Biomedical subjects

László Bene

Publications and source records attributed to László Bene.

15 recordsLinked to original sources

PubMed2005

Detection of receptor trimers on the cell surface by flow cytometric fluorescence energy homotransfer measurements.

Fluorescence energy homotransfer offers a powerful tool for the investigation of the state of oligomerization of cell surface receptors on a cell-by-cell basis by measuring the polarized components of fluorescence intensity of cells labeled with fluorescently stained antibodies. Here we describe homotransfer-based methods for the flow cytometric detection and analysis of hetero- and homo-associations of cell surface receptors. Homotransfer efficiencies for two- and three-body energy transfer interactions are defined and their frequency distribution curves are computed from the fluorescence anisotropy distributions of multiple-labeled cells. The fractions of receptors involved in homo-clustering is calculated based on the dependence of the fluorescence anisotropy on the surface concentration of the fluorescently stained antibodies. A homotransfer analysis of the homo- and hetero-clustering of the MHCI and MHCII glycoproteins, the cytokine receptor IL-2Ralpha, transferrin receptor and the receptor-type tyrosine phosphatase CD45 on JY B and Kit-225-K6 T cells is presented. We investigated how various factors such as the type of dye, rotational mobility of the dye and dye-targeting antibody, as well as the wavelength of the exciting light affect the homotransfer. We show that the homotransfer technique combined with the high statistical resolution of flow cytometry is an effective tool for detecting different oligomeric states of receptors by using fluorophores having restricted rotational mobility on the time scale of fluorescence.

Antibodies, Monoclonal↗

PubMed2005

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins.

BACKGROUND: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. METHODS: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different dual-laser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. RESULTS: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel way to calculate a factor (alpha) that characterized the fluorescence intensity of acceptor molecules relative to the same number of excited donor molecules, which is essential for quantifying FRET efficiency. This was achieved by calculating FRET efficiency in two different ways and minimizing the squared difference between the two results by changing alpha. Our method reliably detected the association of Cdk2 with its inhibitor, Kip1, whereas the nonspecific FRET efficiency between Cdk2 and a random peptide was negligible. We identified and sorted subpopulations of yeast cells showing interaction between the studied proteins. CONCLUSIONS: We have described a straightforward novel calibration method to accurately quantitate FRET efficiency between GFP derivatives in flow cytometry.

Calibration↗

PubMed2005

Detection of channel proximity by nanoparticle-assisted delaying of toxin binding; a combined patch-clamp and flow cytometric energy transfer study.

Gold nanoparticles of 30 nm diameter bound to cell-surface receptor major histocompatibility complex glycoproteins (MHCI and MHCII), interleukin-2 receptor alpha subunit (IL-2Ralpha), very late antigen-4 (VLA-4) integrin, transferrin receptor, and the receptor-type protein tyrosin phosphatase CD45 are shown by the patch-clamp technique to selectively modulate binding characteristics of Pi(2) toxin, an efficient blocker of K(v)1.3 channels. After correlating the electrophysiological data with those on the underlying receptor clusters obtained by simultaneously conducted flow cytometric energy transfer measurements, the modulation was proved to be sensitive to the density and size of the receptor clusters, and to the locations of the receptors as well. Based on the observation that engagement of MHCII by a monoclonal antibody down-regulates channel current and based on the close nanometer-scale proximity of the MHCI and MHCII glycoproteins, an analogous experiment was carried out when gold nanoparticles bound to MHCI delayed down-regulation of the K(v)1.3 current initiated by ligation of MHCII. Localization of K(v)1.3 channels in the nanometer-scale vicinity of the MHC-containing lipid rafts is demonstrated for the first time. A method is proposed for detecting receptor-channel or receptor-receptor proximity by observing nanoparticle-induced increase in relaxation times following concentration jumps of ligands binding to channels or to receptors capable of regulating channel currents.

Cell Line↗

PubMed2004

Membrane topography of HLA I, HLA II, and ICAM-1 is affected by IFN-gamma in lipid rafts of uveal melanomas.

