The Genetically Engineered Heart as a Bridge to Allotransplantation in Infants Just Around the Corner?

BACKGROUND: Mortality for infants on the heart transplant waitlist remains unacceptably high, and available mechanical circulatory support is suboptimal. Our goal is to demonstrate the feasibility of utilizing genetically engineered pig (GEP) heart as a bridge to allotransplantation by transplantation of a GEP heart in a baboon. METHODS: Four baboons underwent orthotopic cardiac transplantation from GEP donors. All donor pigs had galactosyl-1,3-galactose knocked out. Two donor pigs had human complement regulatory CD55 transgene and the other 2 had human complement regulatory CD46 and thrombomodulin. Induction immunosuppression included thymoglobulin, and anti-CD20. Maintenance immunosuppression was rapamycin, anti-CD-40, and methylprednisolone. One donor heart was preserved with University of Wisconsin solution and the other three with del Nido solution. RESULTS: All baboons weaned from cardiopulmonary bypass. B217 received a donor heart preserved with University of Wisconsin solution. Ventricular arrhythmias and depressed cardiac function resulted in early death. All recipients of del Nido preserved hearts easily weaned from cardiopulmonary bypass with minimal inotropic support. B15416 and B1917 survived for 90 days and 241 days, respectively. Histopathology in B15416 revealed no significant myocardial rejection but cellular infiltrate around Purkinje fibers. Histopathology in B1917 was consistent with severe rejection. B37367 had uneventful transplant but developed significant respiratory distress with cardiac arrest. CONCLUSIONS: Survival of B15416 and B1917 demonstrates the feasibility of pursuing additional research to document the ability to bridge an infant to cardiac allotransplant with a GEP heart.

The SRG rat, a Sprague-Dawley Rag2/Il2rg double-knockout validated for human tumor oncology studies.

We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks.

Sprague Dawley Rag2-Null Rats Created from Engineered Spermatogonial Stem Cells Are Immunodeficient and Permissive to Human Xenografts.

The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a preclinical research model including larger sample size collection, lower rates of drug clearance, and relative ease of surgical manipulation. An immunodeficient rat would allow for larger tumor size development, prolonged dosing and drug efficacy studies, and preliminary toxicologic testing and pharmacokinetic/pharmacodynamic studies in the same model animal. Here, we created an immunodeficient rat with a functional deletion of the Recombination Activating Gene 2 (Rag2) gene, using genetically modified spermatogonial stem cells (SSC). We targeted the Rag2 gene in rat SSCs with TALENs and transplanted these Rag2-deficient SSCs into sterile recipients. Offspring were genotyped, and a founder with a 27 bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2 tm1Hera (SDR) knockout rat. We demonstrated that SDR rat lacks mature B and T cells. Furthermore, the SDR rat model was permissive to growth of human glioblastoma cell line subcutaneously resulting in successful growth of tumors. In addition, a human KRAS-mutant non-small cell lung cancer cell line (H358), a patient-derived high-grade serous ovarian cancer cell line (OV81), and a patient-derived recurrent endometrial cancer cell line (OV185) were transplanted subcutaneously to test the ability of the SDR rat to accommodate human xenografts from multiple tissue types. All human cancer cell lines showed efficient tumor uptake and growth kinetics indicating that the SDR rat is a viable host for a range of xenograft studies. Mol Cancer Ther; 17(11); 2481-9. ©2018 AACR.

Furosemide Response Predicts Acute Kidney Injury After Cardiac Surgery in Infants and Neonates.

OBJECTIVE: Cardiac surgery-induced acute kidney injury occurs frequently in neonates and infants and is associated with postoperative morbidity/mortality; early identification of cardiac surgery-induced acute kidney injury may be crucial to mitigate postoperative morbidity. We sought to determine if hourly or 6-hour cumulative urine output after furosemide in the first 24 hours after cardiopulmonary bypass could predict development of cardiac surgery-induced acute kidney injury and other deleterious outcomes. DESIGN: Retrospective chart review. SETTING: Pediatric cardiac ICU. PATIENTS: All infants younger than 90 days old admitted to the cardiac ICU from October 2012 to December 2015 who received at least one dose of furosemide in the first 24 hours after cardiopulmonary bypass surgery. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Ninety-nine patients met inclusion and exclusion criteria. In total, 45.5% developed cardiac surgery-induced acute kidney injury. Median time between cardiopulmonary bypass and furosemide was 7.7 hours (interquartile range, 4.4-9.5). Six-hour cumulative urine output was 33% lower (p = 0.031) in patients with cardiac surgery-induced acute kidney injury. Area under the curve for prediction of cardiac surgery-induced acute kidney injury was 0.69 (p = 0.002). Other models demonstrated urine output response to furosemide had significant area under the curves for prediction of peak fluid over load greater than 15% (0.68; p = 0.047), prolonged peritoneal dialysis (area under the curve, 0.79; p = 0.007), prolonged mechanical ventilation (area under the curve, 0.79; p < 0.001), prolonged hospitalization (area under the curve, 0.62; p = 0.069) and mortality (area under the curve, 0.72; p = 0.05). CONCLUSIONS: Urine output response to furosemide within 24 hours of cardiopulmonary bypass predicts cardiac surgery-induced acute kidney injury development and other important morbidity in children younger than 90 days old; prospective validation is warranted.

