Long-term experience using CNI-free immunosuppression in selected paediatric heart transplant recipients.

BACKGROUND: CNI-free immunosuppression with conversion to mTORi-based immunosuppression has been demonstrated to reduce CNI-toxicity and to exhibit anti-proliferative properties. However, the experience of CNI-free immunosuppression in paediatric heart transplantation is limited. METHODS: A retrospective analysis was conducted of 129 paediatric heart transplants performed between 1997 and 2015. Fifteen patients with clinically indicated conversion from CNI-based to CNI-free immunosuppression were identified. Survival data, rejection episodes, renal function, post-transplantation lymphoproliferative disorder and CAV, including examination with OCT were analysed. RESULTS: Immunosuppression conversion was successful in all patients. Fourteen of 15 patients (93%) are currently living with good graft function. Median post-transplant survival was 15 years (range, 5-23 years), and median follow-up since conversion was 6 years (range, 1-11 years). Mild (grade 1R) ACR was present in three patients after discontinuation of CNIs. The recovery of renal function with a significant increase in eGFR was observed at 1 and 3 years after conversion. No patient had angiographic signs of macroscopic CAV according to the current ISHLT classification; however, OCT showed the signs of angiographically silent CAV in all patients. CAV did not progress in any patient, implying CAV was stabilised by mTORi-based CNI-free immunosuppression. CONCLUSIONS: CNI-free immunosuppression based on mTORis is a safe and appropriate strategy for maintenance therapy in selected paediatric patients, significantly improves renal function and stabilises CAV. OCT revealed early development of angiographically silent CAV.

A Prospective Clinical Trial Measuring the Effects of Cardiopulmonary Bypass Under Mild Hypothermia on the Inflammatory Response and Regulation of Cold-Shock Protein RNA-Binding Motif 3.

Therapeutic hypothermia during cardiac surgery has been widely used for neuroprotection and to attenuate the systemic inflammatory response due to cardiopulmonary bypass (CPB). Experimental data suggest that cold-shock protein RNA-binding motif 3 (RBM3), which is induced in response to hypothermia, plays a key role in hypothermia-induced organ protection. To date, investigation on RBM3 has been performed exclusively in vitro or in animal models, and the detection and regulation of RBM3 in human blood has not been investigated until now. The aim of this study was to investigate the level of RBM3 protein and cytokine expression profile involved in the inflammatory response in patients with congenital heart disease undergoing cardiac surgery involving CPB and therapeutic hypothermia. A single-center prospective trial with 23 patients undergoing cardiac surgery with CPB was performed. RBM3 protein was quantified in blood serum samples collected from patients and healthy individuals employing a new developed enzyme-linked immunosorbent assay. Cytokine levels were analyzed from dry blood spot samples using a Quanterix Simoa Immunoassay. For the first time, RBM3 protein was detected in blood samples of patients with congenital heart disease undergoing cardiac surgery. Hereby, RBM3 protein concentrations were significantly elevated in patients after cardiac surgery with CPB and mild hypothermia as compared with pre-surgery levels. Moreover, a complex immune reaction with significant induction of pro-inflammatory cytokines (interleukin [IL]-1 beta, IL-6, IL-8, IL-16, IL-18, monocyte chemotactic protein 1, CC-chemokine ligand [CCL]3, CCL4, intercellular adhesion molecule-1) in response to CPB was detected. Significantly elevated vascular endothelial growth factor and matrix metallopeptidase 3 concentrations reflecting ischemia/reperfusion-induced injury were observed 24 hours after weaning from CPB. The use of CPB is still associated with a complex inflammatory response. RBM3 protein is measurable in blood samples of patients with significantly higher concentrations after cardiac surgery with CPB and mild-to-moderate hypothermia. RBM3 is a new candidate as a biomarker for therapeutic hypothermia and a possible new therapeutic target for organ protection.

RBM3 and CIRP expressions in targeted temperature management treated cardiac arrest patients-A prospective single center study.

