T-cell Receptor Is a Threshold Detector: Sub- and Supra-Threshold Stochastic Resonance in TCR-MHC Clusters on the Cell Surface.

Stochastic resonance in clusters of major histocompatibility molecules is extended by a more detailed description of adaptive thresholding and by applying the notion of suprathreshold stochastic resonance as a stochastically quantizing encoder of transmembrane signaling downstream of major histocompatibility molecules and T-cell receptors on the side of presenting and recognizing cells, respectively. The adaptive nature of thresholding is partly explained by a mirroring of the noncognate-cognate dichotomy shown by the T-cell receptor structure and the kinetic-segregation model of the onset of T-cell receptor triggering. Membrane clusters of major histocompatibility molecules and T-cell receptors on their host cells are envisioned as places of the temporal encoding of downstream signals via the suprathreshold stochastic resonance process. The ways of optimization of molecular prostheses, such as chimeric antigen receptors against cancer in transmembrane signaling, are suggested in the framework of suprathreshold stochastic resonance. The analogy between Förster resonance energy transfer and suprathreshold stochastic resonance for information transfer is also discussed. The overlap integral for energy transfer parallels the mutual information transferred by suprathreshold stochastic resonance.

Dual-Laser Tetra-Polarization FRET (4polFRET) for Site-Selective Control of Homo-FRET in Hetero-FRET Systems on the Cell Surface: The Homo-FRET Gate.

The effects of donor homo-Förster resonance energy transfer (homo-FRET) taking place in hetero-FRET systems is described in the context of hetero-FRET detection via donor and acceptor fluorescence anisotropies in cell surface receptor clusters. Donor homo-FRET can influence both the efficiency of detection as well as the magnitude of the detectable hetero-FRET. A 4-fold polarized FRET detection scheme-tetrapolarization FRET (4polFRET)-is proposed not only for discriminating the effects of homo-FRET from those of hetero-FRET, but also for correlating homo-associations of the donors and acceptors at different donor-acceptor distances, even beyond the critical Förster distance for hetero-FRET ( R0). The method is based on suppressing homo-FRET at the donor side with red-edge excitation. After the anisotropy effects of physical rotation and homo-FRET were separated by site-selective spectroscopy, the magnitude of the effect of homo-FRET on hetero-FRET has been estimated. It has been found significant, offering a new sensitive technique for detecting conformational dynamics via the homo-FRET mediated component of hetero-FRET, the "homo-FRET enhanced hetero-FRET" or "homo-FRET gate". The method is realizable in flow, as well as in image cytometry equipped with polarization detecting facility.

Confocal microscopic dual-laser dual-polarization FRET (2polFRET) at the acceptor side for correlating rotations at different distances on the cell surface.

Relationship of donor and acceptor fluorescence anisotropies as well as efficiency of fluorescence resonance energy transfer (FRET) has been investigated in a confocal microscope in the context of FRET systems comprised of donor and acceptor-labeled MHCI and MHCII receptors on the surface of Kit-225 K6 human T-cells. The measurements have been carried out in a 2-laser, 5-signal platform where the total donor fluorescence intensity and 2 acceptor fluorescence intensities with their anisotropies - one at the donor's excitation wavelength, the other at the acceptor's excitation wavelength - have been detected. This configuration enabled the determination of FRET efficiency and correlating it with the two acceptor fluorescence anisotropies as a kind of calibration. Estimations for the FRET-enhanced donor fluorescence anisotropy, the directly excited acceptor fluorescence anisotropy, and the fluorescence anisotropy of sensitized emission have been obtained. Procedures for determining FRET by measuring only the total donor intensity and the acceptor intensity and its anisotropy, or two acceptor intensities and their anisotropies have been elaborated, the errors of which have been estimated based on the fluorescence anisotropy values obtained in the calibration with the method of flow cytometric energy transfer (FCET). The combined detection of the donor and acceptor fluorescence anisotropies enabled also the determination of the lower and upper limits of the orientation factor for FRET (κ2). An increase in range for κ2 with increasing FRET efficiency has been observed, with average κ2 values different from the dynamic random average of 2/3. These observations call for the need of κ2 determination in proximity measurements, where the donor and acceptor orientations are not predictable. An increasing range of κ2 with increasing intermolecular proximity of the MHCI and MHCII receptors has been observed. This indicates that molecular flexibility in the clusters of the MHCI and MHCII receptors reduces with increasing cluster density, i.e. a "fluidity gradient" exists in the clusters. More specifically, the local density dependent flexibility can also be taken as a direct proof for that the association of these receptors is non-random, but mediated by some type of physical interaction, a finding as a benefit of FRET detection by polarization spectroscopy. Two new quantities - the quenched donor fluorescence anisotropy and a fluorescence anisotropy analogue, the "dissymmetry index" of the polarized FRET efficiency components - have also been introduced for the characterization of the orientational dynamics of the excited state during FRET.