Adrenal insufficiency in neonates after cardiac surgery with cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass (CPB) may lead to adrenal insufficiency (AI). Emerging evidence supports association of AI with morbidity after cardiac surgery. AIMS: The aim of this study was to define AI incidence in neonates undergoing complex cardiac surgery with CPB and its association with intraoperative post-CPB outcomes. METHODS: Forty subjects enrolled in a prior randomized control trial who received preoperative methylprednisolone as part of our institutional neonatal bypass protocol were included. No intraoperative steroids were given. ACTH stimulation tests were performed: preoperatively and 1 h after separation from CPB. AI was defined as <9 µg·ml-1 increase in cortisol at 30 min post cosyntropin 1 mcg. Clinical outcomes were collected up to 90 min after CPB. RESULTS: 2/40 (5%) subjects had preoperative AI vs 13/40 (32.5%) post-CPB AI, P ≤ 0.001. No significant difference was observed in age, gestational age, weight, CPB time, circulatory arrest, or STAT category between subjects with or without post-CPB AI. ACTH decreased from preoperative values 127.3 vs 35 pcg·ml-1 [median difference = 81.8, 95% CI = 22.7-127.3], while cortisol increased from 18.9 vs 75 µg·dl-1 [median difference = 52.2, 95% CI = 36.3-70.9]. Post-CPB AI was associated with increased median colloid resuscitation, 275 vs 119 ml·kg-1 [median difference = 97.8, 95% CI = 7.1-202.2]; higher median peak lactate, 9.4 vs 6.9 mg·dl-1 [median difference = 3.2, 95% CI = 0.04-6.7]; median post-CPB lactate, 7.9 vs 4.3 mg·dl-1 , [median difference 3.6, 95% CI = 2.1-4.7], and median lactate on admission to CICU, 9.4 vs 6.0 mg·dl-1 [median difference = 3, 95% CI = 1.1-4.9]. No difference was observed in blood pressure or vasoactive inotrope score at any time point measured in operating room (OR). Higher initial post-CPB cortisol correlated with decreased cosyntropin response. CONCLUSIONS: Neonatal cardiac surgery with CPB and preoperative methylprednisolone leads to AI as determined by low-dose ACTH stimulation test in one-third of patients. AI is associated with increased serum lactate and colloid resuscitation in OR. Impact of preoperative methylprednisolone on results is not defined. Benefit of postoperative steroid administration in neonates with post-CPB AI warrants further investigation.

Implementation of a Multidisciplinary Bleeding and Transfusion Protocol Significantly Decreases Perioperative Blood Product Utilization and Improves Some Bleeding Outcomes.

Perioperative transfusion of blood products is associated with increased morbidity and mortality after pediatric cardiac surgery. We report the results of a quality improvement project aimed at decreasing perioperative blood product administration and bleeding after pediatric cardiopulmonary bypass (CPB) surgery. A multidisciplinary team evaluated baseline data from 99 consecutive CPB patients, focusing on the variability in transfusion management and bleeding outcomes, to create a standardized bleeding and transfusion management protocol. A total of 62 subsequent patients were evaluated after implementation of the protocol: 17 with single pass hemoconcentrated (SPHC) blood transfusion and 45 with modified ultrafiltration (MUF). Implementation of the protocol with SPHC blood led to significant decrease in transfusion of every blood product in the cardiovascular operating room and first 6 hours in cardiovascular intensive care unit ([CVICU] p < .05). Addition of MUF to the protocol led to further decrease in transfusion of all blood products compared to preprotocol. Patients <2 months old had 49% decrease in total blood product administration: 155 mL/kg preprotocol, 117 mL/kg protocol plus SPHC, and 79 mL/kg protocol plus MUF (p < .01). There were significant decreases in postoperative bleeding in the first hour after CVICU admission: 6 mL/kg preprotocol, 3.8 mL/kg protocol plus SPHC, and 2 mL/kg protocol plusMUF (p = .02). There was also significantly decreased incidence of severe postoperative bleeding (>10 mL/kg) in the first CVICU hour for protocol plus MUF patients (p < .01). Implementation of a multidisciplinary bleeding and transfusion protocol significantly decreases perioperative blood product transfusion and improves some bleeding outcomes.