BACKGROUND: Management of cardiac arrest patients includes active body temperature control and strict prevention of fever to avoid further neurological damage. Cold-shock proteins RNA-binding motif 3 (RBM3) and cold inducible RNA-binding protein (CIRP) expressions are induced in vitro in response to hypothermia and play a key role in hypothermia-induced neuroprotection. OBJECTIVE: To measure gene expressions of RBM3, CIRP, and inflammatory biomarkers in whole blood samples from targeted temperature management (TTM)-treated post-cardiac arrest patients for the potential application as clinical biomarkers for the efficacy of TTM treatment. METHODS: A prospective single center trial with the inclusion of 22 cardiac arrest patients who were treated with TTM (33°C for 24 hours) after ROSC was performed. RBM3, CIRP, interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), and inducible nitric oxide synthase (iNOS) mRNA expressions were quantified by RT-qPCR. Serum RBM3 protein concentration was quantified using an enzyme-linked immunosorbent assay (ELISA). RESULTS: RBM3 mRNA expression was significantly induced in post-cardiac arrest patients in response to TTM. RBM3 mRNA was increased 2.2-fold compared to before TTM. A similar expression kinetic of 1.4-fold increase was observed for CIRP mRNA, but did not reached significancy. Serum RBM3 protein was not increased in response to TTM. IL-6 and MCP-1 expression peaked after ROSC and then significantly decreased. iNOS expression was significantly increased 24h after return of spontaneous circulation (ROSC) and TTM. CONCLUSIONS: RBM3 is temperature regulated in patients treated with TTM after CA and ROSC. RBM3 is a possible biomarker candidate to ensure the efficacy of TTM treatment in post-cardiac arrest patients and its pharmacological induction could be a potential future intervention strategy that warrants further research.

Immunodepression after CPB: Cytokine dynamics and clinics after pediatric cardiac surgery - A prospective trial.

BACKGROUND: Corrective surgery for congenital heart defects is known to trigger a severe immune reaction. There has been extensive research on the effects of inflammation after cardiopulmonary bypass (CPB). Interestingly, monocytes are observed to be non-responsive to stimulation with lipopolysaccharide (LPS) under these conditions, indicating a state of immunodepression, which lays the ground for second hit infections after cardiosurgery with CPB. OBJECTIVES: The aim of this prospective study was to analyze immunodepression after pediatric cardiopulmonary bypass and to differentiate the effects of monocytic anergy on postoperative outcome. METHODS: In a prospective trial, we quantified the immune responses in 20 pediatric patients (median age 4.9months, range 2.3-38.2months; median weight 7.2kg, range 5.2-11.7kg) with congenital ventricular septal defect undergoing heart surgery with CPB. Ex vivo LPS-induced protein expression of IFN-γ, IL-1ß, IL-1Ra, IL-6, IL-8, IL-10, IL-12, IL-17, TNF-α, and MCP-1 was measured before (T1), immediately after (T2) and 4h after (T3) cardiopulmonary bypass surgery using Luminex technology. RESULTS: The innate immune system responds to CPB with an almost complete depression of monocytic function. Inflammatory IL-12, TNF-α, IL-1ß, IL-6, IL-8 and IFN-y are completely suppressed. IL-10, IL-1Ra and MCP-1 are still produced during suppression with IL-1Ra being overly secreted during reversion. Suppression of TNF-α expression after LPS-stimulation correlates closely with longer mechanical ventilation time (r=-0.619, p=0.004). CONCLUSION: Cardiosurgery with CPB causes a state of immunodepression making pediatric patients more vulnerable to second hit infections. MCP-1, IL-10, and IL-1Ra play an important role in monocyte recovery, eventually permitting new therapeutic options for controlling immunodepression and inflammation. Standardized glucocorticoid therapy should be evaluated carefully for each individual patient.

Results of aortic valve repair using decellularized bovine pericardium in congenital surgery.