Perrin and Förster unified: Dual-laser triple-polarization FRET (3polFRET) for interactions at the Förster-distance and beyond.

Dual laser flow cytometric energy transfer (FCET)--elaborated by Trón et al. in 1984--is an efficient and rapid way of measuring FRET on large cell populations. FRET efficiency and the donor and acceptor concentrations are determined from one donor and two acceptor signals. In this communication this method is extended towards the domain of receptor dynamics by the detection of polarized components of the three intensities. By enabling a complete description of the proximity and dynamics of FRET-systems, the new measuring scheme allows a more refined description of both the structure and dynamics of cell surface receptor clusters at the nano-scale and beyond. Associated donor fraction, limiting anisotropy and rotational correlation time of the donor, acceptor anisotropy and cell-by-cell estimation of the orientation factor for FRET (κ2) are available in the steady state on a single FRET sample in a very rapid and statistically efficient way offered by flow cytometry. For a more sensitive detection of conformational changes the "polarized FRET indices"--quantities composed from FRET efficiency and anisotropies--are proposed. The method is illustrated by measurements on a FRET system with changing FRET-fraction and on a two donor-one acceptor-system, when the existence of receptor trimers are proven by the detection of "hetero-FRET induced homo-FRET relief", i.e. the diminishing of homo-FRET between the two donors in the presence of a donor quencher. The method also offers higher sensitivity for assessing conformational changes at the nano-scale, due to its capability for the simultaneous detection of changes of proximity and relative orientations of the FRET donor and acceptor. Although the method has been introduced in the context of FRET, it is more general: It can be used for monitoring triple-anisotropy correlations also in those cases when FRET actually does not occur, e.g. for interactions occuring beyond the Förster-distance R0. Interpretation of κ2 has been extended.

Depolarized FRET (depolFRET) on the cell surface: FRET control by photoselection.

Sensitivity of FRET in hetero- and homo-FRET systems on the photoselected orientation distribution of donors has been proven by using polarized and depolarized light for excitation. FRET as well as donor and acceptor anisotropies have been simultaneously measured in a dual emission-polarization scheme realized in a conventional flow cytometer by using single laser excitation and applying fluorophore-conjugated mAbs against the MHCI and MHCII cell surface receptors. Depolarization of the originally polarized light have been achieved by using crystal depolarizers based on Cornu's principle, a quarter-wave plate for circular polarization, and a parallel beam splitter acting as a diagonal-polarizer for dual-polarization excitation. Simultaneous analysis of intensity-based FRET efficiency and acceptor depolarization equivocally report that depolarization of light may increase FRET in an amount depending on the acceptor-to-donor concentration ratio. Acceptor depolarization turned to be more sensitive to FRET than donor hyper-polarization and even than intensity-based FRET efficiency. It can be used as a sensitive tool for monitoring changes in the dynamics of the donor-acceptor pairs. The basic observations of FRET enhancement and increased acceptor depolarization obtained for hetero-FRET are paralleled by analog observations of homo-FRET enhancements under depolarized excitation. In terms of the orientation factor for FRET, the FRET enhancements on depolarization in the condition of the macroscopically isotropic orientation distributions such as those of the cell surface bound fluorophores report on the presence of local orientation mismatches of the donor and acceptor preventing the optimal FRET in the polarized case, which may be eliminated by the excitation depolarization. A theory of fluorescence anisotropy for depolarized excitation is also presented.

