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.

[The effect of the I/D polymorphism of the angiotensin-converting enzyme gene on the cardiovascular risk and graft survival of kidney transplant patients]. / Az angiotenzinkonvertálóenzim-gén I/D polimorfizmusának hatása a vesetranszplantált betegek cardiovascularis rizikójára és a grafttúlélésre.

INTRODUCTION: Renal transplantation provides longer life expectancy in patients with renal failure. Nonetheless, this improved life expectancy is still shorter than that for the general population. The main couse of death in renal transplant patients is cardiovascular disease, and chronic allograft nephropathy is the most significant cause of graft loss. Genetic polymorphisms of the renin angiotensin system have been implicated in both chronic allograft nephropathy and fatal cardiovascular diseases. AIM: The long term goal of the authors was to improve the survival of renal transplanted patients. The authors aimed to identify novel biomarkers which correlate with the survival of the transplant organ and the recipient with a special attention to elements of the renin-angiotensin system. METHOD: A retrospective clinical trial was performed involving 72 renal transplanted patients. Angiotensin-converting enzyme I/D genotypes and activity, kidney function and morphological properties of the heart were determined. RESULTS: A significant positive correlation was found between the DD genotype of the angiotensin-converting enzíme gene, and the DD genotype predicted severe left ventricular hypertrophy. CONCLUSIONS: These findings suggest that the I/D genotypes of the angiotensin-converting enzyme gene predict not only the expected survival of the transplanted organ, but also that of the patient. Patients with the DD genotype are more susceptible for transplant failure. These patients should be identified and a special attention should be made on their pharmacological treatment (renin-angiotensin system inhibition), and their complience should also be maintained.

[Favourable effects of kidney transplantation on vascular condition of patients: Potential prognostic significance of non-invasive measurement of arterial stiffness]. / A vesetranszplantáció pozitív hatásai a betegek angiológiai státuszára: Az artériás funkció (stiffness) noninvazív mérésének lehetséges szerepe az elorejelzésben.

INTRODUCTION: Development of atherosclerosis is accelerated in kidney transplant patients. Impaired metabolic pathways have complex effect on the arterial wall which can be measured by non-invasive techniques. Only few data are available on the change of stiffness parameters in the postoperative course. Therefore, in this study the authors analysed the stiffness parameters of kidney transplant recipients during the perioperative period. AIM: Non-invasive clinical trial of the arterial functional parameters in the early postoperative period. METHOD: Seventeen successful primary kidney transplant patients with uneventful postoperative period (8 females, 9 males; age, 46.16 ± 12.19 years) were involved in this short-term prospective longitudinal study. The authors analysed correlations between non-in vasively assessed stiffness parameters (pulse wave velocity PWV, augmentation index - AIx). Stiffness parameters were measured with a TensioMed Arteriograph. These parameters were assessed before the transplantation, as well as 24 hours, 1 and 2 weeks after surgery under standard conditions. RESULTS: It was found that PWV (p = 0.0075) and AIx (p = 0.013) improved significantly. There was no significant change in case of PP and the other monitored parameters. Serum creatinine decreased (p = 0.0008) and glomerular filtration rate increased significantly (p = 0.0005). CONCLUSIONS: Along with the available data in the literature, the findings suggest that kidney transplantation has a positive effect on the arterial function. Improvement can be detected non-invasively with Arteriograph in the early postoperative period.

[Analysis of donor scoring systems in a single Hungarian transplant centre]. / Donorszelekciós kritériumok vizsgálata a debreceni veseátültetési programban.

INTRODUCTION: To ease organ shortage many transplant centres developed different donor scoring systems, however, a general consensus among clinicians on the use of these systems does not still exist. AIM: The aim of the authors was to analyse the effect of expanded criteria donor, deceased donor score and kidney donor risk index on postoperative kidney function and graft survival. METHOD: Analysis of the characteristics of 138 kidney transplantations and 205 donors in a retrospective study of a five-year period. RESULTS: There was a trend towards rejecting donors in higher risk groups; 22.7% of standard criteria donors belonged to the high risk group of deceased donor score. Graft function was worse in high risk patients. High risk donors can be divided due to the use of deceased donor score. Patients with the highest risk had worse graft function and survival. CONCLUSIONS: With the use of these scoring systems grafts with favourable outcome can be selected more precisely.

[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.

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.

Ureteral Complications Requiring Intervention After Kidney Transplant: A Single-Center Experience.

