The TreaT-Assay: A Novel Urine-Derived Donor Kidney Cell-Based Assay for Prediction of Kidney Transplantation Outcome

Donor-reactive immunity plays a major role in rejection after kidney transplantation, but analysis of donor-reactive T-cells is not applied routinely. However, it has been shown that this could help to identify patients at risk of acute rejection. A major obstacle is the limited quantity or quality of the required allogenic stimulator cells, including a limited availability of donor-splenocytes or an insufficient HLA-matching with HLA-bank cells. To overcome these limitations, we developed a novel assay, termed the TreaT (Transplant reactive T-cells)-assay. We cultivated renal tubular epithelial cells from the urine of kidney transplant patients and used them as stimulators for donor-reactive T-cells, which we analyzed by flow cytometry. We could demonstrate that using the TreaT-assay the quantification and characterization of alloreactive T-cells is superior to other stimulators. In a pilot study, the number of pre-transplant alloreactive T-cells negatively correlated with the post-transplant eGFR. Frequencies of pre-transplant CD161+ alloreactive CD4+ T-cells and granzyme B producing alloreactive CD8+ T-cells were substantially higher in patients with early acute rejection compared to patients without complications. In conclusion, we established a novel assay for the assessment of donor-reactive memory T-cells based on kidney cells with the potential to predict early acute rejection and post-transplant eGFR

Prognostic Value of Urinary Calprotectin, NGAL and KIM-1 in Chronic Kidney Disease

Background/Aims: Urinary biomarkers like neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) do not only allow an early diagnosis of acute kidney injury, but also provide prognostic information in this setting. The present prospective study investigates, whether the urinary biomarkers NGAL, KIM-1 and calprotectin have prognostic information in chronic kidney disease (CKD) as well. Methods: Urinary calprotectin, NGAL and KIM-1 concentrations were assessed in a study population of 143 patients with stable CKD comprising diabetic and hypertensive nephropathy, glomerulonephritis/vasculitis, and autosomal dominant polycystic kidney disease. An eGFR fluctuation > 5ml/min/1.73m2 in the past 12 months was defined as an exclusion criterion in order to exclude cases with acute on chronic kidney injury. Renal function was monitored for a median follow-up of 37 months. Results: In the overall study population, all the three biomarkers failed to predict DeGFR and DACR from baseline to follow-up in linear regression analysis adjusted for age, gender, and baseline eGFR. Contrarily, baseline ACR was significantly associated with DeGFR (p< 0.001). In the subgroup of patients with vasculitis and glomerulonephritis, all the three biomarkers were significantly associated with DeGFR, with calprotectin having the highest regression coefficient. Conclusion: In contrast to the traditional biomarker “albuminuria”, neither the inflammatory biomarker calprotectin, nor the tubular biomarkers NGAL and KIM-1, provide robust prognostic information on the loss or renal function in a heterogeneous CKD population. All of them, however, are candidate prognostic biomarkers in primarily inflammatory renal diseases

Detection of pre-existing SARS-CoV-2-reactive T cells in unexposed renal transplant patients

Background: Recent data demonstrate potentially protective pre-existing T cells reactive against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in samples of healthy blood donors, collected before the SARS-CoV-2 pandemic. Whether pre-existing immunity is also detectable in immunosuppressed patients is currently not known. Methods: Fifty-seven patients were included in this case-control study. We compared the frequency of SARS-CoV-2-reactive T cells in the samples of 20 renal transplant (RTx) patients to 20 age/gender matched non-immunosuppressed/immune competent healthy individuals collected before the onset of the SARS-CoV-2 pandemic. Seventeen coronavirus disease 2019 (COVID-19) patients were used as positive controls. T cell reactivity against Spike-, Nucleocapsid-, and Membrane-SARS-CoV-2 proteins were analyzed by multi-parameter flow cytometry. Antibodies were analyzed by neutralization assay. Results: Pre-existing SARS-CoV-2-reactive T cells were detected in the majority of unexposed patients and healthy individuals. In RTx patients, 13/20 showed CD4(+) T cells reactive against at least one SARS-CoV-2 protein. CD8(+) T cells reactive against at least one SARS-CoV-2 protein were demonstrated in 12/20 of RTx patients. The frequency and Th1 cytokine expression pattern of pre-formed SARS-CoV-2 reactive T cells did not differ between RTx and non-immunosuppressed healthy individuals. Conclusions: This study shows that the magnitude and functionality of pre-existing SARS-CoV-2 reactive T cell in transplant patients is non-inferior compared to the immune competent cohort. Although several pro-inflammatory cytokines were produced by the detected T cells, further studies are required to prove their antiviral protection