The lateral distribution and colocalization of HLA I, HLA-DR, and ICAM-1 proteins was studied for the first time in the plasma membrane of two human uveal melanoma cell lines, OCM-1 and OCM-3. Our fluorescence resonance energy transfer and confocal laser scanning microscopic experiments revealed that these molecules are mostly confined to the same membrane regions, where they form similar protein patterns (homo- and hetero-associates) to those found previously on other cell types of lymphoid as well as colorectal carcinoma origin. Confocal microscopic colocalization experiments with GM(1) gangliosides and the GPI-anchored CD59 molecules showed enrichment of HLA I, HLA-DR, and ICAM-1 molecules in specific membrane domains (lipid rafts) excluding the transferrin receptor. IFN-gamma remarkably increased the expression levels of these molecules and rearranged their association patterns, which can affect the adoptive immune response of effector cells.

Flow Cytometry↗

PubMed2004

Raft and cytoskeleton associations of an ABC transporter: P-glycoprotein.

BACKGROUND: A novel flow cytometric assay has been described in an accompanying report (Gombos et al., METHODS: The kinetics of the decrease in immunofluorescence intensity was analyzed after the addition of the raft-preserving Triton X-100 or Nonidet P-40, both of which disrupt the entire membrane. Mild treatments by both detergents leave cells attached to only those proteins that are anchored to the cytoskeleton by rafts or independent of rafts. Agents that affect microfilaments and modulate membrane levels of cholesterol by cyclodextrin were used to distinguish between the raft-mediated and non-raft-related associations of the Pgp. Confocal microscopy and flow cytometric fluorescence energy transfer measurements were used to confirm colocalization of Pgp with raft constituents. RESULTS: The assay was proved to be sensitive enough to resolve differences between the resistance of UIC2-labeled cell-surface Pgps to Triton X-100 versus Nonidet P-40. Approximately 34% of the UIC2 Fab-labeled Pgp molecules were associated with the cytoskeleton through detergent-resistant, cholesterol-sensitive microdomains or directly, whereas approximately 15% were found to be directly linked to the cytoskeleton. Accordingly, confocal microscopy showed that Pgps colocalize with raft markers, mainly in microvilli. Fluorescence resonance energy transfer efficiency data indicating molecular proximity between Pgp and the raft markers CD44, CD59, and G(M1)-gangliosides also suggested that a significant fraction of Pgps resides in raft microdomains. Raft association of Pgp appears to be of functional significance because its modulation markedly affected drug pumping. CONCLUSIONS: By using the flow cytometric detergent resistance assay in kinetic mode, we were able to assess the extent of raft association and actin cytoskeleton anchorage of Pgp expressed at physiologically relevant levels. We demonstrated that a significant fraction of Pgp is raft associated on LS-174-T human colon carcinoma cells and that this localization may influence its transporter function. The kinetic flow cytometric detergent resistance assay presented in this report is considered to be generally applicable for the analysis of molecular interactions of membrane proteins expressed at low levels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Partial protection against dextran sodium sulphate induced colitis in histamine-deficient, histidine decarboxylase knockout mice.

OBJECTIVES: Chemically induced mucosal inflammation in animal models is a suitable tool for studying factors in the pathogenesis of inflammatory bowel disease. The aim of this study was to determine whether absence of histamine has an effect on the development of experimental colitis. METHODS: Histamine-deficient, histidine decarboxylase (HDC) knockout Balb/c mice and genetically identical control animals with intact HDC were studied. Colitis was induced by the administration of 2% dextran sodium sulphate in drinking water. Mice were killed after 5 days and disease activity assessed by clinical, histologic, and immunohistologic parameters. Bacterial components of stool were examined. RESULTS: Clinical disease activity was higher in the mice with intact HDC (disease activity index, 2.21) than in the histamine-deficient knock-out mice (1.88). Histologic findings were similar in the two groups. On day 5, the inflammation score of the HDC sufficient group was 5.25 (+/-1.055) and the crypt score was 5.00 (+/-1.128). The scores in the HDC knock-out group were 4.667 (+/- 0.707) and 4.667 (+/- 0.86), respectively. There was a significant difference in the number of interleukin (IL-10)-producing lymphocytes in colon mucosa. Large numbers of IL-10-positive lymphocytes were observed in wild type mice both those with DSS induced colitis and untreated controls. Only sporadic IL-10 positivity was found in histamine-deficient mice. Significant differences were found in the composition of the fecal bacterial flora between the two groups. CONCLUSION: The reduced number of IL-10-positive lymphocytes in the intestinal mucosa of histamine-deficient, histidine decarboxylase knockout mice and the altered fecal bacterial flora in these animals suggest that histamine may play a role in the pathophysiology of inflammation in the colon of normal animals by upregulating local IL-10 production and stimulating a local shift to Th2 response.

