala6, Pro6-NEt-LHRH), salmon GnRH, (Trp7, Leu8)-LHRH and ... more The effects of mammalian GnRH analog (D-Ala6, Pro6-NEt-LHRH), salmon GnRH, (Trp7, Leu8)-LHRH and its analog, (D-Arg6, Pro9-NEt)-sGnRH on growth hormone (GH) release were examined using a perifusion system for pituitary fragments of the common carp (Cyprinus carpio). Perifusion of 2 min pulses of different concentrations of LHRH-A, sGnRH or sGnRH-A stimulated a rapid and dose-dependent increase in GH release; sGnRH-A
Background: Oxidative stress is an important factor in the pathomechanism of atherosclerosis. Adv... more Background: Oxidative stress is an important factor in the pathomechanism of atherosclerosis. Advanced oxidation protein products (AOPPs) are considered markers of oxidative stress. Thickening of the carotid intima-media layers indicates subclinical atherosclerosis and can be detected by carotid ultrasound. Objective: Our aim was to examine the association between carotid intima-media thickness (CIMT) and the level of AOPPs. Methods: Carotid duplex scans and measurements of AOPPs were performed on 476 participants of a cardiovascular population study. The presence of conventional cardiovascular risk factors was investigated with a questionnaire, physical examination, and laboratory tests. Results: There was a positive correlation between maximum CIMT and the level of AOPPs only in the male population (r = 0.219, p = 0.033). Multivariate analysis has revealed that the association between AOPPs and mean or maximum CIMT was independent of cardiovascular risk factors (OR = 1.458, p = 0.004, and OR = 2.038, p < 0.001). Conclusions: Among males, the elevated level of AOPPs as a marker of oxidative stress may signal the existence of early atherosclerotic alterations.
Bovine insulin had a strong negative effect on the chemotaxis of Tetrahymena at the first encount... more Bovine insulin had a strong negative effect on the chemotaxis of Tetrahymena at the first encounter with the hormone. The crystalline porcine insulin proved to be neutral, while the amorphous porcine insulin had a significant positive chemotactic effect. At the second encounter with the hormone the presence of imprinting was detectable with bovine insulin and induced a significant positive chemotaxis. The chemotactic effect of amorphous porcine insulin was depressed while crystalline porcine insulin expressed the most significant positive chemotactic effect. In the binding of hormones there was unambiguous positive imprinting independently from the species specificity and from the physicochemical condition of the insulins. Tetrahymena discriminated at both the first and second encounters between the bovine and porcine insulins which differed in two amino acids. The imprinter capacity evident in binding was independent of the chemotactic preference and the imprinting embodied in chemotaxis.
International Journal of Laboratory Hematology, Mar 7, 2018
Platelet thrombus formation and platelet dysfunction are behind major cardiovascular and hematolo... more Platelet thrombus formation and platelet dysfunction are behind major cardiovascular and hematological diseases with the significant epidemiological role. Myocardial infarction or stroke contributes to present mortality and morbidity rates to great extent, while genetic platelet function defects being more rare conditions can pose a considerable diagnostic challenge. Antiplatelet and anticoagulant drugs are frequently prescribed drugs in daily clinical practice. Since decades, great effort has been made for investigation of the pathophysiology of platelet thrombus formation, which has been more relevant recently according to the improving number of implantable mechanical circulatory devices.
Dynamic properties of cancer cells, most notably their ability to migrate, have been correlated s... more Dynamic properties of cancer cells, most notably their ability to migrate, have been correlated successfully with their invasive nature in vivo. To establish a stronger experimental basis for such a correlation we subjected five different cancer cell lines of well-defined metastatic potential to a sequence of three independent assays reporting on three different aspects of cell dynamics, namely (1) the kinetics of cell spreading, (2) cell shape fluctuations, and (3) cell migration. The sequentially applied assays correspond to different measuring modes of the well-established ECIS technique that is based on non-invasive and label-free impedance readings of planar gold-film electrodes that serve as the growth substrate for the cells under study. Every individual assay returned a characteristic parameter describing the behavior of the cell lines in that particular assay quantitatively. The parameters of all three assays were ranked to establish individual profiles of cell dynamics for every cell line that correlate favorably with the cells' invasive properties. The sequence of impedance-based assays described here requires only small cell populations (< 10.000 cells), it is highly automated and easily adapted to 96well formats. It provides an in-depth dynamic profile of adherent cells that might be useful in other areas besides cancer research as well.
