Enzymatic activity of extracellular circulating proteasome in the CSF of patients with malignant Intrinsic and metastatic brain tumors: proof of concept.

BACKGROUND: Malignant intrinsic brain tumors are a hazardous disease with limited life expectancy despite intensive research in new targeted treatment options. Lately, proteasome inhibitors have been identified as potent agents causing death in glioma cell lines. It is the aim of the present study to identify proteasomal activity in the CSF of patients suffering from malignant brain tumors. METHODS: A total of 24 patients with histological confirmed brain tumors (12 malignant gliomas, 12 metastases) were included and CSF probes preoperatively analyzed for concentration and enzymatic activity of free circulating proteasome. Tumor volumina were assessed using the preoperative MRI and correlated with the CSF findings. Statistical analyses were performed using SPSS (18.0.3; SPSS Inc., Chicago, IL, USA). RESULTS: Extracellular proteasomes were found in all CSF samples showing enzymatic activity. Proteasome concentrations (28 ng/mL and 23 ng/mL, resp.) were elevated compared to a historical control group. Proteasomal enzymatic chymotrypsin-like activity was significantly raised in patients with gliomas (mean 31 fkat/ mL) compared to controls (P<0.049), whereas the enzymatic activity was not significantly elevated in metastases (P=0.109). In gliomas, neither concentration nor enzymatic activity correlated with the preoperative assessed tumor volume. CONCLUSIONS: This pilot study clearly showed that the proteasomal activity in the CSF is significantly elevated in patients with intrinsic brain tumors. Further studies need to identify the proteasomal concentration and enzymatic activity as a potential biomarker for the effectiveness of any treatment and for the early diagnosis of a possible recurrence of the disease.

Validation of days alive and out of hospital as a new patient-centered outcome to quantify life impact after heart transplantation.

The number of patients waiting for heart transplantation (HTX) is increasing. Thus, identification of outcome-relevant factors is crucial. This study aimed to identify perioperative factors associated with days alive and out of hospital (DAOH)-a patient-centered outcome to quantify life impact-after HTX. This retrospective cohort study screened 187 patients who underwent HTX at university hospital Duesseldorf, Germany from September 2010 to December 2020. The primary endpoint was DAOH at 1 year. Risk factors for mortality after HTX were assessed in univariate analysis. Variables with significant association were entered into multivariable quantile regression. In total, 175 patients were included into analysis. Median DAOH at 1 year was 295 (223-322) days. In univariate analysis the following variables were associated with reduced DAOH: recipient or donor diabetes pre-HTX, renal replacement therapy (RRT), VA-ECMO therapy, recipient body mass index, recipient estimated glomerular filtration rate (eGFR) and postoperative duration of mechanical ventilation. After adjustment, mechanical ventilation, RRT, eGFR and recipient diabetes showed significant independent association with DAOH. This study identified risk factors associated with reduced DAOH at 1-year after HTX. These findings might complement existing data for outcome of patients undergoing HTX.

Impact of Cardiopulmonary Resuscitation of Donors on Days Alive and Out of Hospital after Orthotopic Heart Transplantation.

BACKGROUND: The number of patients waiting for heart transplantation (HTX) is increasing. Optimizing the use of all available donor hearts is crucial. While mortality seems not to be affected by donor cardiopulmonary resuscitation (CPR), the impact of donor CPR on days alive and out of hospital (DAOH) is unclear. METHODS: This retrospective study included adults who underwent HTX at the University Hospital Duesseldorf, Germany from 2010-2020. Main exposure was donor-CPR. Secondary exposure was the length of CPR. The primary endpoint was DAOH at one year. RESULTS: A total of 187 patients were screened and 171 patients remained for statistical analysis. One-year mortality was 18.7%. The median DAOH at one year was 295 days (interquartile range 206-322 days). Forty-two patients (24.6%) received donor-CPR hearts. The median length of CPR was 15 (9-21) minutes. There was no significant difference in DAOH between patients with donor-CPR hearts versus patients with no-CPR hearts (CPR: 291 days (211-318 days) vs. no-CPR: 295 days (215-324 days); p = 0.619). Multivariate linear regression revealed that there was no association between length of CPR and DAOH (unstandardized coefficients B: -0.06, standard error: 0.81, 95% CI -1.65-1.53, p = 0.943). CONCLUSIONS: Donor CPR status and length of CPR are not associated with reduced DAOH at one year after HTX.