The surgical aspect of kidney transplant can be the surgical technique itself or the use of reconstruction techniques in the case of a complication requiring reoperation. In our study, we examined particularly surgical techniques and reconstruction options for ureteral anastomoses. Data from patients who underwent kidney transplant from 2010 to 2020 (N = 433) were examined retrospectively at follow-up of at least 1 year. We sought an association between the type of ureteral anastomoses and parameters considered to be risk factors based on literature data. We did not note the complicated cases that solved spontaneously and only selected cases where the ureteral anastomosis complication (UAcomp) needed urologic, radiological, or surgical intervention. In a smaller group, we examined the correlation between BK polyomavirus and ureteral stenosis. A total of 9.2% (n = 40) of patients developed UAcomp, 67.5% (n = 27) of whom required reoperation. In complicated cases, the rate of primary ureteral anastomosis type was 60.0% (n = 24) ureteroneocystostomy (UNS) and 40.0% (n = 16) ureteroureterostomy (UU) (P = .184). After UNS, 7.7% (n = 17) of cases required reoperation, and this rate was 4.7% (n = 10) after UU (P = .164). After treatment of the UAcomp, 95.0% (n = 38) of the patients were discharged with a functioning graft, and 5.0% (n = 2) required graftectomy. Complications of ureteral anastomosis with appropriate interventions results in good graft function. The type of ureteral anastomosis is not significantly associated with UAcomp. It is important that the operating surgeon is well versed in UNS and UU techniques to be able to adapt to any situation, be it primary surgery or reoperation.

Surgical complications after kidney transplantation based on the Clavien classification, especially with regard to the types of ureteral anastomoses / Veseátültetés utáni sebészeti szövodmények elofordulása a Clavien-beosztás szerint, különös tekintettel a húgyvezeték-anastomosis típusára.

Összefoglaló. Bevezetés: Veseátültetést követoen a graft és a beteg túlélésére hatással lehetnek a posztoperatív intervenciót igénylo szövodmények. Fontos szempont a mutéttechnikai eredményesség monitorozása. Többek között az irodalomban számos lehetoség ismert a veseátültetés sarokpontjának számító ureteranastomosis elkészítésére is, de az éranastomosisok technikája szintén dönto lehet. Célkituzés és módszer: Retrospektíven vizsgáltuk a 2010 és 2020 között végzett veseátültetéseket a Debreceni Egyetem Sebészeti Klinikáján. Célul tuztük ki a sebészeti szövodmények vizsgálatát, melyeket rendszereztünk, a módosított Clavien-féle beosztás alapján. A legnagyobb figyelmet az ureteranastomosisokra fordítottuk. Minden betegnél az adott kategóriában legsúlyosabb szövodményt vettük alapul a beosztáshoz. A minimális utánkövetési ido 1 év volt. Az adatokat az SPSS statisztikai program segítségével elemeztük. Eredmények: A vizsgált periódusban 406 veseátültetés történt, melybol 24,4% (n = 99) vesetranszplantáltnál alakult ki intervenciós (sebészeti, radiológiai, urológiai) szövodmény. A betegek átlagéletkora 49,5 ± 13,7 év, 60,8% férfi volt. A kumulatív mortalitás 10,1% volt. Grade 4-es szövodmény a betegek 6,9%-ánál (n = 28), Grade 3-as a 6,7%-ánál (n = 27), Grade 2-es a 3%-ánál (n = 12), Grade 1-es a 7,9%-ánál (n = 32) jelentkezett. A veseátültetés után 20,4%-ban (n = 83) alakult ki késon induló graftfunkció. Következtetés: A legenyhébb kategóriába (Grade 1.) került a legtöbb beteg, a szövodmények jelentos része sebészi, intervenciós radiológiai és urológiai közremuködéssel megoldható volt. Az ureteranastomosisok mutéti technikája és a releváns szövodmények kialakulása között nincs szignifikáns összefüggés. Megfelelo és idoben alkalmazott korrekciós kezelés mellett a graft- és betegtúlélést nem rontja szignifikánsan az enyhe és középsúlyos (Grade 1-3.) szövodmények kialakulása. Orv Hetil. 2021; 162(26): 1038-1051. INTRODUCTION: Complications associated with postoperative intervention may affect graft and patient survival after kidney transplantation. Monitoring the effectiveness of surgery is an important aspect. Ureter anastomosis can be the pivot of kidney transplant, the same as vascular anastomosis, so efficiency of the surgical technique is important to follow up. OBJECTIVE AND METHOD: We retrospectively examined kidney transplants performed between 2010 and 2020 at the Department of Surgery of the University of Debrecen. Data were analyzed by the SPSS statistical program. We aimed to investigate surgical complications, which were systematized based on the modified Clavien classification. In one patient, the most severe complication was used as the basis for the schedule. The minimum follow-up time was 1 year. RESULTS: 406 kidney transplants were performed in the examined period, of which 24.4% (n = 99) developed renal transplant complications (surgical, radiological, urological). The mean age of the patients was 49.5 ± 13.7 years, and 60.8% were male. The cumulative mortality was 10.1%. Grade 4 complication developed in 6.9% (n = 28) of the recipients, Grade 3 in 6.7% (n = 27), Grade 2 in 3% (n = 12), and Grade 1 in 7.9% (n = 32). 20.4% of the recipients had delayed graft function. CONCLUSION: The Grade 1 group had the biggest case number, so a significant part of the complications could be solved with the help of interventional radiology and urologists. There is no significant association between the surgical technique of ureteral anastomoses and the development of related complications. With appropriate therapy, graft and patient survival are not significantly impaired by the development of Grade 1-3 complications. Orv Hetil. 2021; 162(26): 1038-1051.

