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Crigler-Najjar syndrome is an ultra-rare monogenic recessive liver disease caused by UGT1A1 gene mutations. Complete UGT1A1 deficiency results in severe unconjugated hyperbilirubinemia in newborns that, if not treated, may lead to brain damage and death. Treatment is based on intensive phototherapy, but its efficacy decreases with age, rendering liver transplantation the only curative option. Adeno-associated virus (AAV)-mediated gene therapy has shown long-term correction in adult patients, but loss of viral DNA and therapeutic efficacy are expected in younger patients associated with liver growth. Effective vector re-administration is hindered by anti-AAV neutralizing antibodies generated during the first administration. Here, we investigated AAV vector re-administration by modulating the immune response with rapamycin-loaded nanoparticles (ImmTOR) in Gunn rats ( Ugt1a -/- ) and Ugt1a -/- mice. We administered a liver-specific AAV8 vector expressing a codon-optimized h UGT1A 1 cDNA (1.0E11 vg/kg) in P25-P28 mutant animals and, upon loss of efficacy after 3 to 5 weeks, a higher second dose (1.0E12 or 5.0E12 vg/kg) was given. ImmTOR co-administration reduced anti-AAV neutralizing antibodies and immunoglobulin Gs generation in male animals of both models allowing effective re-dosing, underscored by a significant and long-term decrease in plasma bilirubin, although efficacy was affected by low-titer residual anti-AAV antibodies suggesting that re-administration in patients may require combination with other methods., Competing Interests: T.K.K. is an employee and shareholder of Selecta Bioscience; F.M., G.R., F.C., G.B., and A.F.M. are inventors in patents describing the AAV technology and gene therapy-based treatments for Crigler-Najjar syndrome., (© 2024 The Authors.)

Biliverdin is a secondary metabolite of heme catabolism. It is formed by the reaction catalyzed by heme oxygenase, which converts the heme group contained in proteins such as hemoglobin, myoglobin, cytochromes, and catalase into biliverdin, iron (II) and CO in equimolar amounts, consuming NADPH. Biliverdin is then reduced to bilirubin by biliverdin reductase. Biliverdin and bilirubin form a redox couple and are important for the redox homeostasis of cells. Heme oxygenase-1 is an inducible enzyme that is induced by hypoxic conditions, increased availability of heme or proinflammatory mechanisms such as LPS, UV radiation, etc. In addition, both heme oxygenase-1 and biliverdin reductase play roles other than catalysis by modulating specific metabolic pathways at the transcriptional level. There is a need for affordable assays to analyze these bile pigments in biological and clinical samples. Here we present a method for the combined determination of biliverdin and bilirubin that utilizes the specific binding of bilirubin to the fluorescent recombinant fusion protein HUG and the enzymatic conversion of biliverdin to bilirubin.•This method enables the combined measurement of bilirubin and biliverdin in the nM range.•The method does not require solvent extraction or protein precipitation of the samples., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)

(AAV)-mediated episomal gene replacement therapy for monogenic liver disorders is currently limited in pediatric settings due to the loss of vector DNA, associated with hepatocyte duplication during liver growth. Genome editing is a promising strategy leading to a permanent and specific genome modification that is transmitted to daughter cells upon proliferation. Using genome targeting, we previously rescued neonatal lethality in mice with Crigler-Najjar syndrome. This rare monogenic disease is characterized by severe neonatal unconjugated hyperbilirubinemia, neurological damage, and death. Here, using the CRISPR- Staphylococcus aureus Cas9 ( Sa Cas9) platform, we edited the disease-causing mutation present in the Ugt1a locus of these mice. Newborn mice were treated with two AAV8 vectors: one expressing the Sa Cas9 and single guide RNA, and the other carrying the Ugt1a homology regions with the corrected sequence, while maintained in a temporary phototherapy setting rescuing mortality. We observed a 50% plasma bilirubin reduction that remained stable for up to 6 months. We then tested different Cas9:donor vector ratios, with a 1:5 ratio showing the greatest efficacy in lowering plasma bilirubin, with partial lethality rescue when more severe, lethal conditions were applied. In conclusion, we reduced plasma bilirubin to safe levels and partially rescued neonatal lethality by correcting the mutant Ugt1a1 gene of a Crigler-Najjar mouse model., Competing Interests: G.B. and A.F.M. are inventors of patents describing liver gene transfer approaches for metabolic diseases and/or treatment of hyperbilirubinemia. The remaining authors declare no competing interests., (© 2023 The Author(s).)