Decreasing the Preincision Time for Pulmonary Lobectomy: The Process of Lean and Value Stream Mapping.

BACKGROUND: Our objective was to evaluate our results after the implementation of lean (the elimination of wasteful parts of a process). METHODS: After meetings with our anesthesiologists, we standardized our "in the operating room-to-skin incision protocols" before pulmonary lobectomy. Patients were divided into consecutive cohorts of 300 lobectomy patients. Several protocols were slowly adopted and outcomes were evaluated. RESULTS: One surgeon performed 2,206 pulmonary lobectomies, of which 84% were for cancer. Protocols for lateral decubitus positioning changed over time. We eliminated axillary rolls, arm boards, and beanbags. Monitoring devices were slowly eliminated. Central catheters decreased from 75% to 0% of patients, epidurals from 84% to 3%, arterial catheters from 93% to 4%, and finally, Foley catheters were reduced from 99% to 11% (p ≤ 0.001 for all). A protocol for the insertion of double-lumen endotracheal tubes was established and times decreased (mean, 14 minutes to 1 minute; p = 0.001). After all changes were made, the time between operating room entry and incision decreased from a mean of 64 minutes to 37 minutes (p < 0.001). Outcomes improved, mortality decreased from 3.2% to 0.26% (p = 0.015), and major morbidity decreased from 15.2% to 5.3% (p = 0.042). CONCLUSIONS: Lean and value stream mapping can be safely applied to the clinical algorithms of high-risk patient care. We demonstrate that elimination of non-value-added steps can safely decrease preincision time without increasing patient risk in patients who undergo pulmonary lobectomy. Selected centers may be able to adopt some of these lean-driven protocols.

Surgical and Anesthetic Management of a Mediastinal Fatty Tumor: Lipoblastoma.

Lipoblastoma is a rare fatty tumor that is diagnosed almost exclusively in children. Presentation often consists of respiratory symptoms; chest computed tomography shows a hypodense, low, attenuated mediastinal mass. Surgical approach and anesthetic management are dependent on the location of the tumor and the degree of airway compression; in most cases, a thoracotomy is performed, although a sternotomy is used in selected cases. Final diagnosis can be confirmed using molecular genetic analysis; a genetic hallmark of lipoblastoma is the rearrangement of chromosomal region 8q12 and the PLAG1 gene. Tumor recurrence is rare when a complete resection is performed.

Risk Factors Associated with Reoperation for Bleeding following Liver Transplantation.

Introduction. This study's objective was to identify risk factors associated with reoperation for bleeding following liver transplantation (LTx). Methods. A retrospective study was performed at a single institution between 2001 and 2012. Operative reports were used to identify patients who underwent reoperation for bleeding within 2 weeks following LTx (operations for nonbleeding etiologies were excluded). Results. Reoperation for bleeding was observed in 101/928 (10.8%) of LTx patients. The following characteristics were associated with reoperation on multivariable analysis: recipient MELD score (OR 1.06/MELD unit, 95% CI 1.03, 1.09), number of platelets transfused (OR 0.73/platelet unit, 95% CI 0.58, 0.91), and aminocaproic acid utilization (OR 0.46, 95% CI 0.27, 0.80). LTx patients who underwent reoperation for bleeding had a longer ICU stay (5 days ± 7 versus 2 days ± 3, P < 0.001) and hospitalization (18 days ± 9 versus 10 days ± 18, P < 0.001). The risk of death increased in patients who underwent reoperation for bleeding (HR 1.89, 95% CI 1.26, 2.85). Conclusion. Reoperation for bleeding following LTx was associated with increased resource utilization and recipient mortality. A lower threshold for intraoperative platelet transfusion and antifibrinolytics, especially in patients with high lab-MELD score, may decrease the incidence of reoperation for bleeding following LTx.

Redox therapeutics in hepatic ischemia reperfusion injury.

