Anti-inflammatory and anti-proliferative effects of CBS3830 in arterialized vein grafts in rats.

OBJECTIVE: To investigated whether CBS3830, a highly selectively inhibitor of p38MAPK, could ameliorate inflammation and intimal hyperplasia in arterialized vein grafts (AVGs). METHODS: Sixty male Sprague-Dawley rats underwent a reversed right jugular vein to common carotid artery interposition graft and were randomly treatment with vehicle (control) or single-dose (3 mg/kg, preoperative) or double-dose (3 mg/kg, preoperative and 4 d postoperative) CBS3830. Twenty rats underwent sham operation. The levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were determined by ELISA. Vein grafts were analyzed by intimal/medial morphometry, proliferating cell nuclear antigen (PCNA) expression, and p38MAPK phosphorylation. RESULTS: TNF-α, IL-1ß, and IL-6 gradually increased then slowly decreased in AVG rats. However, at 4 d and 7 d, TNF-α levels decreased by 37.5% and 29.5% (p = 0.003, 0.05, respectively) in the single-dose CBS3830 group, and by 37.6% and 32.5%, respectively (both p = 0.003) in the double-dose group compared with those of control. IL-1ß levels significantly reduced at 4 d and 14 d in both dosage groups. IL-6 levels significantly reduced at 7 d in both groups. Intima and medial thickening were significantly reduced in both dosage treated groups at 7, 14, and 28 d (all p = 0.000) compared to the controls. Further study showed CBS3830 inhibited p38MAPK phosphorylation and decreased PCNA expression. CONCLUSIONS: CBS3830 significantly decreases inflammation and intimal hyperplasia in AVGs.

p38 MAPK inhibitor, CBS3830 limits vascular remodelling in arterialised vein grafts.

OBJECTIVE: Following bypass surgery vein grafts undergo a remodelling process that can lead to restenosis and ultimately vein graft failure. Signalling through mitogen activated protein kinases (MAPKs) is a key mechanism involved in vein graft failure. Here, we investigated whether CBS3830 (c-a-i-r biosciences GmbH, Tübingen, Germany), a new highly selectively inhibitor of p38 MAPK, has a significant effect on inhibiting intimal, medial and adventitial hyperplasia. METHODS: Sixty specific pathogen free Sprague Dawley male rats were randomly divided into three groups. The control group with a reversed right jugular vein, which is common to carotid artery interposition graft, was compared with sham-operated, and CBS3830 treated animals. Intimal, medial and adventitia morphometric examinations and expression of proliferating cell nuclear antigen (PCNA) were analysed after one, two and four weeks for vein grafts. RESULTS: Intimal, medial and adventitia thickening in CBS3830 group were significantly lower than in the control group at each time point. Moreover, CBS3830 significantly reduced the phosphorylation of p38 MAPK and PCNA expression compared to the control. CONCLUSION: On the basis of the present work, intima, media and adventitia of saphenous vein grafts undergo vascular remodelling after surgery. The new, highly selective p38 MAPK inhibitor, CBS3830, ameliorates intimal, medial, and adventitial remodelling by varying degrees.

Kynurenine serves as useful biomarker in acute, Long- and Post-COVID-19 diagnostics.

