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.

[Significance of the computed tomography assisted morphometry in the surgical planning of eventrated abdominal wall hernias]. / A komputertomográfia jelentosége az eventerált, óriás hasfali sérvek mutéteinek tervezésében.

INTRODUCTION: The eventrated, giant abdominal wall hernias represent a considerable challenge in our practice. Presently, preoperative evaluation of the musculo-aponeurotic elements of the abdominal wall by CT imaging is not part of routine planning of surgery. AIM: Evaluation of the abdominal wall hernia progression in time. Moreover, follow up the changes of the abdominal wall structures following series of intraabdominal surgeries. METHOD: Abdominal CT imaging were performed on the 1st, 3rd, 6th, 12th, 18th, and 24th postoperative months after the primary series of surgeries in the cases of 12 patients, whose reconstructive surgeries were not possible. A prospective data collection was applied. Changing of the bilateral rectus muscle morphology, the evolution in time of the midline gap, and the progressive dynamism of the midline wall defects were determined. RESULTS: A characteristic and progressive midline defect enlargement could be settled. Data analysis yielded that the combined width of the bilateral rectus muscles is sufficient to cover the midline abdominal wall defect, although there is an "optimal" timeframe for performing the intervention. CONCLUSION: CT evaluation of abdominal wall prior to reconstructive surgeries of loss of abdominal wall domain has a strong significance on determining and designing the adequate surgical procedure. Orv. Hetil., 2017, 158(7), 257-263.

[Significance of the monitoring and screening for hereditary nonpolyposis colorectal carcinoma syndrome patients by presenting a case of a family tree]. / A hereditaer nonpolyposus colorectalis carcinoma szindrómás betegek szurésének és szoros utánkövetésének fontossága egy családfa bemutatása kapcsán.

INTRODUCTION: Hereditary nonpolyposis colorectal carcinoma (HNPCC) is an autosomal dominant disease, which shows familial clustering. AIM: We would like to emphasize the importance of monitoring the HNPCC syndrome patients by presenting a case of a proven MMR gene mutation carrier and her family tree encompassing 10 years. MATERIALS AND METHOD: To screen a suspected HNPCC Hungarian family member we are taking thorough family histories. If the diagnosis of HNPCC was further supported by immunohistology and the microsatellite status, sequencing of the MMR genes was carried out. RESULTS: A novel mutation in exon 6 of the hMSH2 gene leading to the deletion of two nucleotide pairs [c.969-970delTC] was detected in our patient. During the 10-year follow-up period of our patient new HNPCC-associated tumors have developed in several family members. Conslusion: Close surveillance of the patient and its family members at risk was effective, although it requires compliance from the subjects. Orv Hetil. 2017; 158(30): 1182-1187.

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.

[Allograft nephrectomy - a single-center experience]. / Graftectomiák a debreceni vesetranszplantációs programban.

INTRODUCTION: Indication and timing of allograft nephrectomy is still uncertain in some cases. AIM: The aim of the authors was to summarize their experience with graftectomies. METHOD: Data from patients who underwent kidney transplantation between January 1, 2004 and December 31, 2015 were retrospectively analyzed. Frequency, indications, timing, complications as well as early and late allograft nephrectomies were reviewed. RESULTS: From 480 renal transplants, 55 graftectomies were performed (11%). Frequent indications included chronic allograft nephropathy (47%), arterial blood supply complications (13%), ureter complications (9%). 22 cases (40%) of allograft nephrectomies were urgent while 33 cases (60%) were elective. 24% of graftectomies were performed within 30 days after transplantation and 76% thereafter. CONCLUSIONS: The main indications for early graftectomies were arterial complications (31%) and chronic allograft nephropathy (62%) in cases of late graftectomies. The majority of the graftectomies were elective. Leading indication was chronic allograft nephropathy. Early and late graftectomies have different characteristics.

[First outcomes, since being full member of Eurotransplant. A single center experience of cadaveric kidney transplantation]. / Eredményeink a teljes jogú Eurotransplant-tagság óta. A Debreceni Vesetranszplantációs Központ tapasztalatai.

INTRODUCTION: The first renal transplantation was completed in 1991 at the University of Debrecen. In 2013 Hungary joined Eurotransplant. AIM: The authors retrospectively compared the trends. METHOD: Comparison between Period A (from January 1, 2008 to August 31, 2013) and Period B (from September 1, 2013 to October 22, 2015). RESULTS: The proportion of living transplants rose from 3.5% to 9.1 %. During period B over 25% of utilized donors were over 60 years of age. Recipients with body mass index above 30 kg/m(2) increased from 12% to 31%. Prevalence of diabetes among recipients rose twofold. Uretero-neocystostomy was used during period A (99%) while in period B end to side uretero-ureteral anastomosis has also gained popularity (68%). In 2013 the authors introduced routine use of induction treatment. Acute rejection rate decreased from 34% to 8%. The rate of surgical complications did not change. Acute bacterial infections decreased from 41% to 33%. Cumulative renal allograft 1, 3 and 5 year survival rates were 86.6%, 85% and 82.7% in group A vs. projected rates 88%, 84% and 84% in group B, respectively. CONCLUSIONS: Despite the growing proportion of expanded criteria donors, the authors were able to maintain a low incidence of delayed graft function and a favorable graft survival. Since 2013 the authors introduced treatments for acute humoral rejection according to international standards.