Bilirubin is a heme catabolite and Ugt1a1 is the only enzyme involved in the biological elimination of bilirubin. Partially functional or non-functional Ugt1a1 may result in neuronal damage and death due to the accumulation of unconjugated bilirubin in the brain. The understanding of the role of alternative bilirubin detoxification mechanisms that can reduce bilirubin toxicity risk is crucial for developing novel therapeutic strategies. To provide a proof-of-principle showing whether activation of alternative detoxification pathways could lead to life-compatible bilirubin levels in the absence of Ugt1a1 activity, we used Ugt1 -/- hyperbilirubinemic mice devoid of bilirubin glucuronidation activity. We treated adult Ugt1 -/- mice with TCPOBOP, a strong agonist of the constitutive androstane receptor (CAR). TCPOBOP treatment decreased plasma and liver tissue bilirubin levels by about 38%, and resulted in the transcriptional activation of a vast array of genes involved in bilirubin transport and metabolism. However, brain bilirubin level was unaltered. We observed ~40% degradation of bilirubin in the liver microsomes from TCPOBOP treated Ugt1 -/- mice. Our findings suggest that, in the absence of Ugt1a1, the activation of alternative bilirubin clearance pathways can partially improve hyperbilirubinemic conditions. This therapeutic approach may only be considered in a combinatorial manner along with other treatments.

CentriMag ® extracorporeal VAD support could represent a more physiological choice than conventional ECMO in primary cardiogenic shock. We therefore evaluated the outcome of patients with primary cardiogenic shock who were supported with CentriMag ® extracorporeal VAD implantation versus conventional ECMO. We retrospectively reviewed all extracorporeal life supports implanted for primary cardiogenic shock between January 2009 and December 2018 at our institution. Among 212 patients, 143 cases (67%) were treated exclusively with ECMO (Group 1) and 69 cases (33%) with extracorporeal VAD implantation (Group 2, 48 of whom as conversion of ECMO). ECLS mean duration was 8.37 ± 8.43 days in Group 1 and 14.25 ± 10.84 days in Group 2 ( p = 0.001), while the mean rates of the highest predicted flow were 61.21 ± 16.01% and 79.49 ± 18.42% ( p = 0.001), respectively. Increasing mechanical support flow was related to in-hospital mortality and overall mortality in Group 1 (HR 11.36, CI 95%: 2.19-44.20), but not in Group 2 (HR 1.48, CI 95%: 0.32-6.80). High-flow ECMO patients had lower survival with respect to high-flow extracorporeal VAD patients ( p = 0.027). In the setting of high-flow mechanical circulatory support, CentriMag ® extracorporeal VAD optimized patient survival, granting long-term assistance and physiological circulation patterns.

Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: The optimal revascularization strategy for patients with left main coronary artery disease is still controversial. This is systematic review and meta-analysis aims to evaluate the outcomes of percutaneous coronary intervention (PCI) with drug-eluting stents compared with coronary artery bypass graft (CABG) for LM disease., Methods: Online electronic databases were systematically reviewed until January 2020 for randomized trials comparing PCI with drug-eluting stents and CABG. Primary outcomes were: all-cause mortality, myocardial infarction (MI), stroke, and repeated revascularization. Secondary outcomes included periprocedural and nonperiprocedural MI. The period of follow-up included 30 days, 1 year, and 5 years. Odds ratio and 95% confidence interval were calculated with a fixed-effects model., Results: A total of 4595 patients (5 randomized trials) with left main coronary artery disease were included. At 30 days and 1 year, PCI was associated with lower incidence of stroke, higher repeated revascularization, and similar odds of mortality and MI compared with CABG. At 5 years, PCI was associated with higher rates of MI (odds ratio, 1.43; 95% confidence interval, 1.13-1.79; P = .003) and repeat revascularization (odds ratio, 1.89; 95% CI, 1.58-2.26; P < .001) than CABG. PCI was associated with lower periprocedural MI at 30 days, whereas at 5 years PCI was associated with higher nonperiprocedural MI (odds ratio, 2.32; 95% confidence interval, 1.62-3.31; P < .001). Mortality and stroke rate did not differ at 5-year follow-up., Conclusions: Patients with left main coronary artery disease treated with either PCI or CABG do not show significant difference in early or 5-year mortality. Although CABG was associated with higher stroke rates at 30 days and 1 year, PCI was associated with an increase in MI and need for repeat revascularization at 5 years., (Copyright © 2020 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Accumulation of neurotoxic bilirubin due to a transient neonatal or persistent inherited deficiency of bilirubin glucuronidation activity can cause irreversible brain damage and death. Strategies to inhibit bilirubin production and prevent neurotoxicity in neonatal and adult settings seem promising. We evaluated the impact of Bvra deficiency in neonatal and aged mice, in a background of unconjugated hyperbilirubinemia, by abolishing bilirubin production. We also investigated the disposal of biliverdin during fetal development. In Ugt1 -/- mice, Bvra deficiency appeared sufficient to prevent lethality and to normalize bilirubin level in adults. Although biliverdin accumulated in Bvra -deficient fetuses, both Bvra -/- and Bvra -/- Ugt1 -/- pups were healthy and reached adulthood having normal liver, brain, and spleen histology, albeit with increased iron levels in the latter. During aging, both Bvra -/- and Bvra -/- Ugt1 -/- mice presented normal levels of relevant hematological and metabolic parameters. Interestingly, the oxidative status in erythrocytes from 9-months-old Bvra -/- and Bvra -/- Ugt1 -/- mice was significantly reduced. In addition, triglycerides levels in these 9-months-old Bvra -/- mice were significantly higher than WT controls, while Bvra -/- Ugt1 -/- tested normal. The normal parameters observed in Bvra -/- Ugt1 -/- mice fed chow diet indicate that Bvra inhibition to treat unconjugated hyperbilirubinemia seems safe and effective.

In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy., Competing Interests: The authors have declared that no competing interests exist.

A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (h UGT1A1 ) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-h UGT1A1 , and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs., Competing Interests: F.C., G.B., G.R., A.F.M., and F.M. are inventors of patents describing liver gene transfer approaches for metabolic diseases and/or treatment of hyperbilirubinemia. F.M. is an employee of Spark Therapeutics, a Roche company. The remaining authors declare no competing interests., (© 2020 The Author(s).)

Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human OTC cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTC Spf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic., Competing Interests: F.M. is currently an employee of Spark Therapeutics, a Roche company. P.I. and T.K.K. are employees of Selecta Biosciences., (© 2020 The Authors.)