Animals↗

PubMed2003

Relationship between the tumor necrosis factor alpha polymorphism and the serum C-reactive protein levels in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract, including ulcerative colitis (UC) and Crohn's disease (CD). The aim of the study was to determine the prevalence of the tumor necrosis factor alpha (TNF-alpha) promoter polymorphisms at positions -238 and -308, and to measure the serum CRP levels in CD and UC patients and in a healthy population. The TNF-alpha gene polymorphisms were determined by the PCR-RFLP method. Samples of 74 CD and 50 UC patients and 138 healthy Hungarian volunteers were examined. The G-->A substitution at position -308 (designated the TNF2 allele) was significantly less frequent among IBD patients than in the control group (P=0.0009); 15% of the CD patients and 18% of the UC patients carried the mentioned allele, which was significantly less frequent compared with the healthy population (33%, P=0.0035 and P=0.036, respectively). No difference in the G-->A substitution at position -238 was observed. We found the median CRP levels to be significantly higher in the active phase of the disease than in the inactive phase among the -308A allele carriers (P=0.002), while this difference was not significant when the CRP levels in the active and inactive phases were compared among the -308GG homozygous patients (P=0.084). The decreased frequency of the TNF2 allele (known to be associated with elevated TNF-alpha production) in IBD may determine the severity of IBD through its interaction with plasma CRP levels, and may modify the pathogenesis of this chronic inflammatory disease.

Adult↗

PubMed2003

Signal transduction in T lymphocytes and aging.

Subclasses of cells in different compartments of the immune system possesses all those attributes, that make them suitable though somewhat limited models for the investigation of cellular processes during aging. Blood samples provide relative easily high amount of cells belonging to the same subclass, all of them having complex cascade processes in their signal transduction mechanisms, therefore being excellent targets for such investigations. One such subclass comprises peripheral blood lymphocytes. The signal-transduction cascade across the plasma membrane of lymphocytes displays many of the general features enabling us to draw conclusions for other cellular signaling problems that may arise during aging in other cell types not directly related to the immune system. The advantage of this approach lies in the fact that sometimes it is extremely difficult to study signal transduction processes in certain cell types under physiological conditions. The simultaneous occurrence of physical, chemical and molecular biological regulation of the immune processes at cellular and network levels make them very good examples for focusing our interest also on similar processes in other systems and cells. The fast developing new measuring techniques and the rapidly accumulating experimental data make it relatively easy to provide interesting new aspects, and ideas in this field. Finally, the immune system itself has its great importance and after all, it has an obvious declination with aging, the immune-senescence.

Aged↗

PubMed2003

High normal serum levels of C3 and C1 inhibitor, two acute-phase proteins belonging to the complement system, occur more frequently in patients with Crohn's disease than ulcerative colitis.

Few data are available on measurements of serum concentrations of complement proteins in inflammatory bowel disease (IBD). Therefore we measured serum levels of C3, C4, and C1-esterase inhibitor (C1-INH) as well as C-reactive protein (CRP) in 167 patients with Crohn's disease (CD) and 111 patients with ulcerative colitis (UC). Median serum concentrations of C3 and C1-INH were significantly higher in CD than in UC. According to multiple logistic regression analysis adjusted to age, sex, activity of disease, and presence of extraintestinal manifestations, IBD patients with high-normal (> or = 128%, > or = 75th percentile ) C1-INH concentrations had significantly (0.0275) higher odds ratio to have a diagnosis of CD than UC. Patients with high-normal C3 (> or = 1.40 g/liter) and high (> or =20 mg/liter) CRP concentrations had an even higher odds ratio of a CD diagnosis (P = 0.0132). Our findings indicate that measurement of C3, C1-INH, and CRP can be used as an additional marker to pANCA/ASCA for distinguishing patients with CD and UC.