Sensitivity of Helicobacter pylori detection by Giemsa staining is poor in comparison with immunohistochemistry and fluorescent in situ hybridization and strongly depends on inflammatory activity.

BACKGROUND: Conventional stainings (including H&E and special stains like Giemsa) are the most widely applied histopathologic detection methods of Helicobacter pylori (HP). MATERIALS AND METHODS: We aimed to compare the diagnostic performance of Giemsa staining with immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) on a monocentric cohort of 2896 gastric biopsies and relate results to histologic alterations in order to find such histopathologic subgroups in which these methods underperform. All cases were categorized regarding presence or absence of chronic gastritis, inflammatory activity, and mucosal structural alterations. RESULTS: Giemsa revealed 687 cases (23.7%), IHC 795 cases (27.5%), and FISH 788 cases (27.2%) as being HP positive. Giemsa showed significantly lower overall sensitivity (83.3%) compared to IHC (98.8%) and FISH (98.0%). Moreover, the sensitivity of Giemsa dramatically dropped to 33.6% in the nonactive cases. We found that sensitivity of Giemsa strongly depends on HP density and, accordingly, on the presence of activity. Structural alterations (intestinal metaplasia, atrophy, etc.) had only no or weak effect on sensitivity of the three stainings. Both IHC and FISH proved to be equally reliable HP detecting techniques whose diagnostic performance is minimally influenced by mucosal inflammatory and structural alterations contrary to conventional stainings. CONCLUSIONS: We highly recommend immunohistochemistry for clinically susceptible, nonactive chronic gastritis cases, if the conventional stain-based HP detection is negative. Moreover, we recommend to use IHC more widely as basic HP stain. Helicobacter pylori FISH technique is primarily recommended to determine bacterial clarithromycin resistance. Furthermore, it is another accurate diagnostic tool for HP.

Dual-laser homo-FRET on the cell surface.

Inhomogeneous broadening and red-edge effects have been detected on a highly mobile system of fluorescently conjugated mAbs targeted to cell surface receptors. By exploiting site-selective spectroscopy and the characteristic loss of homo-FRET on increasing excitation and decreasing emission wavelengths, contributions of physical rotation and homo-FRET to the depolarization of fluorescence anisotropy have been separated. Absolute homo-FRET efficiency has been determined by ratioing two anisotropies: a homo-FRET-sensitive one, which is excited at the absorption main band and detected at the long wavelength region of emission, and a homo-FRET-insensitive one, which is excited at the long wavelength region of absorption and detected at the short wavelength region of emission. Because the anisotropies are simultaneously detected in a unified detection scheme of a dual T-format arrangement, the method is applicable for the real-time tracking of dynamical changes of physical rotations and proximities. The utility of the method is demonstrated in the context of the MHCII molecule and the heavy and light chains of the MHCI molecule, a system of three receptors with well-characterized close mutual proximities. Although the method is presented for a flow cytometer, it can also be realized in a fluorescence microscope capable for dual-laser excitation and dual-anisotropy detection.

[Invasive methods in the treatment of obesity]. / Invazív módszerek az elhízás kezelésében.

Treatment of obesity and concomitant diseases have a significant burden on the health care system. Due to the lack of efficacy of conservative treatment methods, attention has shifted towards invasive methods. Surgical and endoscopic treatments of obesity are based on two different methods: restrictive and malabsorptive procedures or their combination. The author reviews the most effective surgical and endoscopic procedures in the treatment of obesity.

Single-laser polarization FRET (polFRET) on the cell surface.