Interventional Medicine and Applied Science, Jul 25, 2018
Periodontal ligament stem cells (PDLSCs) possess extensive regeneration potential. However, their... more Periodontal ligament stem cells (PDLSCs) possess extensive regeneration potential. However, their therapeutic application demands a scaffold with appropriate properties. HydroMatrix (HydM) is a novel injectable peptide nanofiber hydrogel developed recently for cell culture. Our aim was to test whether HydM would be a suitable scaffold for proliferation and osteogenic differentiation of PDLSCs. PDLSCs were seeded on noncoated or HydM-coated surfaces. Both real-time impedance analysis and cell viability assay documented cell growth on HydM. PDLSCs showed healthy, fibroblast-like morphology on the hydrogel. After a 3-week-long culture in osteogenic medium, mineralization was much more intense in HydM cultures compared to control. Alkaline phosphatase activity of the cells grown on the gels reached the non-coated control levels. Our data provided evidence that PDLSCs can adhere, survive, migrate, and proliferate on HydM and this gel also supports their osteogenic differentiation. We first applied impedimetry for dental stem cells cultured on a scaffold. HydM is ideal for in vitro studies of PDLSCs. It may also serve not only as a reference material but also in the future as a promising biocompatible scaffold for preclinical studies.
In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g.... more In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10 −12 M), L-tryptophan (10 −11 M), L-glutamine and L-glutamic acid (10 −6 M), induced positive chemotactic responses, while L-alanine (10 −7 M), L-methionine (10 −11 and 10 −7 M), L-tryptophan (10 −11 M), L-glutamine (10 −12 M) and L-glutamic acid (10 −9 M) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10 −11 M, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.
Background Th17 cells have a central role in inflammation via their pro-inflammatory cytokine pro... more Background Th17 cells have a central role in inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)-17A, -17F, -21, -22, tumor necrosis factor-α and the expression of master regulator transcription factor RORc. The Th17 cells have essential role in many autoimmune diseases via induction of the development of inflammation. Objectives We studied the in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Methods CD45RO- and CD45RO+ cells were isolated from peripheral blood mononuclear cells with a two-step negative magnetic separation. The cells were activated with anti-CD3 and anti-CD28 antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with an anti-IL-4 (10 μg/ml) blocking antibody. After 5 and 10 days IL-17 and IL-22 productions were measured by ELISPOT and ELISA; the RORc and Tbet expression were measured by quantitative real-time PCR methods. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results Although anti-CD3/CD28 activation increased the IL-17 and IL-22 productions, it did not alter the RORc expression of the CD45RO- cells of healthy donors. The IL-22 production and the Tbet expression of the RORc producing cells were inhibited by TGFβ and stimulated by IL-23 treatment. The CD45RO+ cells of healthy donors expressed higher level of RORc, T-bet and IL17 without stimulation or cytokine treatment compared to the CD45RO- cells. However, there was no difference between the RORc and Tbet expression of the freshly separated CD45RO+ and CD45RO- cells of RA patients. IL-23 treatment increased both of IL-17 and IL-22 cytokine production of the differentiated CD45RO- cells. Conclusions IL-17 and IL-22 production are differently regulated during human Th17 differentiation. Our data suggest that the increased baseline RORc expression of CD45RO- cells may contribute to the accelerated Th17 differentiation in RA. Disclosure of Interest None declared
Background and objectives Th17 cells have a central role in the inflammation via their pro-inflam... more Background and objectives Th17 cells have a central role in the inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)-17A, -17F, -21, -22, and tumour necrosis factor-α. We studied in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Materials and methods CD45RO- and CD45RO+ cells were isolated from peripheral blood mononuclear cells with a two-step negative magnetic separation method. The cells were activated with anti-CD3 and anti-CD28 antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with anti-IL-4 (10 μg/ml) blocking antibody. After 5 and 10 days the IL-17 and IL-22 production were measured by ELISPOT and ELISA; the RORc and Tbet expression were measured by quantitative real-time PCR. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results Although the anti-CD3/CD28 activation increased the IL-17 and IL-22 production; it did not alter the RORc expression of the CD45RO- cells of healthy donors. The IL-22 production and the Tbet expression of the RORc producing cells were inhibited by TGFβ and stimulated by IL-23 treatment. The CD45RO+ cells of healthy donors expressed higher level of RORc and T-bet without stimulation or cytokine treatment and also higher amount of IL-17 compared to the CD45RO- cells. By contrast, there was no difference between the RORc expression of the freshly separated CD45RO+ and CD45RO- cells of RA patients. The production of IL-17 and IL-22 of the differentiated CD45RO- cells from RA patients were both increased by IL-23 treatment. Conclusions The IL-17 and IL-22 production are differently regulated during the human Th17 differentiation. Furthermore, our present data suggest that the increased baseline RORc expression of the CD45RO- cells may contribute to the accelerated Th17 differentiation in RA.