Risk factors for Epstein–Barr virus reactivation after renal transplantation: Results of a large, multi‐centre study

Epstein-Barr virus (EBV) reactivation is a very common and potentially lethal complication of renal transplantation. However, its risk factors and effects on transplant outcome are not well known. Here, we have analysed a large, multi-centre cohort (N = 512) in which 18.4% of the patients experienced EBV reactivation during the first post-transplant year. The patients were characterized pre-transplant and two weeks post-transplant by a multi-level biomarker panel. EBV reactivation was episodic for most patients, only 12 patients showed prolonged viraemia for over four months. Pre-transplant EBV shedding and male sex were associated with significantly increased incidence of post-transplant EBV reactivation. Importantly, we also identified a significant association of post-transplant EBV with acute rejection and with decreased haemoglobin levels. No further severe complications associated with EBV, either episodic or chronic, could be detected. Our data suggest that despite relatively frequent EBV reactivation, it had no association with serious complications during the first post-transplantation year. EBV shedding prior to transplantation could be employed as biomarkers for personalized immunosuppressive therapy. In summary, our results support the employed immunosuppressive regimes as relatively safe with regard to EBV. However, long-term studies are paramount to support these conclusions

Low avidity circulating SARS-CoV-2 reactive CD8+ T cells with proinflammatory TEMRA phenotype are associated with post-acute sequelae of COVID-19

The role of adaptive SARS-CoV-2 specific immunity in post-acute sequelae of COVID-19 (PASC) is not well explored, although a growing population of convalescent COVID-19 patients with manifestation of PASC is observed. We analyzed the SARS-CoV-2-specific immune response, via pseudovirus neutralizing assay and multiparametric flow cytometry in 40 post-acute sequelae of COVID-19 patients with non-specific PASC manifestation and 15 COVID-19 convalescent healthy donors. Although frequencies of SARS-CoV-2-reactive CD4+ T cells were similar between the studied cohorts, a stronger SARS-CoV-2 reactive CD8+ T cell response, characterized by IFNγ production and predominant TEMRA phenotype but low functional TCR avidity was detected in PASC patients compared to controls. Of interest, high avidity SARS-CoV-2-reactive CD4+ and CD8+ T cells were comparable between the groups demonstrating sufficient cellular antiviral response in PASC. In line with the cellular immunity, neutralizing capacity in PASC patients was not inferior compared to controls. In conclusion, our data suggest that PASC may be driven by an inflammatory response triggered by an expanded population of low avidity SARS-CoV-2 reactive pro-inflammatory CD8+ T cells. These pro-inflammatory T cells with TEMRA phenotype are known to be activated by a low or even without TCR stimulation and lead to a tissue damage. Further studies including animal models are required for a better understanding of underlying immunopathogensis. Summary: A CD8+ driven persistent inflammatory response triggered by SARS-CoV-2 may be responsible for the observed sequelae in PASC patients

Humoral, endothelial, and mechanical parameters of vasoregulation - detection of new systems and therapeutic options