Adult↗

PubMed2003

Elevated levels of anti-Helicobacter pylori antibodies in Henoch-Schönlein purpura.

OBJECTIVE: Henoch-Schonlein purpura (HSP) is a systemic vasculitis characterized by IgA-containing deposits in the skin, joints, gastrointestinal mucosa and glomeruli. HSP is much rarer in adults than in children. Among a number of other pathogenic factors, Helicobacter pylori (Hp) has recently been implicated in the gastrointestinal and extra-gastrointestinal manifestations underlying HSP. We aimed at studying the occurrence of Hp infections in 11 adult HSP patients with appearance in our clinical practice in the last 5 years. METHODS: Eleven adult HSP and 20 healthy adult patients were recruited for this study. Anti-Hp IgG and IgA antibodies were assessed in sera of HSP patients with active (n = 5) and remittent disease (n = 6) and healthy controls (n = 20) in the context of clinical symptoms, endoscopic evaluation, as well as routine and immunolaboratory observations. Concurrent Hp infection was confirmed by urease test and histology. RESULTS: Anti-Hp antibodies were present in 10/11 of HSP patients, and 11/20 of healthy controls. However, only 4/11 HSP patients had concurrent Hp infection as confirmed by urease test and/or histology. In the healthy controls the actual Hp infection was detectable in 9/20 cases. Patients in the acute phase had significantly higher levels of anti-Hp IgG compared to healthy controls (86.0 +/- 32.0 versus 25.5 +/- 28.5 U/ml, p < 0.05). In contrast, anti-Hp IgA/IgG ratios were significantly higher in the remitting phase compared to the control group (3.1 +/- 1.8 versus 0.8 +/- 0.5 ratio, p < 0.05). Among other immunolaboratory markers, serum CRP, circulating IgA and serum tumor necrosis factor-alpha levels were significantly increased in acute patients compared to healthy group results (45.3 +/- 22.7 versus 4.8 +/- 3.5 mg/l, p < 0,05); (58.9 +/- 18.2 versus 25.2 +/- 6.4pg/ml, p < 0,05); (5.5 +/- 1.1 versus 2.4 +/- 1.2 g/l; respectively, p < 0.05). CONCLUSIONS: Hp infection may be associated with the development and progression of HSP. IgG antibodies to Hp may be present mostly in acute HSP, while IgA antibodies may be involved in sustaining gastrointestinal symptoms underlying the chronic phase of the disease.

Acute Disease↗

PubMed2002

Does mosaicism of the plasma membrane at molecular and higher hierarchical levels in human lymphocytes carry information on the immediate history of cells?

A theoretical analysis of experimental data is presented in this mini-review on non-random homo- and hetero-associations of cell surface receptors, which can be recruited in the plasma membrane or at the surface of the rough endoplasmic reticulum during the protein synthesis. In the latter case, the likely genetic origin of these supramolecular formations is analyzed, contrasting this concept to the mobility of the cell surface proteins. A model is offered which, on the one hand, allows the mobility in a restricted way even among microdomain-confined receptor proteins through 'swapping partners'. On the other hand, the lack of mixing molecular components of protein clusters will be analyzed, when homo-and hetero-associations are studied through cell fusion experiments. The most frequently studied cell surface patterns have included lipid raft organized HLA class I and II, ICAM-1, tetraspan molecules, IL2 and IL15 and other receptors, as well. On the contrary coated pit-associated transferrin receptors would not mix with the above lipid raft associated receptor patterns, although transferrin receptor would readily oligomerize into homo-associates. The functional consequences of these superstructures are also analyzed. On the 30th anniversary of the Singer-Nicolson fluid mosaic membrane model one has to pay tribute to the authors, because of their deep insight emphasizing also the mosaicism of the membranes in general and that of the plasma membrane, in particular.