International Journal of Molecular Sciences, Apr 17, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Chemotactic responses play a significant role during Leishmania (V.) braziliensis differentiation... more Chemotactic responses play a significant role during Leishmania (V.) braziliensis differentiation through its life cycle and during infection. The aim of this description has been to portray the modified “two-chamber capillary chemotaxis assay” as a technique useful for quantitative in vitro evaluation of Leishmania chemotaxis after reviewing the methods described until now to assess chemotaxis in vitro in Leishmania sp. This valued simple and reproducible method convenient for parasite migration determination, was tested by the use of controlled changes in monosaccharide (D-glucose and D-fructose) concentrations as referent ligands. The validation of the method demonstrates that this technique is useful to evaluate the relationship existing between parasite migration towards the monosaccharides and sugar concentration. This means that within specific ranges, parasites attracted by the monosaccharide migrate towards more concentrated solutions and accumulate (higher number of parasites) at that spot. Interestingly, both the time course of the experiment and the osmolality of the solution influence parasite migration capacity. Our validation suggests that this improved methodology quantitatively evaluates taxis of Leishmania towards/against different substances. On the basis of our herein presented data, we conclude that this technique is a novel, rapid and reliable screening method to evaluate chemotaxis in Leishmania.• The two-chamber capillary chemotaxis assay was standardized for Leishmania.• The technique is useful to quantitatively evaluate in vitro chemotaxis in Leishmania.• Parasite migration was characterized by monosaccharide chemical gradients.• This assay is a novel, rapid and reliable screening method to evaluate chemotaxis. Contain between 1 and 3 bullet points highlighting the customization rather than the steps of the procedure.
By means of copper(I)-and ruthenium(II)-catalyzed click reactions of quinine-and quinidinederived... more By means of copper(I)-and ruthenium(II)-catalyzed click reactions of quinine-and quinidinederived alkynes with azide-substituted chalcones a systematic series of novel cinchona-chalcone hybrid compounds, containing 1,4-disubstituted-and 1,5-disubstituted 1,2,3-triazole linkers, were synthesized and evaluated for their cytotoxic activity on four human malignant cell lines (PANC-1, COLO-205, A2058 and EBC-1). In most cases, the cyclization reactions were accompanied by the transition-metal-catalyzed epimerization of the C9-stereogenic centre in the cinchona fragment. The results of the in vitro assays disclosed that all the prepared hybrids exhibit marked cytotoxicity in concentrations of low micromolar range, while the C9-epimerized model comprising quinidineand (E)-1-(4-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenyl) fragments, connected by 1,5-disubstituted 1,2,3-triazole linker, and can be regarded as the most potent lead of which activity is probably associated with a limited conformational space allowing for the adoption of a relatively rigid well-defined conformation identified by DFT modelling. The mechanism of action of this hybrid along with that of a model with markedly decreased activity were approached by comparative cell-cycle analyses in PANC-1 cells. These studies disclosed that the hybrid of enhanced antiproliferative activity exerts significantly more extensive inhibitory effects in subG1, S and G2/M phases than does the less cytotoxic counterpart.