Die Regulation des arteriellen Vasotonus wird durch ein dynamisches Zusammenspiel verschiedener lokaler und systemischer Komponenten gewährleistet. Ein Ungleichgewicht dieser Komponenten spielt in der Pathogenese der Hypertonie und der Atherosklerose eine zentrale Rolle. Tierexperimentelle Daten deuten darauf hin, dass für die Entstehung der essentiellen Hypertonie noch unentdeckte vasoregulatorische Systeme eine zentrale Rolle spielen. Der erste Teil der vorliegenden Arbeit widmet sich der Identifizierung und pharmakologischen Charakterisierung neuer humoraler, vasoaktiver Substanzen sowie der Suche nach neuen blutdruckregulierenden differentiell exprimierten Genen. Im zweiten Teil der Arbeit wird anhand von klinischen Studien untersucht, wie sich die arterielle Gefäßfunktion messen und therapeutisch beeinflussen lässt. Mit der Isolierung der Adenosinguanosinpolyphosphate (ApnG) aus menschlichen Thrombozyten gelang der Nachweis einer neuen Klasse von humanen vasoaktiven Substanzen, die über die Variation der Anzahl von Adenosin- und Phosphatgruppen eine Feinregulation der lokalen Perfusion eines Gefäßes ermöglichen. Die pharmakologische Charakterisierung dieser Substanzen zeigte, dass die vasoaktiven Effekte über den vaskulären P2X1-Rezeptor und einen kürzlich identifizierten P2X- Rezeptorsubtypen vermittelt werden. Die chromatographische Aufreiningung humanen Myokards führte zum erstmaligen Nachweis von Adenosintetraphosphat (Ap4) im Herzen. Es konnte gezeigt werden, dass sich Ap4 in den sekretorischen Granula der Myozyten befindet. Die Untersuchungen am Rattenherzen zeigten, dass Ap4 ein potenter Vasoregulator der Koronararterien ist. Bei intaktem Endothel kommt es zu einer P2Y1-vermittelten Vasodilatation. Bei geschädigtem Endothel kommt es hingegen zu einem Überwiegen der P¬2X-vermittelten Vasokonstriktion, was abermals die zentrale Rolle eines intakten Endothels für die physiologische Vasoregulation unterstreicht. Diese Daten zeigen, dass die endokrinen Funktionen des Herzens über die Synthese des atrialen natriuretischen Peptids hinausgehen. Im nächsten Teil der Arbeit gelang es, ein physiogenomisches Modell zur microarray-gestützten transkriptomweiten Detektion differerentiell exprimierter, vasoregulatorischer Gene zu etablieren. Mit Hilfe dieses Modells konnte ich nachweisen, dass eine vermehrte Produktion von Sauerstoffradikalen mit einer konsekutiven Verminderung der NO-Bioverfügbarkeit nicht zwingend Zeichen einer endothelialen Dysfunktion mit Krankheitswert sein muss, sondern auch Teil eines physiologischen Regelkreises sein kann. Neben der Identifizierung von neuen Vasoregulationssystemen ist dieses Modell für die Detektion von Kandidatengenen der essentiellen Hypertonie von Interesse. Eine Vielzahl von Studien haben die zentrale Rolle der endothelialen Dysfunktion als unabhängigen kardiovaskulären Risikofaktor beschrieben. Die nicht-invasive Messung der endothelialen Funktion über den derzeitigen Goldstandard, die sonographisch gestützte Untersuchung der FMD nach Unterarmkompression, ist jedoch aufwendig und für den klinischen Alltag ungeeignet. Von einigen Autoren wurde daher postuliert, dass eine statische, automatisierte Pulswellenanalyse zur Messung der Compliance der kleinen arteriellen Gefäße in der Lage sei, die Endothelfunktion mit hinreichender Genauigkeit zu reflektieren. Diese These wurde von mir überprüft und an einem großen Kollektiv hypertensiver und normotensiver Patienten widerlegt. Die Messung der NO-abhängigen Vasodilatation bedarf eines dynamischen Messvorgangs, in dem die Reaktion des Endothels auf einen definierten Stimulus dargestellt wird. Die Endothelfunktion und die arterielle Compliance haben einen hohen prädiktiven Wert für die kardiovaskuläre Morbidität und Mortalität. Es besteht daher ein großes Interesse an der therapeutischen Beeinflussbarkeit dieser Parameter. Im letzten Teil der vorliegenden Arbeit wurde nach Therapiekonzepten für Patienten mit besonders stark ausgeprägter Gefäßschädigung gesucht: Patienten mit terminaler Niereninsuffizienz und Patienten mit isolierter systolischer Hypertonie. Es wurden zunächst die Auswirkungen einer de novo Nierentransplantation auf die arterielle Compliance untersucht. Es zeigte sich, dass die Transplantation lediglich zu einer transienten Besserung