Cell Fusion↗

PubMed2002

INF-gamma rearranges membrane topography of MHC-I and ICAM-1 in colon carcinoma cells.

Flow-cytometric fluorescence energy transfer (FCET) measurements between fluorescently labeled cell surface MHC-I and ICAM-1 molecules indicated similar receptor patterns in the plasma membrane of interferon-gamma (INF-gamma)-treated colon carcinoma cells as those observed earlier at the surface of lymphoid cells. INF-gamma activation significantly increased the density of MHC-I and ICAM-1 proteins in the membrane. This increase in receptor density was accompanied by decreased proximity level of the homo-associated MHC-I receptors. Hetero-association of MHC-I and ICAM-1 molecules was increased by INF-gamma treatment. INF-gamma changed neither hetero- nor homo-association of transferrin receptors. By staining the sphingomyelin/cholesterol-enriched lipid microdomains with fluorescently labeled cholera toxin B subunit, we found an increase in the amount of lipid-raft associated G(M1)-gangliosides due to INF-gamma treatment. Confocal microscopic results and FCET measurements show that MHC-I and ICAM-1 are components of G(M1)-ganglioside containing lipid-rafts and also support an increase in the size of these lipid-rafts upon INF-gamma treatment.

Carcinoma↗

PubMed2002

GPI-microdomains (membrane rafts) and signaling of the multi-chain interleukin-2 receptor in human lymphoma/leukemia T cell lines.

Subunits (alpha, beta and gamma) of the interleukin-2 receptor complex (IL-2R) are involved in both proliferative and activation-induced cell death (AICD) signaling of T cells. In addition, the signaling beta and gamma chains are shared by other cytokines (e.g. IL-7, IL-9, IL-15). However, the molecular mechanisms responsible for recruiting/sorting the alpha chains to the signaling chains at the cell surface are not clear. Here we show, in four cell lines of human adult T cell lymphoma/leukemia origin, that the three IL-2R subunits are compartmented together with HLA glycoproteins and CD48 molecules in the plasma membrane, by means of fluorescence resonance energy transfer (FRET), confocal microscopy and immuno-biochemical techniques. In addition to the beta and gamma(c) chains constitutively expressed in detergent-resistant membrane fractions (DRMs) of T cells, IL-2Ralpha (CD25) was also found in DRMs, independently of its ligand-occupation. Association of CD25 with rafts was also confirmed by its colocalization with GM-1 ganglioside. Depletion of membrane cholesterol using methyl-beta-cyclodextrin substantially reduced co-clustering of CD25 with CD48 and HLA-DR, as well as the IL-2 stimulated tyrosine-phosphorylation of STATs (signal transducer and activator of transcription). These data indicate a GPI-microdomain (raft)-assisted recruitment of CD25 to the vicinity of the signaling beta and gamma(c) chains. Rafts may promote rapid formation of a high affinity IL-2R complex, even at low levels of IL-2 stimulus, and may also form a platform for the regulation of IL-2 induced signals by GPI-proteins (e.g. CD48). Based on these data, the integrity of these GPI-microdomains seems critical in signal transduction through the IL-2R complex.

Antigens, CD↗

PubMed2002

[Model investigation of push-out test used for quantitative evaluation of dental implant osseointegration].