[The role of autografts in the treatment of complicated incisional hernias]. / Az autograftok szerepe a komplikált hasfali sérvek kezelésében.

INTRODUCTION: Complicated incisional hernias (at least one time recurrent and/or multilocular and/or infected synthetic mesh) still represent a significant problem. AIM: Presentation of operating techniques desribed so far, as well as publication of a novel procedure and results invented by the authors. METHOD: Between 01/2011 and 09/2015, 41 patients with recurrent and/or infected incisional hernias with or without entero- and subcutaneous fistulas were operated using the method of the s.c. double-layer autologous tension free dermal flap technique. An accurate follow-up method and a continuous registration of the results was conducted in case of every patient. The body mass index (BMI) and presence of diabetes mellitus (DM) were distinguished factors out of the patients' clinical data. Surgical complications, bulking or laxity, recurrence and the patients' satisfactory index - among other things - were recorded considering the procedure. Patients' clinical data and results: Average age was 59.2 years (13 male / 28 female) in the cohort. 1, 2, 3 times recurrent incisional hernias had 12, 23, 6 patients, respectively. Average BMI was 32,1 kg/m2. 12 patients were treated with type II diabetes. 13 patients had entero- or subcutaneous fistulas and/or infected synthetic meshes at the time of operations. Average follow-up time was 32 months (2-58 months). Seroma formation was registered in 13 cases (31.7%). Fistula formation was registered in one case (2.4%). Bulking formation or laxity was observed in 3 patients (7.3%) and recurrence was noticed in 3 patients (7.3%), 13, 17 and 19 months later in the postoperative period. All the entero- and subcutaneous fistulas developed prior to the last procedure were completely healed. There was no lethal outcome. CONCLUSION: The method developed by the authors is recommended and suitable for the solution to complicated and/or infected incisional abdominal wall hernias especially in cases of obese (BMI ≥ 25 kg/m(2)) and diabetic, high risk patients. After acquiring the precise operative technique, the method is safe, feasible and it comes along with lower complication and recurrence rate, compared to other applied and well established ones. Further clinical trials need to be conducted in the future in order to be evaluated definetely this procedure.

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.

Primary angiosarcoma of the pancreas mimicking severe acute pancreatitis--case report.

Primary angiosarcoma of the pancreas is an extremely rare neoplasm that often mimicks severe acute pancreatitis. A 58-year-old man was admitted with clinical and laboratory signs of severe acute pancreatitis. Contrast enhanced CT scan demonstrated haemorrhagic necrotizing inflammation of the pancreas involving the pancreatic tail, splenic hilum and small bowels with multiple peripancreatic and free abdominal fluid collection. Percutaneous drainage was performed. After 13 days, laparotomy was indicated because of persistent intra-abdominal bleeding, fever and a palpable, rapidly growing mass in the left upper quadrant of the abdomen. During the operation a necrotic, haemorrhagic mass was found in the pancreatic tail; a frozen section showed malignancy, although the tumour was unresectable. Despite all conservative and surgical therapeutic attempts, the patient died within four weeks after diagnosis. Final histology justified primary angiosarcoma of the pancreas. If a patient with signs of severe acute pancreatitis has fever without elevated PCT, the presence of a malignant tumour of the pancreas should be considered.

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.

Prospective, comparative study for the evaluation of lymph node involvement in gastric cancer: Maruyama computer program versus sentinel lymph node biopsy.

BACKGROUND: Stage-adapted surgery guarantees the best outcome for patients with gastric cancer. Successful identification of lymph node involvement may help to reduce the number of extended lymphadenectomies. Preoperative diagnostic tools have low sensitivity and specificity for determining lymph node involvement. Evaluation of sentinel lymph nodes (SLNs) intraoperatively has good results, while the accuracy of the Maruyama computer program (MCP) is controversial. METHODS: We investigated 40 patients by the Maruyama computer model and labeled lymph nodes with blue dye for SLN mapping. To compare the probability calculations by MCP and the results of SLN mapping, we had to define a cutoff level; we did this using receiver-operating characteristics analysis. Sentinel lymph nodes were examined in frozen sections intraoperatively and by standard hematoxylin and eosin staining postoperatively. RESULTS: A total of 795 lymph nodes were removed and examined. The Maruyama computer model had a sensitivity of 91.3 %, specificity of 64 %, and accuracy of 80 % by the best cutoff point. The false-negative rate was 8.7 %. The sensitivity of SLN mapping was 95.7 %, the false-negative rate was 4.3 %, and the specificity was 100 %. The accuracy of SLN mapping was 97.4 %. Only the sensitivity of MCP and SLN biopsy was proven equivalent. CONCLUSIONS: Our results suggest that intraoperative SLN examination is superior to preoperative estimation with the MCP. Correct definition of lymph node involvement helps in planning the best stage-adapted surgery in gastric cancer.