Tumor-cell infiltration is a major obstacle to successful therapy for brain tumors. Membrane-type matrix metalloproteinases (MT-MMPs), a metzincin subfamily of six proteases, are important mediators of infiltration. The cellular source of MT-MMPs and their role in glioma biology, however, remain controversial. Thus, we comprehensively analyzed the expression of MT-MMPs in primary brain tumors. All MT-MMPs were differentially expressed in primary brain tumors. In diffuse gliomas, MT-MMP1, -3, and -4 were predominantly expressed by IDH1 mutated tumor cells, while macrophages/microglia contributed significantly less to MT-MMP expression. For functional analyses, individual MT-MMPs were expressed in primary mouse p53 -/- astrocytes. Invasion and migration potential of MT-MMP-transduced astrocytes was determined via scratch, matrigel invasion, and novel organotypic porcine spinal slice migration (OPoSSM) and invasion assays. Overall, MT-MMP-transduced astrocytes showed enhanced migration compared to controls. MMP14 was the strongest mediator of migration in scratch assays. However, in the OPoSSM assays, the glycosylphosphatidylinositol (GPI)-anchored MT-MMPs MMP17 and MMP25, not MMP14, mediated the highest infiltration rates of astrocytes. Our data unequivocally demonstrate for the first time that glioma cells, not microglia, are the predominant producers of MT-MMPs in glioma and can act as potent mediators of tumor-cell infiltration into CNS tissue. These proteases are therefore promising targets for therapeutic interventions., Competing Interests: The authors declare no conflict of interest.

Potency assessment of clinical-grade vector lots is crucial to support adeno-associated virus (AAV) vector release and is required for future marketing authorization. We have developed and validated a cell-based, quantitative potency assay that detects both transgenic expression and activity of an AAV8-h UGT1A1 vector, which is currently under clinical evaluation for the treatment of Crigler-Najjar syndrome. Potency of AAV8-h UGT1A1 was evaluated in vitro . After transduction of human hepatoma 7 (Huh7) cells, transgene-positive cells were quantified using flow cytometry and transgenic activity by a bilirubin conjugation assay. The in vitro potency of various AAV8-hUGT1A1 batches was compared with their potency in vivo . After AAV8-h UGT1A1 transduction, quantification of UGT1A1-expressing cells shows a linear dose-response relation (R 2  = 0.98) with adequate intra-assay and inter-day reproducibility (coefficient of variation [CV] = 11.0% and 22.6%, respectively). In accordance, bilirubin conjugation shows a linear dose-response relation (R 2  = 0.99) with adequate intra- and inter-day reproducibility in the low dose range (CV = 15.7% and 19.7%, respectively). Both in vitro potency assays reliably translate to in vivo efficacy of AAV8-h UGT1A1 vector lots. The described cell-based potency assay for AAV8-h UGT1A1 adequately determines transgenic UGT1A1 expression and activity, which is consistent with in vivo efficacy. This novel approach is suited for the determination of vector lot potency to support clinical-grade vector release., (© 2020 The Author(s).)

The influence of registries in medicine is large. However, there has been no systematic assessment conducted to quantify the impact of benchmarking with registries focused on cardiothoracic surgery. Numerous publications conclude that registry participation leads to improvement of outcomes for patients. A large number of registries provide evidence sub-structured by statistics that show decreases in morbidity and mortality in the participants' clinical units. Many authors praise the benchmarking method making use of databases of registries as having a positive effect on outcome of care. However, studies proving the direct causal relation between the use of cardiothoracic surgery-oriented registries and improvement of clinical in-hospital outcomes are extremely scarce. We aimed to analyse the causal relation between the use of cardiothoracic surgery-oriented registries and improvement of clinical outcomes. In a systematic literature review, publications demonstrating the use of registry data to obtain consolidated quality improvements were selected. After analysis of 2990 scientific publications, 6 studies filled the inclusion criteria. The selected studies acknowledged that benchmarking of data against registries was used for a focused and methodologically organized improvement in cardiothoracic departments. In conjunction with the impact of the applied methods on healthcare, their results demonstrate quantifiable enhanced local outcomes over time., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Crigler Najjar Syndrome type I (CNSI) is a rare recessive disorder caused by mutations in the Ugt1a1 gene. There is no permanent cure except for liver transplantation, and current therapies present several shortcomings. Since stem cell-based therapy offers a promising alternative for the treatment of this disorder, we evaluated the therapeutic potential of human liver stem cells (HLSC) in immune-compromised NOD SCID Gamma (NSG)/Ugt1 -/- mice, which closely mimic the pathological manifestations in CNSI patients. To assess whether HLSC expressed UGT1A1, decellularised mouse liver scaffolds were repopulated with these cells. After 15 days' culture ex vivo, HLSC differentiated into hepatocyte-like cells showing UGT1A1 expression and activity. For the in vivo human cell engraftment and recovery experiments, DiI-labelled HLSC were injected into the liver of 5 days old NSG/Ugt1 -/- pups which were analysed at postnatal Day 21. HLSC expressed UGT1A1 in vivo, induced a strong decrease in serum unconjugated bilirubin, thus significantly improving phenotype and survival compared to untreated controls. A striking recovery from brain damage was also observed in HLSC-injected mutant mice versus controls. Our proof-of-concept study shows that HLSC express UGT1A1 in vivo and improve the phenotype and survival of NSG/Ugt1 -/- mice, and show promises for the treatment of CNSI.