Days alive and out of hospital after left ventricular assist device implantation.

AIMS: Implantation of left ventricular assist devices (LVADs) as a bridge to transplant or as destination therapy is increasing. The selection of suitable patients and outcome assessment belong to the key challenges. Mortality has traditionally been a focus of research in this field, but literature on quality of life is very limited. This study aimed to identify perioperative factors influencing patients' life as measured by days alive and out of hospital (DAOH) in the first year after LVAD implantation. METHODS AND RESULTS: This retrospective single-centre cohort study screened 227 patients who underwent LVAD implantation at the University Hospital Duesseldorf, Germany, between 2010 and 2020. First, the influence of 10 prespecified variables on DAOH was investigated by univariate analysis. Second, multivariate quantile regression was conducted including all factors with significant influence on DAOH in the univariate model. Additionally, the impact of all variables on 1 year mortality was investigated using Kaplan-Meier curves to oppose DAOH and mortality. In total, 221 patients were included into analysis. As pre-operative factors, chronic kidney disease (CKD), pre-operative mechanical circulatory support (pMCS), and Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) stadium < 3 were associated with lower DAOH at 1 year [CKD: 280 (155-322) vs. 230 (0-219), P = 0.0286; pMCS: 294 (155-325) vs. 243 (0-293), P = 0.0004; INTERMACS 1: 218 (0-293) vs. INTERMACS 2: 264 (6-320) vs. INTERMACS 3: 299 (228-325) vs. INTERMACS 4: 313 (247-332), P ≤ 0.0001]. Intra-operative additional implantation of a right ventricular assist device (RVAD) was also associated with lower DAOH [RVAD: 290 (160-325) vs. 174 (0-277), P ≤ 0.0001]. As post-operative values that were associated with lower DAOH, dialysis and tracheotomy could be identified [dialysis: 300 (252-326) vs. 186 (0-300), P ≤ 0.0001; tracheotomy: 292 (139-325) vs. 168 (0-269), P ≤ 0.0001]. Multivariate analysis revealed that all of these factors besides pMCS were independently associated with DAOH. According to Kaplan-Meier analysis, only post-operative dialysis was significantly associated with increased mortality at 1 year (survival: no dialysis 89.4% vs. dialysis 70.1%, hazard ratio: 0.56, 95% confidence interval: 0.33-0.94; P = 0.031). CONCLUSIONS: The results of this study indicate that there can be a clear discrepancy between hard endpoints such as mortality and more patient-centred outcomes reflecting life impact. DAOH may relevantly contribute to a more comprehensive selection process and outcome assessment in LVAD patients.

Life impact of VA-ECMO due to primary graft dysfunction in patients after orthotopic heart transplantation.

AIMS: Primary graft dysfunction (PGD) is a feared complication after heart transplantation (HTX). HTX patients frequently receive veno-arterial extracorporeal membrane oxygenation (VA-ECMO) until graft recovery. Long-term mortality of patients weaned from VA-ECMO after HTX is comparable with non-ECMO patients. However, impact on quality of life is unknown. This study investigated days alive and out of hospital (DAOH) as patient-centred outcome in HTX patients at 1 year after surgery. METHODS AND RESULTS: This retrospective single-centre cohort study included patients who underwent HTX at the University Hospital Düsseldorf, Germany, from 2010 to 2020. Main exposure was VA-ECMO due to PGD. VA-ECMO and non-VA-ECMO patients were compared regarding the primary endpoint DAOH at 1 year after HTX. Subgroup analysis for patients weaned from VA-ECMO was performed. In total, 144 patients were included into analysis; 1 year mortality was significantly lower in non-ECMO patients [non-ECMO 14.3% (14/98) vs. VA-ECMO 34.8% (16/46), adjusted hazard ratio: 0.32, 95% confidence interval: 0.15-0.74; P = 0.002]. Mortality did not differ significantly between patients weaned from VA-ECMO and non-ECMO patients [non-ECMO 14.3% (14/98) vs. VA-ECMO (weaned) 18.9% (7/37), adjusted hazard ratio: 0.72, 95% confidence interval: 0.27-1.90; P = 0.48]. DAOH were significantly higher in non-ECMO patients compared with VA-ECMO patients and patients weaned from VA-ECMO [non-ECMO vs. VA-ECMO: median 310 (inter-quartile range 277-327) days vs. 243 (0-288) days; P < 0.0001; non-ECMO vs. VA-ECMO (weaned): 310 (277-327) days vs. 253 (208-299) days; P < 0.0001]. These results were still significant after multivariable adjustment with forced entry of predefined covariables. CONCLUSIONS: Despite similar survival rates, VA-ECMO due to PGD has a relevant life impact as defined by DAOH in the first year after HTX. As a more patient-centred endpoint, DAOH may contribute to a more comprehensive assessment of outcome in HTX patients.