One of the most focused-on point in biomedical and biotechnological research is the fabrication o... more One of the most focused-on point in biomedical and biotechnological research is the fabrication of complex tissue engineering scaffolds, with the ultimate objective being a functional, biocompatible and biodegradable implant that could facilitate and enhance tissue regeneration. Creating such implants is a highly challenging task. Physical and chemical characteristics have to be optimized and the balance between biodegradability, mechanical strength and overall practicality cannot be easily obtained. In this regard, composite materials have been regularly used in numerous areas of science and engineering as they incorporate advantages from two or more component materials. Our objective was to fabricate a composite, fibrous mesh composed of both degradable and non-degradable elements, that could be applicable as an implant with reliable mechanical properties without hindering in vivo tissue integration. In the manuscript, we present the fabrication, chemical, physical, mechanical and cytotoxic evaluation of co-electrospun polysuccinimide/poly(vinyl alcohol) (PSI/PVA) meshes. Results confirmed the presence and random distribution of both PSI and PVA fibres in the fabricated meshes. Mechanical studies indicate that meshes are competent for implantation while cell viability study revealed no cytotoxic effects.
Tetrahymena pyriformis is one of the most frequently used unicellular, eukaryotic models in molec... more Tetrahymena pyriformis is one of the most frequently used unicellular, eukaryotic models in molecular and cell biology. The suitability of the model is supported by several molecular level homologies to higher ranked vertebrates, like (i) identical and inducible receptor pools of the surface membrane (e.g., insulin receptor) and cytoplasm (e.g., steroids); (ii) similar elements of signaling pathways (e.g., cyclic nucleotide phosphates, Ca 2+ -calmodulin system, phosphatidylinositol metabolism); (iii) homologous cell physiological responsiveness (e.g., chemotaxis, phagocytosis, proliferation, and metabolic processes) induced by natural ligands (e.g., peptide hormones, chemokines, drugs, and artificial signals); (iv) high-level sensitivity and molecular level distinctiveness are shown by diverse activity or structurally closely related molecules (e.g., bradykinins, crystalline, and amorphous insulins). Practical properties like the size of the cells (20 × 50 μm), short generation time (about 150 min), chemically defined media, fast and easy handling as well as the potential of accurate application of test substances all support using Tetrahymena cells not only as an advantageous model in biology and medicine but also as a good candidate of microbiorobotics too. Motion itself and the wide range of random and vectorial forms of locomotion represent one of the most essential and physiological responses of T. pyriformis . However, these cells – as regular ciliates – show no cell adhesion. The axis of “cell adhesion – chemotaxis – phagocytosis” is still observed as creeping on solid surfaces represent a functionally analogous process to adhesion, which provides the possibility of reorientation followed by chemotaxis which directs cells to nourishment and finished by phagocytosis, the target reaction of chemotaxis. The structural backgrounds of motion in Tetrahymena are well detectable both on microscopic and molecular biological levels. In small scale, about 600 cilia beating on the surface of the cells cover the force generation requirements, while coordinated interaction of several families of cytoskeletal proteins (e.g., tubulins, nexins, dyneins, etc.) support the structural basis of migration. The changes in swimming behavior of our model provide a good index to be analyzed. Computer-based path evaluation of consecutive swimming patterns (run, rest, and turn) and mathematical simulations of chemoattractant induced, concentration dependent migration (e.g., time-delayed model) are sensible and high-tech investigations of the classic phenomena of motion (chemotaxis, chemokinesis, and necrotaxis), while the model is also a proper one to evaluate a list of other migratory responses elicited by transient or definitive shifts of environmental stimuli (e.g., phototaxis, magnetotaxis, galvanotaxis). It is obvious that majority of the above listed migratory responses are elicited via specific signaling pathways. Investigation of these processes was more detailed in the case of chemotaxis, where groups of professional signal molecules were identified as strong chemoattractants (e.g., fMLF and IL-8) or chemorepellents (e.g., serotonin); however, several other non-professional chemotaxis inducer molecules are also capable of eliciting strong migratory responses (e.g., vasoactive peptides, melatonin). It is worth mentioning that classes of membrane-receptors identical to vertebrate ones were demonstrated by tools of molecular genetics as significant ones in evolving chemotactic responses of Tetrahymena and more intracellular signaling pathways (e.g., PI3K pathway) were also described as triggered ones. On the basis of processes described above, more migration-specific phenomena like (i) chemotactic range fitting, (ii) chemotactic selection, and (iii) chemotactic drug-targeting were also reported using Tetrahymena chemotaxis as a referent one. In each case, the increased ability for molecular (membrane) level discrimination of the individual cells provides the possibility to gain distinct groups of cells with increased responsiveness to a specific concentration of chemicals. However, the possibility to form long-term sub-populations of cells by the help of their migratory responses shows that genetic/nuclear level mechanisms have also significant role in development of migratory responsiveness of cells as individual ones and in populations, too. A theoretically underlined aspect of experiments on Tetrahymena motion is that, due to its basic cell physiological properties, investigations of chemotaxis provide data significant in respect of phylogenetics. Evaluation of chemotactic properties of amino acids and comparison of these data with consensus sequences describing order of appearance of amino acids in the primordial soup show that the first ones in molecular phylogeny (e.g., G, E, P) are chemoattractant while the latest ones (e.g., W, F, Y) are chemorepellent.
The effects of mammalian GnRH analog (D-Ala6, Pro6-NEt-LHRH), salmon GnRH, (Trp7, Leu8)-LHRH and ... more The effects of mammalian GnRH analog (D-Ala6, Pro6-NEt-LHRH), salmon GnRH, (Trp7, Leu8)-LHRH and its analog, (D-Arg6, Pro9-NEt)-sGnRH on growth hormone (GH) release were examined using a perifusion system for pituitary fragments of the common carp (Cyprinus carpio). Perifusion of 2 min pulses of different concentrations of LHRH-A, sGnRH or sGnRH-A stimulated a rapid and dose-dependent increase in GH release; sGnRH-A
Background: Oxidative stress is an important factor in the pathomechanism of atherosclerosis. Adv... more Background: Oxidative stress is an important factor in the pathomechanism of atherosclerosis. Advanced oxidation protein products (AOPPs) are considered markers of oxidative stress. Thickening of the carotid intima-media layers indicates subclinical atherosclerosis and can be detected by carotid ultrasound. Objective: Our aim was to examine the association between carotid intima-media thickness (CIMT) and the level of AOPPs. Methods: Carotid duplex scans and measurements of AOPPs were performed on 476 participants of a cardiovascular population study. The presence of conventional cardiovascular risk factors was investigated with a questionnaire, physical examination, and laboratory tests. Results: There was a positive correlation between maximum CIMT and the level of AOPPs only in the male population (r = 0.219, p = 0.033). Multivariate analysis has revealed that the association between AOPPs and mean or maximum CIMT was independent of cardiovascular risk factors (OR = 1.458, p = 0.004, and OR = 2.038, p < 0.001). Conclusions: Among males, the elevated level of AOPPs as a marker of oxidative stress may signal the existence of early atherosclerotic alterations.
Bovine insulin had a strong negative effect on the chemotaxis of Tetrahymena at the first encount... more Bovine insulin had a strong negative effect on the chemotaxis of Tetrahymena at the first encounter with the hormone. The crystalline porcine insulin proved to be neutral, while the amorphous porcine insulin had a significant positive chemotactic effect. At the second encounter with the hormone the presence of imprinting was detectable with bovine insulin and induced a significant positive chemotaxis. The chemotactic effect of amorphous porcine insulin was depressed while crystalline porcine insulin expressed the most significant positive chemotactic effect. In the binding of hormones there was unambiguous positive imprinting independently from the species specificity and from the physicochemical condition of the insulins. Tetrahymena discriminated at both the first and second encounters between the bovine and porcine insulins which differed in two amino acids. The imprinter capacity evident in binding was independent of the chemotactic preference and the imprinting embodied in chemotaxis.