A new method for the simultaneous detection of rotational mobility and proximity of cell surface receptors is presented based on cell-by-cell basis measurement of polarized fluorescence intensity components of the donor and acceptor of a FRET system. In addition to the FRET efficiency and the donor and acceptor concentrations, the method makes also possible the determination of the rotational characteristics and the associated fraction of the donors (FRET-fraction). The method is illustrated with flow cytometric and rFLIM measurements on donor-acceptor systems comprising fluorescently labeled whole antibodies and their Fab fragments against epitopes of the MHCI and MHCII cell surface receptors on human lymphoblast cells. Fluorescence anisotropy of donor and acceptor and FRET efficiency were measured for samples of different acceptor-to-donor concentration ratios. Acceptor anisotropy proved to be more sensitive than the donor anisotropy for sensing FRET. After determining the rotational constants of the donor-conjugated antibodies by measurements of FRET in the steady state, and by rFLIM as a reference, the associated fractions of the MHCI and MHCII molecules in their clusters were determined. Besides the flow cytometer and the wide-field rFLIM used in this study, the method can be applied also in other devices capable of dual-anisotropy detection.

Intensity correlation-based calibration of FRET.

Dual-laser flow cytometric resonance energy transfer (FCET) is a statistically efficient and accurate way of determining proximity relationships for molecules of cells even under living conditions. In the framework of this algorithm, absolute fluorescence resonance energy transfer (FRET) efficiency is determined by the simultaneous measurement of donor-quenching and sensitized emission. A crucial point is the determination of the scaling factor α responsible for balancing the different sensitivities of the donor and acceptor signal channels. The determination of α is not simple, requiring preparation of special samples that are generally different from a double-labeled FRET sample, or by the use of sophisticated statistical estimation (least-squares) procedures. We present an alternative, free-from-spectral-constants approach for the determination of α and the absolute FRET efficiency, by an extension of the presented framework of the FCET algorithm with an analysis of the second moments (variances and covariances) of the detected intensity distributions. A quadratic equation for α is formulated with the intensity fluctuations, which is proved sufficiently robust to give accurate α-values on a cell-by-cell basis in a wide system of conditions using the same double-labeled sample from which the FRET efficiency itself is determined. This seemingly new approach is illustrated by FRET measurements between epitopes of the MHCI receptor on the cell surface of two cell lines, FT and LS174T. The figures show that whereas the common way of α determination fails at large dye-per-protein labeling ratios of mAbs, this presented-as-new approach has sufficient ability to give accurate results. Although introduced in a flow cytometer, the new approach can also be straightforwardly used with fluorescence microscopes.

Crohn's disease alters MHC-rafts in CD4+ T-cells.

Clusters of MHCI, ICAM-1, CD44, CD59, IL-2R, and IL-15R molecules have been studied on the surface of CD4(+) T-cells from peripheral blood and lymph nodes of patients in Crohn's disease and healthy individuals as controls by using a dual-laser flow cytometric fluorescence resonance energy transfer (FRET) technique and fluorescently stained Fabs. When cells from patients in Crohn's disease are compared to those of controls, the surface expression level for the MHCI reduced by ∼45%, for CD44 enhanced by ∼100%, and for IL-2Rα, IL-15Rα, and common γ(c) enhanced by ∼50%, ∼70%, and ∼130%, respectively. Efficiencies of FRET monitoring homoassociation for the MHCI and CD44 reduced, that for IL-2Rα enhanced. While efficiencies of FRET monitoring the association of γ(c) and ICAM-1 with the MHCI reduced, those monitoring association of IL-2/15Rα, CD44, and CD59 with MHCI enhanced. Efficiencies of FRET measured between the MHCI and IL-2Rα, IL-15Rα differently enhanced to the advantage of IL-15Rα, the one measured between γ(c) and IL-2Rα reduced, suggesting modulations in the strength of interaction of MHCI with IL-2R, IL-15R, and γ(c). The increases in density of surface bound cTx and in the associations of the receptors with the G(M1)-ganglioside lipid molecules suggest stronger lipid raft interactions of the receptors. The observed alterations of MHC-rafts in Crohn's disease--summarized in models of receptor patterns of diseased and control cells--may have functional consequences regarding signaling by the raft components.

[Treatment adherence and use of complementary and alternative medicine in patients with inflammatory bowel disease]. / A terápiás adherencia, valamint a komplementer és alternatív gyógymódok használata gyulladásos bélbetegek kezelésében.