Risk Factors for Acute Kidney Injury Requiring Renal Replacement Therapy after Orthotopic Heart Transplantation in Patients with Preserved Renal Function.

Acute kidney injury (AKI), requiring renal replacement therapy (RRT). is a serious complication after orthotopic heart transplantation (HTX). In patients with preexisting impaired renal function, postoperative AKI is unsurprising. However, even in patients with preserved renal function, AKI requiring RRT is frequent. Therefore, this study aimed to identify risk factors associated with postoperative AKI requiring RRT after HTX in this sub-cohort. This retrospective cohort study included patients ≥ 18 years of age with preserved renal function (defined as preoperative glomerular filtration rate ≥ 60 mL/min) who underwent HTX between 2010 and 2021. In total, 107 patients were included in the analysis (mean age 52 ± 12 years, 78.5% male, 45.8% AKI requiring RRT). Based on univariate logistic regression, use of extracorporeal membrane oxygenation, postoperative infection, levosimendan therapy, duration of norepinephrine (NE) therapy and maximum daily increase in tacrolimus plasma levels were chosen to be included into multivariate analysis. Duration of NE therapy and maximum daily increase in tacrolimus plasma levels remained as independent significant risk factors (NE: OR 1.01, 95%CI: 1.00-1.02, p = 0.005; increase in tacrolimus plasma level: OR 1.18, 95%CI: 1.01-1.37, p = 0.036). In conclusion, this study identified long NE therapy and maximum daily increase in tacrolimus plasma levels as risk factors for AKI requiring RRT in HTX patients with preserved renal function.

Osteopontin as novel biomarker for reversibility of pressure overload induced left ventricular hypertrophy.

Aim: The aim of this study was to evaluate the prognostic value of osteopontin (OPN) as a marker for left ventricular (LV) hypertrophy and its reversibility after surgical aortic valve replacement (SAVR). Patients & methods: Echocardiographic data and OPN plasma levels of 149 consecutive patients undergoing SAVR were obtained preoperatively and 3 months postoperatively. OPN was measured by Quantikine Human OPN immunoassay. Results: There was a significant correlation between higher OPN plasma levels and lower LV-mass regression. In patients receiving SAVR combined with coronary artery bypass grafting, high OPN plasma levels were also an indicator for eccentric hypertrophy phenotype. Conclusion: OPN may be a useful indicator for LV hypertrophy phenotype and could have a prognostic value to estimate LV-mass regression after SAVR.

Impact of Ca2+-Sensitive Potassium Channels in Levosimendan-Induced Postconditioning.

PURPOSE: Small and big conductance Ca2+-sensitive potassium (KCa) channels are involved in cardioprotective measures aiming at reducing myocardial reperfusion injury. For levosimendan, infarct size-reducing effects were shown. Whether activation of these channels is involved in levosimendan-induced postconditioning is unknown. We hypothesized that levosimendan exerts a concentration-dependent cardioprotective effect and that both types of Ca2+-sensitive potassium channels are involved. METHODS: In a prospective blinded experimental laboratory investigation, hearts of male Wistar rats were randomized and placed on a Langendorff system, perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. All hearts were subjected to 33 min of global ischemia and 60 min of reperfusion. At the onset of reperfusion, hearts were perfused with various concentrations of levosimendan (0.03-1 µM) in order to determine a concentration-response relationship. To elucidate the involvement of KCa-channels for the observed cardioprotection, in the second set of experiments, 0.3 µM levosimendan was administered in combination with the subtype-specific KCa-channel inhibitors paxilline (1 µM, big KCa-channel) and NS8593 (0.1 µM, small KCa-channel) respectively. Infarct size was determined by tetrazolium chloride (TTC) staining. RESULTS: Infarct size in controls was 60 ± 7% and 59 ± 6% respectively. Levosimendan at a concentration of 0.3 µM reduced infarct size to 30 ± 5% (P < 0.0001 vs. control). Higher concentrations of levosimendan did not induce a stronger effect. Paxilline but not NS8593 completely abolished levosimendan-induced cardioprotection while both substances alone had no effect on infarct size. CONCLUSIONS: Cardioprotection by levosimendan-induced postconditioning shows a binary phenomenon, either ineffective or with maximal effect. The cardioprotective effect requires activation of big but not small KCa channels.