The molecular and cellular events leading to bilirubin-induced neurotoxicity, the mechanisms regulating liver and intestine expression in neonates, and alternative pathways of bilirubin catabolism remain incompletely defined. To answer these questions, researchers have developed a number of model systems to closely recapitulate the main characteristics of the disease, ranging from tissue cultures to engineered mouse models. In the present review we describe in vitro, ex vivo, and in vivo models developed to study bilirubin metabolism and neurotoxicity, with a special focus on the use of engineered animal models. In addition, we discussed the most recent studies related to potential therapeutic approaches to treat neonatal hyperbilirubinemia, ranging from anti-inflammatory drugs, activation of nuclear receptor pathways, blockade of bilirubin catabolism, and stimulation of alternative bilirubin-disposal pathways.

Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination rate and therapeutic efficacy, here we used CRISPR/SaCas9. Neonatal mice were transduced with two AAVs: one expressing the SaCas9 and sgRNA, and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased ~26-fold with an eGFP reporter cDNA, reaching up to 24% of eGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were ~6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.

Objectives: Administrative databases with dedicated coding systems in healthcare systems where providers are funded based on services recorded have been shown to be useful for clinical research, although their reliability is still questioned. We devised a custom classification of procedures and algorithms based on OPCS, enabling us to identify open heart surgeries from the English administrative database, Hospital Episode Statistics, with the objective of comparing the incidence of cardiac procedures in administrative and clinical databases., Design: A comparative study of the incidence of cardiac procedures in administrative and clinical databases., Setting: Data from all National Health Service Trusts in England, performing cardiac surgery., Participants: Patients classified as having cardiac surgery across England between 2004 and 2015, using a combination of procedure codes, age >18 and consultant specialty, where the classification was validated against internal and external benchmarks., Results: We identified a total of 296 426 cardiac surgery procedures, of which majority of the procedures were coronary artery bypass grafting (CABG), aortic valve replacement (AVR), mitral repair and aortic surgery. The matching at local level was 100% for CABG and transplant, >90% for aortic valve and major aortic procedures and >80% for mitral. At national level, results were similar for CABG (IQR 98.6%-104%), AVR (IQR 105%-118%) and mitral valve replacement (IQR 86.2%-111%)., Conclusions: We set up a process which can identify cardiac surgeries in England from administrative data. This will lead to the development of a risk model to predict early and late postoperative mortality, useful for risk stratification, risk prediction, benchmarking and real-time monitoring. Once appropriately adjusted, the system can be applied to other specialties, proving especially useful in those areas where clinical databases are not fully established., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Adeno-associated viruses (AAVs) are among the most efficient vectors for liver gene therapy. Results obtained in the first hemophilia clinical trials demonstrated the long-term efficacy of this approach in humans, showing efficient targeting of hepatocytes with both self-complementary (sc) and single-stranded (ss) AAV vectors. However, to support clinical development of AAV-based gene therapies, efficient and scalable production processes are needed. In an effort to translate to the clinic an approach of AAV-mediated liver gene transfer to treat Crigler-Najjar (CN) syndrome, we developed an (ss)AAV8 vector carrying the human UDP-glucuronosyltransferase family 1-member A1 (hUGT1A1) transgene under the control of a liver-specific promoter. We compared our construct with similar (sc)AAV8 vectors expressing hUGT1A1, showing comparable potency in vitro and in vivo . Conversely, (ss)AAV8-hUGT1A1 vectors showed superior yields and product homogeneity compared with their (sc) counterpart. We then focused our efforts in the scale-up of a manufacturing process of the clinical product (ss)AAV8-hUGT1A1 based on the triple transfection of HEK293 cells grown in suspension. Large-scale production of this vector had characteristics identical to those of small-scale vectors produced in adherent cells. Preclinical studies in animal models of the disease and a good laboratory practice (GLP) toxicology-biodistribution study were also conducted using large-scale preparations of vectors. These studies demonstrated long-term safety and efficacy of gene transfer with (ss)AAV8-hUGT1A1 in relevant animal models of the disease, thus supporting the clinical translation of this gene therapy approach for the treatment of CN syndrome.