Introduction: In patients with SARS-CoV-2, innate immunity is playing a central role, depicted by hyperinflammation and longer lasting inflammatory response. Reliable inflammatory markers that cover both acute and long-lasting COVID-19 monitoring are still lacking. Thus, we investigated one specific inflammatory marker involved as one key player of the immune system, kynurenine (Kyn), and its use for diagnosis/detection of the Long-/Post-COVID syndrome in comparison to currently used markers in both serum and saliva samples. Material and methods: The study compromised in total 151 inpatients with a SARS-CoV-2 infection hospitalized between 03/2020 and 09/2021. The group NC (normal controls) included blood bank donors (n=302, 144f/158m, mean age 47.1 ± 18.3 years (range 18-75)). Two further groups were generated based on Group A (n=85, 27f/58m, mean age 63.1 ± 18.3 years (range 19-90), acute admission to the hospital) and Group B (n=66, 22f/44m, mean age 66.6 ± 17.6 years (range 17-90), admitted either for weaning or for rehabilitation period due to Long-COVID symptoms/syndrome). Plasma concentrations of Kyn, C-Reactive Protein (CRP) and interleukin-6 (IL-6) were measured on admission. In Group B we determined Kyn 4 weeks after the negative PCR-test. In a subset of patients (n=11) concentrations of Kyn and CRP were measured in sera and saliva two, three and four months after dismission. We identified 12 patients with Post-COVID symptoms >20 weeks with still significant elevated Kyn-levels. Results: Mean values for NC used as reference were 2.79 ± 0.61 µM, range 1.2-4.1 µM. On admission, patients showed significantly higher concentrations of Kyn compared to NC (p-values < 0.001). Kyn significantly correlated with IL-6 peak-values (r=0.411; p-values <0.001) and CRP (r=0.488, p-values<0.001). Kyn values in Group B (Long-/Post-COVID) showed still significant higher values (8.77 ± 1.72 µM, range 5.5-16.6 µM), whereas CRP values in Group B were in the normal range. Conclusion: Serum and saliva Kyn are reflecting the acute and long-term pathophysiology of the SARS-CoV-2 disease concerning the innate immune response and thus may serve a useful biomarker for diagnosis and monitoring both Long- and Post-COVID syndrome and its therapy.

Differences in Immune Response During Competition and Preparation Phase in Elite Rowers.

Background: Metabolic stress is high during training and competition of Olympic rowers, but there is a lack of biomedical markers allowing to quantify training load on the molecular level. We aimed to identify such markers applying a complex approach involving inflammatory and immunologic variables. Methods: Eleven international elite male rowers (age 22.7 ± 2.4 yrs.; VO2max 71 ± 5 ml·min-1·kg-1) of the German National Rowing team were monitored at competition phase (COMP) vs. preparation phase (PREP), representing high vs. low load. Perceived stress and recovery were assessed by a Recovery Stress Questionnaire for Athletes (RESTQ-76 Sport). Immune cell activation (dendritic cell (DC)/macrophage/monocytes/T-cells) was evaluated via fluorescent activated cell sorting. Cytokines, High-Mobility Group Protein B1 (HMGB1), cell-free DNA (cfDNA), creatine kinase (CK), uric acid (UA), and kynurenine (KYN) were measured in venous blood. Results: Rowers experienced more general stress and less recovery during COMP, but sports-related stress and recovery did not differ from PREP. During COMP, DC/macrophage/monocyte and T-regulatory cells (Treg-cell) increased (p = 0.001 and 0.010). HMGB1 and cfDNA increased in most athletes during COMP (p = 0.001 and 0.048), while CK, UA, and KYN remained unaltered (p = 0.053, 0.304, and 0.211). Pro-inflammatory cytokines IL-1ß (p = 0.002), TNF-α (p < 0.001), and the chemokine IL-8 (p = 0.001) were elevated during COMP, while anti-inflammatory Il-10 was lower (p = 0.002). Conclusion: COMP resulted in an increase in biomarkers reflecting tissue damage, with plausible evidence of immune cell activation that appeared to be compensated by anti-inflammatory mechanisms, such as Treg-cell proliferation. We suggest an anti-inflammatory and immunological matrix approach to optimize training load quantification in elite athletes.

Dynamics and Diagnostic Relevance of Kynurenine Serum Level after Kidney Transplantation.