Preconditioning by Levosimendan is Mediated by Activation of Mitochondrial Ca2+-Sensitive Potassium (mBKCa) Channels.

PURPOSE: Activation of mitochondrial large-conductance Ca2+-sensitive potassium (mBKCa)-channels is a crucial step for cardioprotection by preconditioning. Whether activation of these channels is involved in levosimendan-induced preconditioning is unknown. We investigated if cardioprotection by levosimendan requires activation of mBKCa-channels in the rat heart in vitro. METHODS: In a prospective blinded experimental laboratory investigation, hearts of male Wistar rats were randomized and placed on a Langendorff system, perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. All hearts were subjected to 33 min of global ischemia and 60 min of reperfusion. Before ischemia, hearts were perfused with different concentrations of levosimendan (0.03-1 µM) for determination of a dose-effect curve. In a second set of experiments, 0.3 µM levosimendan was administered in combination with the mBKCa-channel inhibitor paxilline (1 µM). Infarct size was determined by TTC staining. RESULTS: In control, animal's infarct size was 58 ± 7%. Levosimendan at a concentration of 0.3 µM reduced infarct size to 30 ± 7% (P < 0.05 vs. control). Higher concentrations with 1 µM levosimendan did not confer stronger protection. Paxilline completely blocked levosimendan-induced cardioprotection while paxilline alone had no effect on infarct size. CONCLUSIONS: This study shows that activation of mBKCa-channels plays a pivotal role in levosimendan-induced preconditioning.

CCL18 in serum, BAL fluid and alveolar macrophage culture supernatant in interstitial lung diseases.

BACKGROUND: CCL18 is a CC chemokine produced mainly by antigen-presenting cells, and is chemotactic predominantly for T-lymphocytes. CCL18 can stimulate pulmonary fibroblasts and increase the collagen production in vitro. OBJECTIVES: This study aimed to compare the CCL18 levels in a variety of human biological fluids between various interstitial lung diseases (ILDs), and to reveal potential correlations with BAL cell differentials. METHODS: Serum and bronchoalveolar lavage fluid (BALF) samples were collected from 199 patients with idiopathic pulmonary fibrosis (IPF), idiopathic non-specific interstitial pneumonia (iNSIP), respiratory bronchiolitis interstitial lung disease/desquamative interstitial pneumonia (RB-ILD/DIP), cryptogenic organizing pneumonia (COP), hypersensitivity pneumonitis (HP) or sarcoidosis. Alveolar macrophage (AM) culture was performed in 44 patients with IPF, iNSIP, COP, HP, sarcoidosis or non-ILDs. The CCL18 levels in serum, BALF and AM culture supernatant were measured with ELISA. RESULTS: Both serum and BALF CCL18 levels in all ILDs were higher than in controls (all p < 0.005). In HP, CCL18 serum levels were the highest of all ILDs, and its BALF levels were significantly higher than in other ILDs except iNSIP. The BALF CCL18 levels markedly correlated with BAL cell differentials, especially with the percentage of BAL lymphocytes. In AM culture supernatant, the spontaneous CCL18 production was higher in HP and COP than in IPF and controls. CONCLUSION: CCL18 levels in serum, BALF and AM culture supernatant are markedly increased in various inflammatory and fibrotic ILDs. However, the CCL18 level being highest in HP among the investigated ILDs suggests that CCL18 may be more profoundly involved in inflammatory immune responses.

Angiogenic and angiostatic chemokines in idiopathic pulmonary fibrosis and granulomatous lung disease.