OBJECTIVES: The search for an optimal patch material for aortic valve reconstruction (AVR) is an ongoing challenge. In this study, we report our experience of AVR using decellularized bovine pericardial patch material in congenital heart surgery. METHODS: Data of 40 consecutive patients who underwent AVR using the CardioCel® patch (Admedus Regen Pty Ltd, Perth, WA, Australia) between February 2014 and August 2016 were retrospectively reviewed. The median age of the patients at operation was 9 (2-34) years, and 18 patients were younger than 7 years. Twenty-six patients initially presented with aortic valve insufficiency (AI) and 14 with stenosis. Clinical and echocardiographic data were available until August 2017 for a median postoperative follow-up (FU) of 22 (6-42) months. RESULTS: Nine of 40 (23%) patients experienced an event during FU (death: n = 1, 2.5%; reoperation: n = 8, 20%). Overall, the probability of freedom from reoperation or death was 97 ± 3%, 76 ± 9% and 57 ± 12% at 12, 24 and 36 months of FU, respectively. Reason for reoperation was stenosis in 3 (37.5%) patients, insufficiency in 4 (50%) patients and 1 (12.5%) patient was diagnosed with aortic valve endocarditis. Of the remaining 31 patients, 2 patients are scheduled for reoperation (aortic valve stenosis: n = 1 and AI: n = 1) and 9 patients exhibit worsening of aortic valve function with moderate AI. Freedom from developing combined end point [death/reoperation/moderate degree of aortic valve dysfunction (aortic valve stenosis, AI)] after AVR was 92 ± 5%, 55 ± 9% and 28 ± 9% at 12, 24 and 36 months, respectively. CONCLUSIONS: AVR using decellularized bovine pericardial patch material in patients with congenital aortic valve disease show unsatisfactory results within the first 3 years of FU.

Neuroprotection via RNA-binding protein RBM3 expression is regulated by hypothermia but not by hypoxia in human SK-N-SH neurons.

OBJECTIVE: Therapeutic hypothermia is an established treatment for perinatal asphyxia. Yet, many term infants continue to die or suffer from neurodevelopmental disability. Several experimental studies have demonstrated a beneficial effect of mild-to-moderate hypothermia after hypoxic injury, but the understanding of hypothermia-induced neuroprotection remains incomplete. In general, global protein synthesis is attenuated by hypothermia, but a small group of RNA-binding proteins including the RNA-binding motif 3 (RBM3) is upregulated in response to cooling. The aim of this study was to establish an in vitro model to investigate the effects of hypoxia and hypothermia on neuronal cell survival, as well as to examine the kinetics of concurrent cold-shock protein RBM3 gene expression. METHODS: Experiments were performed by using human SK-N-SH neurons exposed to different oxygen concentrations (21%, 8%, or 0.2% O2) for 24 hours followed by moderate hypothermia (33.5°C) or normothermia for 24, 48, or 72 hours. Cell death was determined by quantification of lactate dehydrogenase and neuron-specific enolase releases into the cell cultured medium, and cell morphology was assessed by using immunofluorescence staining. The regulation of RBM3 gene expression was assessed by reverse transcriptase-quantitative polymerase chain reaction and Western blot analysis. RESULTS: Exposure to hypoxia (0.2% O2) for 24 hours resulted in significantly increased cell death in SK-N-SH neurons, whereas exposure to 8% O2 had no significant impact on cell viability. Post-hypoxia treatment with moderate hypothermia for 48 or 72 hours rescued the neurons from hypoxia-induced cell death. Moreover, exposure to severe hypoxia led to observable cell swelling, which was also attenuated by moderate hypothermia. Finally, moderate hypothermia but not hypoxia led to the induction of RBM3 expression on both transcriptional and translational levels. CONCLUSION: Moderate hypothermia protects neurons from hypoxia-induced cell death. The expression of the cold-shock protein RBM3 is induced by moderate hypothermia and could be one possible mediator of hypothermia-induced neuroprotection.

Moderate therapeutic hypothermia induces multimodal protective effects in oxygen-glucose deprivation/reperfusion injured cardiomyocytes.