der Gefäßelastizität führte. Da der Grund für die erneute Abnahme der Compliance am ehesten in den vasotoxischen Nebenwirkungen der Immunsuppressiva zu finden war, untersuchte ich in einer weiteren Arbeit die Auswirkungen des alternativen Immunmodulators FTY720 auf die funktionellen und mechanischen Gefäßeigenschaften. In Tierexperimenten hatte diese neu entwickelte Substanz bewiesen, dass sie eine S1P3-Rezeptor-vermittelte NO-abhängige Vasodilatation induziert. Die von mir innerhalb einer Phase III Studie erhobenen Daten zeigten jedoch, dass FTY720 in-vivo nicht in der Lage war, die Endothelfunktion zu verbessern. Vielmehr kam es zu einer Verbesserung der NO- abhängigen Vasodilation nach Konversion auf Mykophenolatmofetil. Diese Ergebnisse stellen den bisher einzigen Erklärungsansatz für die in der Phase III Studie aufgefallene reduzierte glomeruläre Filtrationsrate unter FTY720 dar. Regelmäßige sportliche Aktivität kann zu einer Verbesserung der Endothelfunktion und einer Abnahme des totalen peripheren Widerstands führen. Es wurde jedoch bezweifelt, dass körperliche Aktivität auch beim alten Menschen mit limitierter körperlicher Fitness, vermehrten muskuloskelettalen Problemen und einer fortgeschrittenen Atherosklerose mit dem Bild einer isolierten systolischen Hypertonie zu einer messbaren Verbesserung der Gefäßfunktion und einer Blutdrucksenkung führen kann. Unsere randomisierte, kontrollierte Studie konnte jedoch zeigen, dass auch bei diesen Patienten eine Verbesserung der Endothelfunktion erzielt wird, die zu einer klinisch relevanten Blutdrucksenkung führt. Die sportinduzierte Abnahme des Blutdrucks erwies sich als unabhängig vom Pulsdruck als Maß für arterielle Steifigkeit. In einer weiteren Studie wurde erstmalig der Einfluss einer chronischen Betablockade auf den kardiovaskulären Benefit durch Sport untersucht. Es zeigte sich, dass weder die Verbesserung der Endothelfunktion, noch die Abnahme des Blutdrucks durch Betablocker reduziert wurde. Diese Studie stellt einen ersten Ansatz zur Festlegung einer Trainingsherzfrequenz für Patienten mit Betablockern dar. In einer abschließenden kontrollierten, randomisierten Studie ließ sich nachweisen, dass auch ein Ausdauertraining der oberen Extremität zu messbaren Veränderungen der arteriellen Compliance und einer Blutdrucksenkung führt. Somit stellen für den älteren Hypertoniker weder eine fortgeschrittene Atherosklerose mit isolierter systolischer Hypertonie, noch eine Betablockertherapie oder Gehprobleme ein Hindernis dar, um durch regelmäßige sportliche Aktivität einen kardiovaskulären Benefit zu erzielen. Zusammenfassend gelang es in der vorliegenden Habilitationsschrift, neue vasoregulatorische Systeme zu detektieren und klinische Therapieoptionen zur Verbesserung der funktionellen und mechanischen Gefäßeigenschaften aufzuzeigen.The regulation of vasomotor tone is mediated by a dynamic interplay of various local and systemic parameters. Imbalances in this complex system may play a role in the pathogenesis of hypertension. The first part of the present work describes the identification and pharmacological characterization of new humoral vasoactive substances and the search for new differentially expressed blood pressure regulating genes. The second part describes clinical trials on the measurement of arterial function in vivo and therapeutic options. Adenosineguanosinepolyphosphates were isolated from human platelets. The activation of P(2x)-receptors in the rat renal vasculature by dinucleoside polyphosphates with variable phosphate group chain length (Xp(n)X; X=Adenin (A) /Guanin (G)) was studied by measuring their effects on perfusion pressure of the isolated perfused rat kidney. The experiments showed that the differential effects of dinucleoside polyphosphates allow a fine tuning of local perfusion via composition of Xp(n)Xs. Human myocardial tissue was fractionated by several chromatographic studies. A substance purified to homogeneity was identified as adenosine 5'-tetraphosphate (Ap(4). Ap(4) was also identified in ventricular specific granules. In the isolated perfused rat heart, Ap(4) elicited dose-dependent vasodilations by a P2Y1-receptor. After removal of the endothelium Ap(4) induced P2X-receptor-mediated vasoconstrictions. Thus, Ap(4) acts as an endogenous extracellular