BACKGROUND: Angiogenesis-angiostasis balance and leukocyte recruitment are influenced by different concentrations of distinct chemokines. OBJECTIVE: To investigate the relative contribution of angiogenic and angiostatic CXC chemokines to the pathogenesis of idiopathic pulmonary fibrosis (IPF) and granulomatous lung diseases, we examined the in vitro production of an angiogenic chemokine (IL-8), and 2 angiostatic chemokines (IP-10 and MIG) by alveolar macrophages. METHODS: Alveolar macrophages from 16 patients with granulomatous lung diseases [8 with sarcoidosis, 8 with extrinsic allergic alveolitis (EAA)], 16 patients with IPF, and 8 control subjects were cultured for 24 h. IL-8, IL-18, IP-10 and MIG in the culture supernatants were measured by a fluorescent bead-based multiplex technique. RESULTS: In IPF patients, IL-8 was increased and correlated with bronchoalveolar lavage (BAL) neutrophils, whereas the levels of IP-10 and MIG were normal. In sarcoidosis and EAA patients, IL-8, IP-10, and MIG were all increased and IP-10 and MIG correlated with IL-18, a Th1 cytokine, and the percentage and number of BAL lymphocytes. CONCLUSIONS: The difference in the expression of CXC chemokines and a Th1 cytokine may contribute to the different immunopathogenesis, clinical course and responsiveness to treatment of these diseases.

Proteasomes in human bronchoalveolar lavage fluid after burn and inhalation injury.

The purpose of this study was to determine whether 26S proteasome is detectable in human bronchoalveolar lavage fluid (BALF) and whether burn and inhalation injury is accompanied by changes in BALF proteasome content or activity. BALF was obtained on hospital admission from 28 patients with burn and inhalation injury (controls: 10 healthy volunteers). Proteasome concentrations were quantified by enzyme-linked immunosorbent assay, and their native molecular mass was assessed by gel filtration. Proteasome peptidase activity was measured using a chymotryptic-like peptide substrate in combination with epoxomicin (specific proteasome inhibitor). BALF protein was increased in patients (P<.001) and correlated positively with the degree of inhalation injury. The 20S/26S proteasomes were detectable in all BALF by enzyme-linked immunosorbent assay. Gel filtration confirmed the presence of intact 20S and 26S proteasome that was stable without soluble ATP/Mg. In all BALF chymotryptic-like activity was detectable and could be inhibited with epoxomicin by 60 to 70% (P<.01). Absolute amounts of 20S/26S proteasomes and proteasome activity were increased in patients (P<.001 for all). The relative BALF composition after injury was characterized by increased concentrations of 20S proteasome/mg protein (P=.0034 vs volunteers), decreased concentrations of 26S proteasome/mg protein (P=.041 vs volunteers), and reduced specific proteasome activity (P=.044 vs volunteers). The 26S proteasome per milligram and specific proteasome activity were even further reduced in patients who developed ventilator-associated pneumonia (P=.045 and P=.03 vs patients without ventilator-associated pneumonia). This study supports the novel concept that extracellular proteasomes could play a pathophysiological role in the injured lung and suggests that insufficient proteasome function may increase susceptibility for pulmonary complications.

Extracellular, circulating proteasomes and ubiquitin - incidence and relevance.

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and is also deeply involved in the regulation of most basic cellular processes. Its proteolytic core, the 20S proteasome, has found to be attached also to the cell plasma membrane and certain observations are interpreted as to suggest that they may be released into the extracellular medium, e.g. in the alveolar lining fluid, epididymal fluid and possibly during the acrosome reaction. Proteasomes have also been detected in normal human blood plasma and designated circulating proteasomes; these have a comparatively low specific activity, a distinct pattern of subtypes and their exact origin is still enigmatic. In patients suffering from autoimmune diseases, malignant myeloproliferative syndromes, multiple myeloma, acute and chronic lymphatic leukaemia, solid tumour, sepsis or trauma, respectively, the concentration of circulating proteasomes has been found to be elevated, to correlate with the disease state and has even prognostic significance. Similarly, ubiquitin has been discovered as a normal component of human blood and seminal plasma and in ovarian follicular fluid. Increased concentrations were measured in diverse pathological situations, not only in blood plasma but also in cerebrospinal fluid, where it may have neuroprotective effects. As defective spermatozoa are covered with ubiquitin in the epididymal fluid, extracellular ubiquitination is proposed to be a mechanism for quality control in spermatogenesis. Growing evidence exists also for a participation of extracellular proteasomes and ubiquitin in the fertilization process.