BACKGROUND: Inflammatory events after kidney transplantation (Tx) may lead to activation of the tryptophane-catabolizing enzyme indoleamine 2,3-dioxygenase followed by the formation of kynurenine (KYN). Post-transplant KYN serum levels in kidney allograft recipients were analyzed for their diagnostic value. MATERIAL AND METHODS: This was a retrospective analysis of KYN levels (normal value: 2.7±0.6 nmol/ml) measured in 4083 blood samples collected from 355 kidney graft recipients in connection with uncomplicated courses, acute rejections (ARs), infections, and type of immunosuppression. We performed descriptive data analysis and analysis of variance. RESULTS: In 212 recipients with immediately functioning grafts, the KYN levels dropped from pre-Tx 13.3±5.9 nmol/ml to nearly normal values at day 5 (5.8±3.0 nmol/ml). In patients with delayed graft function, the KYN reduction started only after the last hemodialysis treatment. With respect to ARs in recipients with creatinine values <300 µmol/l pre-AR, the increase of KYN levels depended on the severity of ARs (steroid-sensitive ARs: from 4.5±1.4 to 6.0±6.1 nmol/ml; steroid-resistant ARs: from 6.1±3.1 to 12.9±7.1 nmol/ml; vascular rejections: from 5.8±3.0 to 16.9±9.1 nmol/ml). In patients with creatinine values ≥300 µmol/l pre-AR, a further increase of the KYN level (from 10.1 to 13.2 nmol/ml) was only observed in severe, steroid-resistant ARs. With respect to infections evaluated, the KYN levels before diagnosis/start of treatment were 5.7±3.4 nmol/ml in asymptomatic CMV infections, 7.5±4.4 nmol/ml in CMV diseases, 8.3±3.3 nmol/ml in pneumonia, and 10.4±6.5 nmol/ml in bacterial sepsis. CONCLUSIONS: Serum KYN seems to be a reliable diagnostic tool for the assessment of post-transplant inflammatory complications, already in an early stage, and for monitoring the efficacy of therapeutic interventions. Prospective studies are recommended.

Effect of ischemia/reperfusion on telomere length and CDKI genes expression in a concordant ex-vivo hemoperfusion model of primate kidneys.

OBJECTIVES: The telomere (T) length, p21(WAF1/CIP1) and p27(Kip1) cyclin dependent kinase inhibitor (CDKI) genes are considered the markers of cell senescence and DNA damage. The aim of the study was to evaluate the influence of renal ischemia/reperfusion (I/R) on the value of above-mentioned markers. METHODS: 13 Macaque cynomolgus monkey kidneys were harvested and placed in Eurocollins solution. 9 kidneys were ex-vivo perfused with human blood and 4 kidneys were not perfused at all (control group). Tissue expression of p21(WAF1/CIP1) and p27(Kip1) was evaluated immunohistochemically and the T lengths were measured by southern blotting technique. RESULTS: Significantly higher levels of p21 and p27 were expressed by the glomeruli (p = 0.001 and 0.0001), tubules (p = 0.0065 and 0.0006) and interstitial cells (p = 0.0017 and 0.0022, respectively) of the xenoperfused kidneys. The mean T length was higher in the control group (5.56 +/- 0.60 kbp) than in the study group kidneys (5.46 +/- 0.36 kbp) (P = NS). CONCLUSION: Renal I/R is associated with telomere shortening and an over-expression of p21 and p27 genes indicating substantial DNA damage and/or accelerated tissue senescence.

Causes and prognostic value of pre-transplant elevated kynurenine level in kidney allograft recipients.

BACKGROUND: The activation of the tryptophane catabolizing enzyme Indoleamine 2,3-dioxygenase leads to the formation of kynurenine and other metabolites that counter-regulate immune activation resulting in restoration of immune homeostasis. But in chronic immune activation, as in hemodialysed patients, the immunosuppressive feedback mechanisms continue as indicated by elevated kynurenine concentrations. However, its relevance is still a matter of debate. MATERIAL/METHODS: This retrospective analysis presents the pre-transplant kynurenine levels (quantified photometrically) of 307 kidney graft recipients in connection with some pre- and post-transplant variables and the type of immunosuppression (cyclosporine-based triple drug therapy without/with ATG-Fresenius-induction). Statistical analyses performed were analysis of variance, Scheffé's test for pairwise comparisons, Cox regression, Spearman's rank correlation, and extended segmentation analysis. RESULTS: The pre-transplant kynurenine level was significantly elevated as compared to healthy adults (14.1±5.9 vs. 2.7±0.6 nmol/ml, p<0.0001), significantly higher in PRA positive than in PRA negative patients (16.1 vs. 12.9 nmol/ml, p<0.001) and, supporting this observation, also higher (p<0.0001) in a cohort with predominant (89.7%) pre-sensitized patients (16.4±6.4 nmol/ml) having the longest time on the waiting list (median 39 months) as compared to cohorts with fewer (16.8-22%) pre-sensitized patients (12.7±4.4 resp. 13.4±5.8 nmol/ml) having shorter times on the waiting list (16-24 months). Patients with immediately functioning grafts showed a lower pre-transplant kynurenine level than patients with non-immediately functioning grafts (13.5±6.0 vs. 14.9±5.7 nmol/ml, p=0.053). No associations were found with basic diseases, rejections, or graft survival. CONCLUSIONS: The pre-transplant elevated serum kynurenine levels were highly associated with the patient's pre-sensitization status and their longer time on hemodialysis treatments, but did not allow prognostic assessments.