Non-Hodgkin Lymphoma in a Kidney Transplant Patient: A Case Report.

Post-transplant lymphoproliferative disorders are a possible complication of kidney transplant due to chronic immunosuppressive therapy, and they can elevate the mortality rate. Furthermore, the type of clinical appearance has a wide range. We describe a case of a 38-year-old male recipient who developed post-transplant lymphoproliferative disorders and received successful treatment. The recipient had received a kidney with 1 HLA-B and 1 HLA-DR match, and the deceased donor allotransplant was performed successfully on December 9, 2012. The cause of kidney failure was membranoproliferative-glomerulonephritis proved by biopsy results. The induction therapy was antithymocyte globulin; the basic immunosuppressive therapy consisted of tacrolimus, steroid, and mycophenolate mofetil. After 2 months the patient had elevated serum creatinine level, and biopsy results revealed cellular rejection (Banff grade I). We applied steroid bolus therapy. After that the graft worked properly for 5 years, and the patient had no symptoms or complaints; then he had right lower abdomen pain. After urgent procedures (laboratory diagnostics, abdominal ultrasonography, computed tomography), we operated on the patient in a short time, and after a few weeks the fluorescence in situ hybridization confirmed the translocation of region C-myc; the diagnosis was diffuse large B-cell lymphoma. With the assistance of hematologists, the patient received adequate therapy. He was asymptomatic half a year after the rituximab with cyclophosphamide, vincristine, doxorubicin, methotrexate/ifosfamide, etoposide, and high-dose cytarabine protocol therapy; the lymphoma is in remission. Our case is worth presenting because immunosuppressive drugs can modify the clinical picture, complicating the diagnosis and delaying treatment.

Pregnancy management of women with kidney transplantation.

Women with renal disease, besides many dysfunctions, face increasing infertility and high-risk pregnancy due to uremia and changes of the hormonal functions. After renal transplantation, sexual dysfunction improves, providing the possibility of successful pregnancy for women of childbearing age. However, kidney transplanted patients are high-risk pregnant patients with increased maternal and fetal risks, and the graft also may be compromised during pregnancy; most studies report on several successive deliveries due to multidisciplinary team management. In clinical practice, the graft is rarely affected during the period of gestation. Fetal development disorders are also rare although preterm delivery and intrauterine growth retardation are common. For now, several studies and clinical investigations proved that, under multidisciplinary control, kidney transplanted female patients are also possible to have safe pregnancy and successful delivery. There are conflicting data in the literature about the prevention of complications and the timing of pregnancy. Herein, we would like to present some experience of our centre. A total of 847 kidney transplantations have been performed between June 1993 and December 2013 with 163 childbearing aged females (18-45 years) in our center. We report on three kidney transplanted patients who have given birth to healthy newborns. In our practice, severe complications have not been observed.

New perspectives in the renin-angiotensin-aldosterone system (RAAS) IV: circulating ACE2 as a biomarker of systolic dysfunction in human hypertension and heart failure.

BACKGROUND: Growing evidence exists for soluble Angiotensin Converting Enzyme-2 (sACE2) as a biomarker in definitive heart failure (HF), but there is little information about changes in sACE2 activity in hypertension with imminent heart failure and in reverse remodeling. METHODS, FINDINGS: Patients with systolic HF (NYHAII-IV, enrolled for cardiac resynchronisation therapy, CRT, n = 100) were compared to hypertensive patients (n = 239) and to a healthy cohort (n = 45) with preserved ejection fraction (EF>50%) in a single center prospective clinical study. The status of the heart failure patients were checked before and after CRT. Biochemical (ACE and sACE2 activity, ACE concentration) and echocardiographic parameters (EF, left ventricular end-diastolic (EDD) and end-systolic diameter (ESD) and dP/dt) were measured. sACE2 activity negatively correlated with EF and positively with ESD and EDD in all patient's populations, while it was independent in the healthy cohort. sACE2 activity was already increased in the hypertensive group, where signs for imminent heart failure (slightly decreased EF and barely increased NT-proBNP levels) were detected. sACE2 activities further increased in patients with definitive heart failure (EF<50%), while sACE2 activities decreased with the improvement of the heart failure after CRT (reverse remodeling). Serum angiotensin converting enzyme (ACE) concentrations were lower in the diseased populations, but did not show a strong correlation with the echocardiographic parameters. CONCLUSIONS: Soluble ACE2 activity appears to be biomarker in heart failure, and in hypertension, where heart failure may be imminent. Our data suggest that sACE2 is involved in the pathomechanism of hypertension and HF.