OBJECTIVE: Therapeutic hypothermia has been shown to attenuate myocardial cell death due to ischemia/reperfusion injury. However, cellular mechanisms of cooling remain to be elucidated. Especially during reperfusion, mitochondrial dysfunction contributes to cell death by releasing apoptosis inductors. The aim of the present study was to investigate the effects of moderate therapeutic hypothermia (33.5°C) on mitochondrial mediated apoptosis in ischemia/reperfusion-injured cardiomyocytes. METHODS: Ischemic injury was simulated by oxygen-glucose deprivation for 6h in glucose/serum-free medium at 0.2% O2 in mouse atrial HL-1 cardiomyocytes. Simulation of reperfusion was achieved by restoration of nutrients in complete supplemented medium and incubation at 21% O2. Early application of therapeutic hypothermia, cooling during the oxygen-glucose deprivation phase, was initiated after 3h of oxygen-glucose deprivation and maintained for 24h. Mitochondrial membrane integrity was assessed by cytochrome c and AIF protein releases. Furthermore, mitochondria were stained with MitoTracker Red and intra-cellular cytochrome c localization was visualized by immunofluorescence staining. Moreover, anti-apoptotic Bcl-2 and Hsp70 as well as phagophore promoting LC3-II protein expressions were analyzed by Western-blot analysis. RESULTS: Therapeutic hypothermia initiated during oxygen-glucose deprivation significantly reduced mitochondrial release of cytochrome c and AIF in cardiomyocytes during reperfusion. Secondly, anti-apoptotic Bcl-2/Bax ratio and Hsp70 protein expressions were significantly upregulated due to hypothermia, indicating an inhibition of both caspase-dependent and -independent apoptosis. Furthermore, cardiomyocytes treated with therapeutic hypothermia showed increased LC3-II protein levels associated with the mitochondria during the first 3h of reperfusion, indicating the initiation of phagophores formation and sequestration of presumably damaged mitochondrion. CONCLUSION: Early application of therapeutic hypothermia effectively inhibited cardiomyocyte cell death due to oxygen-glucose deprivation/reperfusion-induced injury via multiple pathways. As hypothermia preserved mitochondrial membrane integrity, which resulted in reduced cytochrome c and AIF releases, induction of both caspase-dependent and -independent apoptosis was minimized. Secondly, cooling attenuated intrinsic apoptosis via Hsp70 upregulation and increasing anti-apoptotic Bcl-2/Bax ratio. Moreover, therapeutic hypothermia promoted mitochondrial associated LC3-II during the early phase of reperfusion, possibly leading to the sequestration and degradation of damaged mitochondrion to attenuate the activation of cell death.

Hypothermia During Cardiopulmonary Bypass Increases Need for Inotropic Support but Does Not Impact Inflammation in Children Undergoing Surgical Ventricular Septal Defect Closure.

Minimizing the systemic inflammatory response caused by cardiopulmonary bypass is a major concern. It has been suggested that the perfusion temperature affects the inflammatory response. The aim of this prospective study was to compare the effects of moderate hypothermia (32°C) and normothermia (36°C) during cardiopulmonary bypass on markers of the inflammatory response and clinical outcomes (time on ventilator) after surgical closure of ventricular septal defects. During surgical closure of ventricular septal defects under cardiopulmonary bypass, 20 children (median age 4.9 months, range 2.3-38 months; median weight 7.2 kg, range 5.2-11.7 kg) were randomized to a perfusion temperature of either 32°C (Group 1, n = 10) or 36°C (Group 2, n = 10). The clinical data and blood samples were collected before cardiopulmonary bypass, directly after aortic cross-clamp release, and 4 and 24 h after weaning from cardiopulmonary bypass. Time on ventilation as primary outcome did not differ between the two groups. Other clinical outcome parameters like fluid balance or length of stay in the intensive care were also similar in the two groups. Compared with Group 2, Group 1 needed significantly higher and longer inotropic support (P < 0.001). In Group 1, two infants had junctional ectopic tachycardia, and another had a pulmonary hypertensive crisis. Perfusion temperature did not influence cytokine release, organ injury, or coagulation. Cardiopulmonary bypass temperature does not influence time on ventilation or inflammatory marker release. However, in the present study, with a small patient cohort, patients operated under hypothermic bypass needed higher and longer inotropic support. The use of hypothermic cardiopulmonary bypass in infants and children should be approached with care.

Moderate hypothermia initiated during oxygen-glucose deprivation preserves HL-1 cardiomyocytes.