Customized mycophenolate dosing based on measuring inosine-monophosphate dehydrogenase activity significantly improves patients' outcomes after renal transplantation.

BACKGROUND: Significant relationships have been reported between the uptake of mycophenolic acid (MPA) and the risk of acute rejection. In a prospective study after renal transplantation, we assessed the value of measuring inosine-monophosphate dehydrogenase (IMPDH) activity as a predictive indicator of an acute rejection episode in the initial postoperative period. PATIENTS AND METHODS: Fifty-two patients received 360 mg enteric-coated mycophenolate-sodium two times per day with concomitant tacrolimus/cyclosporine A, providing a total of 122 pharmacodynamic profiles. IMPDH activity was measured by a validated high-performance liquid chromatography method in four plasma samples collected at predose, 30 and 60 min, 2 and 4 hr, and preoperative, during weeks 1 and 2 and 3 months after transplantation. MPA concentrations were measured by mass spectrometry. Inhibition of IMPDH was correlated to the MPA values, MPA area under the curves, and predose levels of the different calcineurin inhibitors. RESULTS: Comparing the two groups (group I: rejection; n=17; mean age 51±15 years vs. group II: no rejection; n=35; mean age 51±14 years), we found a significantly (P<0.001) lower inhibition of IMPDH in group I (26.5%±11% vs. 56.7%±18%) already in the first week after transplantation. There was no correlation of MPA values (6.85±4 vs. 4.1±3 mg/L; first week) nor with the calcineurin inhibitor trough blood levels. Area under the curves for MPA did not differ significantly. Furthermore, IMPDH activity was a reliable predictor of rejection episodes and inflammation. CONCLUSION: The data suggest that measuring biologic response may be a more valuable indicator than traditional therapeutic drug monitoring of MPA. Patients at risk for rejection could be earlier identified, and the therapeutic potential of MPA will be optimized.

Up-regulation of cell cycle regulatory genes after renal ischemia/reperfusion: differential expression of p16(INK4a), p21(WAF1/CIP1) and p27(Kip1) cyclin-dependent kinase inhibitor genes depending on reperfusion time.

The aim of this study was to evaluate the influence of renal ischemia, cold preservation and reperfusion on the degree of renal kidney senescence. An experimental model of ex vivo renal hemoperfusion was used. Expression of p16(INK4a), p21(WAF1/CIP1) and p27(Kip1) cyclin-dependent kinase inhibitor genes (CDKIGs) was studied immunohistochemically in kidney biopsy samples at baseline and different time points after reperfusion. All three markers were up-regulated in kidney tissue after the reperfusion; however, their activation in different renal cells varied according to the reperfusion time. Expression of p16 was significantly increased in tubular cells at 180 min of reperfusion when compared with the baseline. Activation of p27 was detected in glomerular cells at 15 min and was significantly higher at 60, 120 and 180 min of reperfusion. The marker started increasing in tubular cells at 15 min and was elevated at every time point afterwards. p21 was significantly over-expressed in all renal cells after the reperfusion. It has been shown by the results of the current study that renal ischemia/reperfusion is associated with over-expression of CDKIGs indicating on substantial DNA damage and/or accelerated tissue senescence. For the first time it has been shown that tissue expression of CDKIGs is positively related with the reperfusion time.

Severe graft rejection, increased immunosuppression, and active CMV infection in renal transplantation.