UNLABELLED: Previous studies have suggested an increasing use of complementary and alternative medicine (CAM) in patients with inflammatory bowel disease (IBD). Furthermore, a significant number of IBD patients fail to comply with treatment. The aim of our study was to evaluate the prevalence of non-adherence the use of CAM in Hungarian patients with IBD. METHODS: A total of 655 consecutive IBD patients (Crohn's disease [CD]: 344, age: 38.2 + or - 12.9 years; ulcerative colitis [UC]: 311, age: 44.9 + or - 15.3 years) were interviewed during the visit at specialists by self-administered questionnaire including demographic and disease-related data, as well as items analyzing the extent of non-adherence and CAM use. Patients taking more then 80% of each prescribed medicine were classified as adherent. RESULTS: The overall rate of self reported non-adherence (CD: 20.9%, UC: 20.6%) and CAM (CD: 31.7%, UC: 30.9%) use was not different between CD and UC. The most common causes of non-adherence were: forgetfulness (47.8%), too many/unnecessary pills (39.7%), being afraid of side effects (27.9%) and too frequent dosing. Most common forms of CAM were herbal tee (47.3%), homeopathy (14.6%), special diet (12.2%), and acupuncture (5.8%). In CD, disease duration, date of last follow-up visit, educational level and previous surgeries were predicting factors for non-adherence. Alternative medicine use was associated in both diseases with younger age, higher educational level and immunosuppressant use. In addition, CAM use in UC was more common in females and in patients with supportive psychiatric/psychological therapy. CONCLUSIONS: Non-adherence and CAM use is common in patients with IBD. Special attention should be paid to explore the identified predictive factors during follow-up visits to improve adherence to therapy and improving patient-doctor relationship.

Adaptive threshold-stochastic resonance (AT-SR) in MHC clusters on the cell surface.

Highly conserved 2D receptor clusters (membrane rafts) of immunological signaling molecules with MHCI and MHCII antigens as their cores have been observed in the past on the surface of T- and B-cell lines of lymphoid origin, as well as on cells from patients with colon tumor and Crohn's disease. Conservativity is related to the ever presence of MHCI molecules. Although they are suspected to play a role in maintaining these clusters and facilitating transmembrane signaling, their exact role has been left largely enigmatic. Here we are suggesting stochastic resonance (SR), or "noise-assisted signal detection", as a general organizing principle for transmembrane signaling events evoked by processes like immune recognition and cytokine binding taking place in these clusters. In the conceptual framework of SR, in immune recognition as a prototype of transmembrane signaling, the sea of self-peptide-MHC complexes around a nonself-peptide presenting MHC is conceived as a source of quickly fluctuating unspecific signal ("athermal noise") serving the extra energy for amplifying the weak sub-threshold specific signal of the nonself-peptide presenting MHC. This same noise is also utilized for a readjustment of the threshold - and also the sensitivity and specificity - of detection by a closed loop feedback control of the TcR-CD8 (CD4) proximity on the detecting T-cell. The weak sub threshold specific signal of nonself-peptide presenting MHC is amplified by the superposing unspecific signals of the neighboring self peptide-MHC complexes towards the T-cell receptor as the detector. Because in a successful detection event both self- and nonself-peptides are detected simultaneously, the principle of coincidence (or lock-in) detection is also realized. The ever presence of MHC islands gets a natural explanation as a source of extra power - in a form of "athermal noise" - needed for coincidence detection and frequency encoding the evoked downstream signals. The effect is quite general, because the actual type of molecules surrounding a chief signaling molecule - like nonself-peptide holding MHC, interleukin-2 and -15 cytokine receptors (IL-2R/15R) - as the fluctuating interaction energy sources is immaterial. The model applies also for other types of signaling, such as those evoked by cytokine binding. The phenomenon of SR can also be interpreted as sampling of a low frequency, specific signal with a high frequency unspecific signal, the "noise". Recipes for identifying other forms of SR in membrane clusters with biophysical tools are recommended.