International Journal of Laboratory Hematology, Mar 7, 2018
Platelet thrombus formation and platelet dysfunction are behind major cardiovascular and hematolo... more Platelet thrombus formation and platelet dysfunction are behind major cardiovascular and hematological diseases with the significant epidemiological role. Myocardial infarction or stroke contributes to present mortality and morbidity rates to great extent, while genetic platelet function defects being more rare conditions can pose a considerable diagnostic challenge. Antiplatelet and anticoagulant drugs are frequently prescribed drugs in daily clinical practice. Since decades, great effort has been made for investigation of the pathophysiology of platelet thrombus formation, which has been more relevant recently according to the improving number of implantable mechanical circulatory devices.
Dynamic properties of cancer cells, most notably their ability to migrate, have been correlated s... more Dynamic properties of cancer cells, most notably their ability to migrate, have been correlated successfully with their invasive nature in vivo. To establish a stronger experimental basis for such a correlation we subjected five different cancer cell lines of well-defined metastatic potential to a sequence of three independent assays reporting on three different aspects of cell dynamics, namely (1) the kinetics of cell spreading, (2) cell shape fluctuations, and (3) cell migration. The sequentially applied assays correspond to different measuring modes of the well-established ECIS technique that is based on non-invasive and label-free impedance readings of planar gold-film electrodes that serve as the growth substrate for the cells under study. Every individual assay returned a characteristic parameter describing the behavior of the cell lines in that particular assay quantitatively. The parameters of all three assays were ranked to establish individual profiles of cell dynamics for every cell line that correlate favorably with the cells' invasive properties. The sequence of impedance-based assays described here requires only small cell populations (< 10.000 cells), it is highly automated and easily adapted to 96well formats. It provides an in-depth dynamic profile of adherent cells that might be useful in other areas besides cancer research as well.
Interventional Medicine and Applied Science, Jul 25, 2018
Periodontal ligament stem cells (PDLSCs) possess extensive regeneration potential. However, their... more Periodontal ligament stem cells (PDLSCs) possess extensive regeneration potential. However, their therapeutic application demands a scaffold with appropriate properties. HydroMatrix (HydM) is a novel injectable peptide nanofiber hydrogel developed recently for cell culture. Our aim was to test whether HydM would be a suitable scaffold for proliferation and osteogenic differentiation of PDLSCs. PDLSCs were seeded on noncoated or HydM-coated surfaces. Both real-time impedance analysis and cell viability assay documented cell growth on HydM. PDLSCs showed healthy, fibroblast-like morphology on the hydrogel. After a 3-week-long culture in osteogenic medium, mineralization was much more intense in HydM cultures compared to control. Alkaline phosphatase activity of the cells grown on the gels reached the non-coated control levels. Our data provided evidence that PDLSCs can adhere, survive, migrate, and proliferate on HydM and this gel also supports their osteogenic differentiation. We first applied impedimetry for dental stem cells cultured on a scaffold. HydM is ideal for in vitro studies of PDLSCs. It may also serve not only as a reference material but also in the future as a promising biocompatible scaffold for preclinical studies.
In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g.... more In the sand-fly mid gut, Leishmania promastigotes are exposed to acute changes in nutrients, e.g. amino acids (AAs). These metabolites are the main energy sources for the parasite, crucial for its differentiation and motility. We analysed the migratory behaviour and morphological changes produced by aliphatic, monocarboxylic, dicarboxylic, heterocyclic and sulphur-containing AAs in Leishmania amazonensis and Leishmania braziliensis and demonstrated that L-methionine (10 −12 M), L-tryptophan (10 −11 M), L-glutamine and L-glutamic acid (10 −6 M), induced positive chemotactic responses, while L-alanine (10 −7 M), L-methionine (10 −11 and 10 −7 M), L-tryptophan (10 −11 M), L-glutamine (10 −12 M) and L-glutamic acid (10 −9 M) induced negative chemotactic responses. L-proline and L-cysteine did not change the migratory potential of Leishmania. The flagellum length of L. braziliensis, but not of L. amazonensis, decreased when incubated in hyperosmotic conditions. However, chemo-repellent concentrations of L-alanine (Hypo-/hyper-osmotic conditions) and L-glutamic acid (hypo-osmotic conditions) decreased L. braziliensis flagellum length and L-methionine (10 −11 M, hypo-/hyper-osmotic conditions) decreased L. amazonensis flagellum length. This chemotactic responsiveness suggests that Leishmania discriminate between slight concentration differences of small and structurally closely related molecules and indicates that besides their metabolic effects, AAs play key roles linked to sensory mechanisms that might determine the parasite's behaviour.