OBJECTIVES: Therapeutic hypothermia (TH) is an acknowledged strategy for neuroprotection for patients suffering from hypoxic-anoxic brain injury (HAI). Albeit similar pathomechanisms of HAI for both brain and heart, moderate TH (32-34°C) has not been established as a heart-protective measure. Therefore, we investigated the cardioprotective effects of moderate TH on oxygen-glucose deprivation/re-oxygenation (OGD/R)-induced injury in HL-1 cardiomyocytes. METHODS: Cardiac OGD/R injury was induced by exposing HL-1 cardiomyocytes to 0.2% oxygen in serum/glucose-free medium for 6h. OGD injured cells were subsequently re-oxygenated with 21% oxygen in complete medium. Two hypothermic protocols were investigated: Post-OGD cooling to 33.5°C for 24 h initiated at the start of re-oxygenation and intra-OGD cooling to 33.5°C for 24 h initiated after 3 h of OGD and maintained throughout the re-oxygenation phase. Cell viability was determined by LDH and cTnT releases. Mitochondria dysfunction was evaluated by intracellular ATP content and cellular metabolic activity was accessed by MTT reduction. Activation of caspase 3 was analyzed by Western blot. RESULTS: OGD/R-induced injury resulted in increased cell death (higher LDH and cTnT releases), mitochondrial impairment (decreased ATP content), and decreased cellular metabolic activity (decreased MTT reduction). Only intra-OGD cooling attenuated both OGD and OGD-R-induced injuries (significantly decreased LDH and cTnT releases and increased ATP contents and MTT reduction). Furthermore, caspase 3 activation was abated by intra-OGD cooling. No protective effects were observed by post-OGD cooling. CONCLUSIONS: Moderate TH initiated during OGD is a promising intervention for the protection of cardiomyocytes from OGD/R-induced injury. The attenuation of mitochondrial dysfunction and apoptosis by intra-OGD cooling are beneficial effects of hypothermia-induced cardioprotection, resulting in minimized myocardial cell death after OGD and OGD-R-induced injuries.

Mechanisms of hypothermia-induced cell protection in the brain.

Therapeutic hypothermia is an effective cytoprotectant and promising intervention shown to improve outcome in patients following cardiac arrest and neonatal hypoxia-ischemia. However, despite our clinical and experimental experiences, the protective molecular mechanisms of therapeutic hypothermia remain to be elucidated. Therefore, in this brief overview we discuss both the clinical evidence and molecular mechanisms of therapeutic hypothermia in order to provide further insights into this promising intervention.

Effects of moderate and deep hypothermia on RNA-binding proteins RBM3 and CIRP expressions in murine hippocampal brain slices.

Therapeutic hypothermia has emerged as an effective neuroprotective therapy for cardiac arrest survivors. There are a number of purported mechanisms for therapeutic hypothermia, but the exact mechanism still remains to be elucidated. Although hypothermia generally down-regulates protein synthesis and metabolism in mammalian cells, a small subset of homologous (>70%) cold-shock proteins (RNA-binding motif protein 3, RBM3 and cold-inducible RNA-binding protein, CIRP) are induced under these conditions. In addition, RBM3 up-regulation in neuronal cells has recently been implicated in hypothermia-induced neuroprotection. Therefore, we compared the effects of moderate (33.5°C) and deep (17°C) hypothermia with normothermia (37°C) on the regulation of RBM3 and CIRP expressions in murine organotypic hippocampal slice cultures (OHSC), hippocampal neuronal cells (HT-22), and microglia cells (BV-2). Moderate hypothermia resulted in significant up-regulation of both RBM3 and CIRP mRNA in murine OHSC, but deep hyporthermia did not. RBM3 protein regulation was also significantly up-regulated by 33.5°C, but no significant up-regulation of CIRP protein was observed in the OHSC. Additionally, OHSC exposed to 17°C for 24h were positive for Propidium Iodide (PI) immunostaining, indicating the onset of cell death. Similarly, RBM3 gene expression in a HT-22 neuronal cells mono-culture and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were also up-regulated at 33.5°C but only in the co-culture at 17°C. No significant up-regulation of RBM3 nor CIRP gene expression were observed in a BV-2 mono-culture at either temperature. We observed that RBM3 mRNA and protein expressions in murine OHSC, as well as in mono-culture of HT-22 neuronal cells and direct co-culture of HT-22 neuronal cells with BV-2 microglia cells were significantly up-regulated by exposure to moderate hypothermia. These findings further support the implication of RBM3 as a potential effector for hypothermia-induced neuroprotection.