A frequently used method for quantitative evaluation of implant-bone interface strength is the push-out test. The objective of the authors' study was to determine the effect of implants' geometric characteristics on the results of push-out method. The degree of osseointegration is determined by a complicated interaction of mechanical, biological and chemical factors. As this study focused on the effects of geometrical characteristics of dental implants, the authors tried to reduce the influence of biological and chemical factors as much as possible. For this reason a model of push-out test was set up. Cylindrical metal rods were imbedded in acrylic resin and were pushed out, meanwhile the force needed to disrupt the connection between the surface of the metal rods and the resin was measured and push-out strengths were calculated. Metal rods with three different lengths (7, 9, and 13 mm) and three different diameters (3.4, 4, 4.5 mm) were investigated. No correlation was found between implant length and maximum push-out strength. The mean push-out strengths were 17.32 MPa, 15.99 MPa and 15.56 MPa in groups, with 3.4, 4 and 4.5 mm diameter, thus the push-out strength decreases with increasing metal rod diameter. This may be attributed to the different distance between the outer circumference of the metal rods and the hole of the supporting table. Supporting this theory metal rods with identical diameter were examined, while the diameter of the hole on the supporting table was changed. The results demonstrated that larger holes on the supporting table resulted in higher push-out strengths. This indicates that comparisons of this variable should only be performed with standardized clearance of the hole on the supporting table. Push-out strengths of groups of cylindrical and conical implants with 9 degrees convergence were also investigated. The mean values were 16.57 MPa and 16.43 MPa, respectively. There was no statistically significant difference between the two groups. This means that results of experiments with all the above mentioned implant types are comparable, at least to that degree of conicity. As masticatory forces act usually on a surface, which is not perpendicular to the implant axis, we compared forces and push-out strengths that acted on surfaces with 10 degrees and 15 degrees inclination. The calculated average push-out strengths were 24.89 MPa and 22.56 MPa, respectively. More than twice as high push-out strengths were measured when threaded metal rods were compared to unthreaded ones (30.15 MPa, 14.89 MPa, respectively). Examining the effect of pitch of threads we compared the mean push-out strengths in three groups of threaded metal rods with pitch of threads 0.7, 1.0 and 1.25. The results were 28.86 MPa, 30.48 MPa, 31.12 MPa, respectively. The differences can be explained with the alteration in the dimension of the interface covered by the acrylic resin. Thus the effect of diameter, inclined opposing surface and threads must not be ignored if one compares the osseointegration capacity of different implants.

Dental Implantation, Endosseous↗

PubMedDate not supplied

Nanoparticle energy transfer on the cell surface.

Membrane topology of receptors plays an important role in shaping transmembrane signalling of cells. Among the methods used for characterizing receptor clusters, fluorescence resonance energy transfer between a donor and acceptor fluorophore plays a unique role based on its capability of detecting molecular level (2-10 nm) proximities of receptors in physiological conditions. Recent development of biotechnology has made possible the usage of colloidal gold particles in a large size range for specific labelling of cells for the purposes of electron microscopy. However, by combining metal and fluorophore labelling of cells, the versatility of metal-fluorophore interactions opens the way for new applications by detecting the presence of the metal particles by the methods of fluorescence spectroscopy. An outstanding feature of the metal nanoparticle-fluorophore interaction is that the metal particle can enhance spontaneous emission of the fluorophore in a distance-dependent fashion, in an interaction range essentially determined by the size of the nanoparticle. In our work enhanced fluorescence of rhodamine and cyanine dyes was observed in the vicinity of immunogold nanoparticles on the surface of JY cells in a flow cytometer. The dyes and the immunogold were targetted to the cell surface receptors MHCI, MHCII, transferrin receptor and CD45 by monoclonal antibodies. The fluorescence enhancement was sensitive to the wavelength of the exciting light, the size and amount of surface bound gold beads, as well as the fluorophore-nanoparticle distance. The intensity of 90 degrees scattering of the incident light beam was enhanced by the immunogold in a concentration and size-dependent fashion. The 90 degrees light scattering varied with the wavelength of the incident light in a manner characteristic to gold nanoparticles of the applied sizes. A reduction in photobleaching time constant of the cyanine dye was observed in the vicinity of gold particles in a digital imaging microscope. Modulations of 90 degrees light scattering intensity and photobleaching time constant indicate the role of the local field in the fluorescence enhancement. A mathematical simulation based on the electrodynamic theory of fluorescence enhancement showed a consistency between the measured enhancement values, the inter-epitope distances and the quantum yields. The feasibility of realizing proximity sensors operating at distance ranges larger than that of the conventional Forster transfer is demonstrated on the surface of living cells.

Antibodies, Monoclonal↗