Background Th17 cells have a central role in inflammation via their pro-inflammatory cytokine pro... more Background Th17 cells have a central role in inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)-17A, -17F, -21, -22, tumor necrosis factor-α and the expression of master regulator transcription factor RORc. The Th17 cells have essential role in many autoimmune diseases via induction of the development of inflammation. Objectives We studied the in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Methods CD45RO- and CD45RO+ cells were isolated from peripheral blood mononuclear cells with a two-step negative magnetic separation. The cells were activated with anti-CD3 and anti-CD28 antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with an anti-IL-4 (10 μg/ml) blocking antibody. After 5 and 10 days IL-17 and IL-22 productions were measured by ELISPOT and ELISA; the RORc and Tbet expression were measured by quantitative real-time PCR methods. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results Although anti-CD3/CD28 activation increased the IL-17 and IL-22 productions, it did not alter the RORc expression of the CD45RO- cells of healthy donors. The IL-22 production and the Tbet expression of the RORc producing cells were inhibited by TGFβ and stimulated by IL-23 treatment. The CD45RO+ cells of healthy donors expressed higher level of RORc, T-bet and IL17 without stimulation or cytokine treatment compared to the CD45RO- cells. However, there was no difference between the RORc and Tbet expression of the freshly separated CD45RO+ and CD45RO- cells of RA patients. IL-23 treatment increased both of IL-17 and IL-22 cytokine production of the differentiated CD45RO- cells. Conclusions IL-17 and IL-22 production are differently regulated during human Th17 differentiation. Our data suggest that the increased baseline RORc expression of CD45RO- cells may contribute to the accelerated Th17 differentiation in RA. Disclosure of Interest None declared
Background and objectives Th17 cells have a central role in the inflammation via their pro-inflam... more Background and objectives Th17 cells have a central role in the inflammation via their pro-inflammatory cytokine production, such as interleukin (IL)-17A, -17F, -21, -22, and tumour necrosis factor-α. We studied in vitro Th17 cell differentiation of healthy donors and patients with rheumatoid arthritis (RA). Materials and methods CD45RO- and CD45RO+ cells were isolated from peripheral blood mononuclear cells with a two-step negative magnetic separation method. The cells were activated with anti-CD3 and anti-CD28 antibodies and treated with TGFβ (2.5 ng/ml), IL-6 (25 ng/ml), IL-1β (10 ng/ml) and IL-23 (10 ng/ml) cytokines and with anti-IL-4 (10 μg/ml) blocking antibody. After 5 and 10 days the IL-17 and IL-22 production were measured by ELISPOT and ELISA; the RORc and Tbet expression were measured by quantitative real-time PCR. Cell viability was monitored by impendance-based cell analyzer (CASY-TT). Results Although the anti-CD3/CD28 activation increased the IL-17 and IL-22 production; it did not alter the RORc expression of the CD45RO- cells of healthy donors. The IL-22 production and the Tbet expression of the RORc producing cells were inhibited by TGFβ and stimulated by IL-23 treatment. The CD45RO+ cells of healthy donors expressed higher level of RORc and T-bet without stimulation or cytokine treatment and also higher amount of IL-17 compared to the CD45RO- cells. By contrast, there was no difference between the RORc expression of the freshly separated CD45RO+ and CD45RO- cells of RA patients. The production of IL-17 and IL-22 of the differentiated CD45RO- cells from RA patients were both increased by IL-23 treatment. Conclusions The IL-17 and IL-22 production are differently regulated during the human Th17 differentiation. Furthermore, our present data suggest that the increased baseline RORc expression of the CD45RO- cells may contribute to the accelerated Th17 differentiation in RA.
International Journal of Molecular Sciences, Apr 17, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
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