mediator and might contribute to the regulation of coronary perfusion. In the next project a physiogenomic screening model for differentially expressed genes in the regulation of blood pressure was established. By means of this model it could be shown that acute hypotension induces a diminished vasodilator potency of the endothelium due to an accelerated degradation of nitric oxide by reactive oxygen species (ROS). Thus, ROS do not only play a role in cardiovascular pathology but also in the physiological regulation of vasomotor tone. Determination of endothelial function has emerged as a crucial factor in the assessment of individual cardiovascular risk. Some authors speculated that a reduction of small artery compliance (C2) measured by pulse wave analysis might be an indicator of endothelial dysfunction. We investigated the correlation of pulse wave analysis parameters and endothelial function with special regard to patients who are at increased risk for endothelial dysfunction. It was shown that static measurement of vascular parameters by an automated pulse-wave analysis procedure cannot be used to assess endothelial function. Endothelial function and arterial compliance are of crucial relevance for cardiovascular morbidity and mortality. The last part of the present work therefore deals with clinical trials on therapeutic options. At first, the impact of renal transplant on arterial function was examined. We showed that transplantation led to a transient improvement of arterial compliance. With regard to a potential impact of immunosuppressive drugs on arterial function, the effects of the novel immunomodulator FTY720 on functional and mechanical vascular properties was investigated. In animal experiments FTY induced a S1P3-receptor mediated NO-dependent vasodilation. Participants of a phase III study, however, showed no improvement of endothelial function by FTY. Indeed, NO-dependent vasodilation improved after conversion to mycophenolatmofetil. To date, these findings constitute the only approach to a possible explanation for the reduced glomerular filtration rate in the phase III study on the use of FTY720 after renal transplantation. Physical exercise on a regular basis can induce an improvement of endothelial function and a decrease of peripheral vascular resistance. With regard to advanced arteriosclerosis and limited physical fitness, doubt was casted whether elderly patients still achieve relevant cardiovascular benefits by physical exercise. The present work examines the impact of pulse pressure as a footprint of vascular ageing on cardiovascular benefits of endurance training in elderly hypertensives. In a prospective randomized controlled study we showed that the exercise-induced reduction of BP, which is mediated by improved endothelial function, is independent of pulse pressure. Thus, physical exercise is a helpful adjunct to control BP even in old hypertensives with markedly increased arterial stiffness and isolated systolic hypertension. In another prospective randomized controlled study, we compared the cardiovascular effects of an endurance training programme in elderly hypertensives with or without beta-blockers and developed a first approach to determine a lactate-based training heart rate in presence of beta-blockade. A lactate-based aerobic endurance training evoked comparable cardiovascular benefits in the presence and absence of beta-blockade including a marked improvement of endothelial function. In this study target training heart rate with beta-blockers was about 18% lower than without. Several hypertensive patients are limited by musculoskeletal complaints or vascular occlusive disease from lower-limb exercise. In a final randomized-controlled study, we evaluate we showed that regular arm aerobic exercise leads to a marked reduction in systolic and diastolic blood pressures and an improvement in small artery compliance. In summary, the elderly hypertensive may have exercise-induced cardiovascular benefits despite of advanced atherosclerosis with isolated systolic hypertension, beta blocker therapy or musculoskeletal complaints. In the present work it was able to detect new vasoregulatory systems and to describe therapeutic options for an improvement of functional and mechanical vascular properties