Hypothermia protects H9c2 cardiomyocytes from H2O2 induced apoptosis.

The purpose of our study was to investigate underlying basic mechanisms of hypothermia-induced cardioprotection during oxidative stress in a cardiomyocyte cell culture model. For hypothermic treatment we cooled H9c2 cardiomyocytes to 20°C, maintained 20min at 20°C during which short-term oxidative damage was inflicted with 2mM H(2)O(2,) followed by rewarming to 37°C. Later on, we analyzed lactate dehydrogenase (LDH), caspase-3 cleavage, reactive oxygen species (ROS), mitochondrial activity, intracellular ATP production, cytoprotective signal molecules as well as DNA damage. Hypothermia decreased H(2)O(2) damage in cardiomyocytes as demonstrated in a lower LDH release, less caspase-3 cleavage and less M30 CytoDeath staining. After rewarming H(2)O(2) damaged cells demonstrated a significantly higher reduction rate of intracellular ROS compared to normothermic H(2)O(2) damaged cardiomyocytes(.) This was in line with a significantly greater mitochondrial dehydrogenase activity and higher intracellular ATP content in cooled and rewarmed cells. Moreover, hypothermia preserved cell viability by up-regulation of the anti-apoptotic protein Bcl-2 and a reduction of p53 phosphorylation. DNA damage, proven by PARP-1 cleavage and H2AX phosphorylation, was significantly reduced by hypothermia. In conclusion, we could demonstrate that hypothermia protects cardiomyocytes during oxidative stress by preventing apoptosis via inhibiting mitochondrial dysfunction and DNA damage.

Methylprednisolone and tacrolimus prevent hypothermia-induced endothelial dysfunction.

BACKGROUND: Hypothermia is used to preserve organs for transplantation and is the oldest method to protect organs during complex pediatric cardiac surgery. Loss of tissue function and tissue edema are common complications in children undergoing corrective cardiac surgery and heart transplantation. The present study was designed to examine the effects of methylprednisolone and tacrolimus on endothelial cell function and morphology after deep hypothermia and rewarming. METHODS: Human umbilical vein endothelial cells were pre-treated with methylprednisolone or tacrolimus, or both, incubated within a specially designed bioreactor or in monolayers, and then exposed to a dynamic cooling and rewarming protocol. Immunocytochemistry, time-lapse video microscopy, cell permeability and adherence assays, and Western blot analysis were performed. RESULTS: Confluent endothelial cells exposed to hypothermia displayed elongated cell shapes with intercellular gap formation, increased endothelial cell-layer permeability, and loss in adherence. Upon rewarming, however, endothelial cell integrity was restored. Opening and closing of intercellular gaps was dependent on extracellular signal-regulated kinase 1 and 2 (ERK 1/2) activation and connexin 43 expression. The combined treatment with methylprednisolone and tacrolimus inhibited these hypothermia-induced changes. CONCLUSIONS: These results suggest that methylprednisolone and tacrolimus inhibit hypothermia-induced endothelial gap formation by phosphorylated ERK 1/2 inhibition and connexin 43 stabilization. Application of combined drugs that affect multiple targets may therefore be considered as a possible new therapeutic strategy to prevent endothelial dysfunction after hypothermia and rewarming.

Hypothermia suppresses inflammation via ERK signaling pathway in stimulated microglial cells.

Hypothermic perfusion is a standard method for neuroprotection during cardiac surgery in children. However, the cellular responses underlying these mechanisms have not been clearly elucidated. In the present study we demonstrated that the inflammatory response of stimulated microglial cells is significantly reduced after moderate hypothermia. Continuous hypothermia caused a diminished NO release. Moderate hypothermia and rewarming caused a downregulation of phosphorylated MEK, ERK and iNOS-expression, diminished cytokine release and reduced CD-11a and ICAM-1 expression. Thus, neuroprotection offered by hypothermia could be attributed to reduced cytotoxic products released from stimulated microglial cells mediated by the MEK/ERK signal transduction pathway.