Associations between active cytomegalovirus (CMV) infection, graft rejection, rejection therapy, and clinical signs and symptoms have been shown repeatedly. However, the causes and the sequence of events remain an area of debate. Two hundred twenty five patients with cadaveric renal transplant were included in the present study. Clinical signs and symptoms, and the development of active CMV infections were recorded during the first 3 months after renal transplantation. CMV monitoring by pp65-antigenemia was performed followed by preemptive antiviral therapy. Delayed graft function and severe graft rejection followed by anti T-cell antibody therapy was associated with the development of active CMV infection. In contrast, the induction therapy with anti-T-cell antibodies was not associated with more active CMV infections. Post-transplant morbidity determined by fever, pneumonia, and duration of hospital stay was increased significantly in patients with active CMV infection. However, in times of preemptive antiviral therapy an increased morbidity occurred in association with severe graft rejection and not with active CMV infection alone. In patients with renal transplantation and preemptive antiviral therapy, the morbidity was no more influenced by the CMV serostatus although the prevalence of active CMV infection was obviously different between CMV exposed (D+/R+,D+/R-, D-/R+) and unexposed (D-/R-) patients. Severe graft rejection and increased immunosuppression could stimulate cooperatively active CMV infections whereas immunosuppression alone may not be as effective. Prevention of severe graft rejection may be important to decrease early post-transplant morbidity and also the development of active CMV infections after renal transplantation.

Rapamycin rescue therapy in patients after kidney transplantation: first clinical experience.

This study was aimed at analysing rapamycin (RAPA) rescue therapy with calcineurin inhibitor (CNI) withdrawal in renal transplant patients primarily presenting with CNI-nephrotoxicity (CNI-Neph), chronic allograft nephropathy (CAN) without [CAN(a)] and with histological changes suggestive of chronic rejection [CAN(b)]. In 36 patient with CNI-Neph (n = 6), CAN(b) (n = 21), CAN(a) (n = 7), and others (n = 2) RAPA therapy was started 4.4-115 months (median 30.6 months) after renal transplantation. During a follow up of 3-33 months (median 19 months) parameters of kidney function were recorded. Three patients on haemodialysis did not show any recovery of graft function. Of the remaining 33 patients renal function improved in 22 (66.7%), was stable in three (9%) but deteriorated in eight (24%) patients, of whom seven (21%) required haemodialysis thereafter. Success rate of RAPA therapy differed with respect to the histological diagnosis: 70% in CAN(b), 80% in CNI-Neph and 33% in CAN(a). Furthermore, in patients with creatinine levels above 400 mum (n = 6) graft function rarely improved (n = 2, 33%). The RAPA rescue therapy with CNI withdrawal appears promising in a special cohort of patients with chronic renal allograft dysfunction even late after transplantation.

Influence of ischaemia/reperfusion and LFA-1 inhibition on telomere lengths and CDKI genes in ex vivo haemoperfusion of primate kidneys.

The telomere (T) length, p21(WAF1/CIP1) and p27(Kip1) cyclin-dependent kinase inhibitor (CDKI) genes are the markers of cell senescence and DNA damage. The aim of the study was to determine the influence of renal ischaemia/reperfusion (I/R) and anti-lymphocyte function-associated antigen-1 (LFA-1) monoclonal antibody (mAb) treatment on the value of the above-mentioned markers. Significantly higher levels of p21 and p27 were expressed by the glomeruli (P=0.001 and P=0.0001), tubules (P=0.0065 and P=0.0006), and interstitial cells (P=0.0017 and P=0.0022, respectively) of the xenoperfused kidneys. The mean T length of non-perfused renal specimens (5.56+/-0.60 kbp) was longer than that of the xenoperfused kidneys (5.46+/-0.36 kbp) [P= non-significant (NS)]. Addition of anti-LFA-1 mAb did not significantly influence the gene expression profile in the xenoperfused kidneys. The mean T length was longer in the kidneys with anti-LFA-1 mAb than in those without the medication (5.7+/-0.11 vs 5.13+/-0.31 kbp) (P=0.0661). Kidney I/R is associated with telomere shortening and an over-expression of p21 and p27 CDKIs, which indicates substantial DNA damage and/or accelerated tissue senescence. Although anti-LFA-1 mAb had some protective effect on the telomeres, it did not influence the gene expression profile in this study.