Ischemia-reperfusion plays a major role in the injury experienced by the liver during transplantation. Much work has been done recently investigating the role of redox species in hepatic ischemia-reperfusion. As animal models are better characterized and developed, and more insights are gained into the pathophysiology of hepatic ischemia reperfusion injury in humans the questions into exactly how oxidants participate in this injury are becoming more refined. These questions include effects of cellular location, timing of injury, and ability of therapeutics to access this site are increasing our appreciation of the complexity of ischemia reperfusion and improving attempts to ameliorate its effects. In this review, we aim to discuss the various methods to alter redox chemistry during ischemia reperfusion injury and future prospects for preventing organ injury during hepatic ischemia reperfusion.

Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation.

Local vasodilation in response to hypoxia is a fundamental physiologic response ensuring oxygen delivery to tissues under metabolic stress. Recent studies identify a role for the red blood cell (RBC), with hemoglobin the hypoxic sensor. Herein, we investigate the mechanisms regulating this process and explore the relative roles of adenosine triphosphate, S-nitrosohemoglobin, and nitrite as effectors. We provide evidence that hypoxic RBCs mediate vasodilation by reducing nitrite to nitric oxide (NO) and ATP release. NO dependence for nitrite-mediated vasodilation was evidenced by NO gas formation, stimulation of cGMP production, and inhibition of mitochondrial respiration in a process sensitive to the NO scavenger C-PTIO. The nitrite reductase activity of hemoglobin is modulated by heme deoxygenation and heme redox potential, with maximal activity observed at 50% hemoglobin oxygenation (P(50)). Concomitantly, vasodilation is initiated at the P(50), suggesting that oxygen sensing by hemoglobin is mechanistically linked to nitrite reduction and stimulation of vasodilation. Mutation of the conserved beta93cys residue decreases the heme redox potential (ie, decreases E(1/2)), an effect that increases nitrite reductase activity and vasodilation at any given hemoglobin saturation. These data support a function for RBC hemoglobin as an allosterically and redox-regulated nitrite reductase whose "enzyme activity" couples hypoxia to increased NO-dependent blood flow.

The red blood cell and vascular function in health and disease.

Nitric oxide (NO) is widely accepted as a central regulator of vascular tone and a vast array of other cardiovascular signaling mechanisms. An emerging player in these mechanisms is hemoglobin (Hb), an erythrocytic protein that serves as the archetypical model for an allosteric protein. Specifically, red blood cells (RBC) are suggested to be integral in matching blood flow to tissue oxygen demands. The mechanisms proposed involve the ability of Hb to sense changes in oxygen concentrations and coupling this process to modulating vascular NO levels. The molecular basis of these mechanisms remains under investigation, but is clearly diverse and discussed in this article from the basis of the blood flow responses to hypoxia. Another emerging theme in RBC biology is the role of these cells during inflammatory disease in which disease processes promote the interaction of vascular NO and the RBC. This is exemplified in hemolytic diseases, in which released Hb has drastic affects on vascular homeostasis mechanisms. Additionally, it is becoming evident that RBC express numerous molecules that mediate interactions with the extracellular matrix and cellular mediators of inflammation. The functional implications for such interactions remain unclear but highlight potential roles of the RBC in modulating inflammatory disease.

Down-regulation of hepatic CYP1A2 plays an important role in inflammatory responses in sepsis.

OBJECTIVE: Although hepatic cytochrome P-450 protein concentrations are altered following endotoxin shock, changes in P-450 isoforms in sepsis have not been fully investigated. The aim of this study was to determine whether the major P-450 isoform in rat liver (i.e., CYP1A2) is down-regulated during the progression of sepsis and, if so, whether reduction of P-450 enzyme system plays an important role in the inflammatory response. DESIGN: Prospective, controlled, and randomized animal study. SETTING: A university/institute research laboratory. SUBJECTS: Male adult Sprague-Dawley rats were subjected either to polymicrobial sepsis by cecal ligation and puncture (CLP) or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). INTERVENTIONS: P-450 isoforms in the liver (i.e., CYP1A2 and 4A1) were determined using reverse transcription polymerase chain reaction and Western blot analysis at various time points after CLP. MEASUREMENTS AND MAIN RESULTS: The results indicate that CYP1A2 messenger RNA expression decreased significantly at 10 and 20 hrs whereas its protein concentrations decreased at 20 hrs after the induction of sepsis. In contrast, CYP4A1 messenger RNA and protein concentrations were not altered even at 20 hrs after CLP. In an additional experiment, all P-450 isoforms were inhibited by pretreatment with 1-aminobenzotriazole to determine the effect of cytochrome P-450 blockade on inflammatory responses by assessing proinflammatory cytokines. The results show further increases in serum concentrations of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 in aminobenzotriazole-treated animals at 10 hrs after CLP, which was associated with elevated concentrations of circulating lactate and severe morphologic alterations in the liver. These results suggest that the integrity of the cytochrome P-450 enzyme system plays an important role in septic inflammatory response. CONCLUSION: The major hepatic P-450 isoform CYP1A2 is down-regulated and inhibition of P-450 enzyme system is associated with an exacerbated inflammatory response in sepsis. Treatment with pharmaceutical agents that regulate or are metabolized by P-450 enzymes might be approached cautiously in the septic patient if this holds true in a clinical setting.

Mechanisms of the interaction of nitroxyl with mitochondria.

It is now thought that NO* (nitric oxide) and its redox congeners may play a role in the physiological regulation of mitochondrial function. The inhibition of cytochrome c oxidase by NO* is characterized as being reversible and oxygen dependent. In contrast, peroxynitrite, the product of the reaction of NO* with superoxide, irreversibly inhibits several of the respiratory complexes. However, little is known about the effects of HNO (nitroxyl) on mitochondrial function. This is especially important, since HNO has been shown to be more cytotoxic than NO*, may potentially be generated in vivo, and elicits biological responses with some of the characteristics of NO and peroxynitrite. In the present study, we present evidence that isolated mitochondria, in the absence or presence of substrate, convert HNO into NO* by a process that is dependent on mitochondrial concentration as well as the concentration of the HNO donor Angeli's salt. In addition, HNO is able to inhibit mitochondrial respiration through the inhibition of complexes I and II, most probably via modification of specific cysteine residues in the proteins. Using a proteomics approach, extensive modification of mitochondrial protein thiols was demonstrated. From these data it is evident that HNO interacts with mitochondria through mechanisms distinct from those of either NO* or peroxynitrite, including the generation of NO*, the modification of thiols and the inhibition of complexes I and II.

Redox signalling: from nitric oxide to oxidized lipids.

Cellular redox signalling is mediated by the post-translational modification of proteins in signal-transduction pathways by ROS/RNS (reactive oxygen species/reactive nitrogen species) or the products derived from their reactions. NO is perhaps the best understood in this regard with two important modifications of proteins known to induce conformational changes leading to modulation of function. The first is the addition of NO to haem groups as shown for soluble guanylate cyclase and the newly discovered NO/cytochrome c oxidase signalling pathway in mitochondria. The second mechanism is through the modification of thiols by NO to form an S-nitrosated species. Other ROS/RNS can also modify signalling proteins although the mechanisms are not as clearly defined. For example, electrophilic lipids, formed as the reaction products of oxidation reactions, orchestrate adaptive responses in the vasculature by reacting with nucleophilic cysteine residues. In modifying signalling proteins ROS/RNS appear to change the overall activity of signalling pathways in a process that we have termed 'redox tone'. In this review, we discuss these different mechanisms of redox cell signalling, and give specific examples of ROS/RNS participation in signal transduction.

Vasoactivity of S-nitrosohemoglobin: role of oxygen, heme, and NO oxidation states.

The mechanisms by which S-nitrosohemoglobin (SNOHb) stimulates vasodilation are unclear and underlie the controversies surrounding the proposal that this S-nitrosothiol modulates blood flow in vivo. Among the mechanistic complexities are the nature of vasoactive species released from SNOHb and the role heme and oxygen play in this process. This is important to address since hemoglobin inhibits NO-dependent vasodilation. We compared the vasodilatory properties of distinct oxidation and ligation states of SNOHb at different oxygen tensions. The results show that SNOHb in the oxygenated state (SNOoxyHb) is significantly less efficient than SNOHb in the ferric or met oxidation state (SNOmetHb) at stimulating relaxation of isolated rat aortic rings. Using pharmacologic approaches to modulate nitrogen monoxide radical (.NO)-dependent relaxation, our data suggest that SNOoxyHb promotes vasodilation in a.NO-independent manner. In contrast, both SNOmetHb and S-nitrosoglutathione (GSNO), a putative intermediate in SNOHb reactivity, elicit vasodilation in a.NO-dependent process. Consistent with previous observations, an increase in sensitivity of SNOHb vasodilation at low oxygen tensions also was observed. However, this was not exclusive for this protein but applied to a range of nitrosovasodilators (including a.NO donor [DeaNonoate], an S-nitrosothiol [GSNO], and the nitroxyl anion donor, Angelis salt). This suggests that oxygen-dependent modulation of SNOHb vasoactivity does not occur by controlling the allosteric state of Hb but is a property of vessel responsiveness to nitrosovasodilators at low oxygen tensions.