Unconjugated bilirubin is considered a potent antioxidant when present at moderate levels. However, at high concentrations, it produces severe neurological damage and death associated with kernicterus due to oxidative stress and other mechanisms. While it is widely recognized that oxidative stress by different toxic insults results in severe damage to cellular macromolecules, especially to DNA, no data are available either on DNA damage in the brain triggered by hyperbilirubinemia during the neonatal period or on the activation of DNA repair mechanisms. Here, using a mouse model of neonatal hyperbilirubinemia, we demonstrated that DNA damage occurs in vivo in the cerebellum, the brain region most affected by bilirubin toxicity. We studied the mechanisms associated with potential toxic action of bilirubin on DNA in in vitro models, which showed significant increases in DNA damage when neuronal and nonneuronal cells were treated with 140 nM of free bilirubin (Bf), as determined by γ H2AX Western blot and immunofluorescence analyses. Cotreatment of cells with N-acetyl-cysteine, a potent oxidative-stress inhibitor, prevented DNA damage by bilirubin, supporting the concept that DNA damage was caused by bilirubin-induced oxidative stress. Bilirubin treatment also activated the main DNA repair pathways through homologous recombination (HR) and nonhomologous end joining (NHEJ), which may be adaptive responses to repair bilirubin-induced DNA damage. Since DNA damage may be another important factor contributing to neuronal death and bilirubin encephalopathy, these results contribute to the understanding of the mechanisms associated with bilirubin toxicity and may be of relevance in neonates affected with severe hyperbilirubinemia.

Crigler-Najjar syndrome type I (CNSI) is a rare monogenic disease characterized by severe neonatal unconjugated hyperbilirubinemia with a lifelong risk of neurological damage and death. Liver transplantation is the only curative option, which has several limitations and risks. We applied an in vivo gene targeting approach based on the insertion, without the use of nucleases, of a promoterless therapeutic cDNA into the albumin locus of a mouse model reproducing all major features of CNSI Neonatal transduction with the donor vector resulted in the complete rescue from neonatal lethality, with a therapeutic reduction in plasma bilirubin lasting for at least 12 months, the latest time point analyzed. Mutant mice, which expressed about 5-6% of WT Ugt1a1 levels, showed normal liver histology and motor-coordination abilities, suggesting no functional liver or brain abnormalities. These results proved that the promoterless gene therapy is applicable for CNSI, providing therapeutic levels of an intracellular ER membrane-bound enzyme responsible for a lethal liver metabolic disease., (© 2017 International Centre for Genetic Engineering and Biotechnology Published under the terms of the CC BY 4.0 license.)

Adeno-associated virus (AAV) -mediated gene therapy is a promising strategy to treat liver-based monogenic diseases. However, two major obstacles limit its success: first, vector dilution in actively dividing cells, such as hepatocytes in neonates/children, due to the non-integrating nature of the vector; second, development of an immune response against the transgene and/or viral vector. Crigler-Najjar Syndrome Type I is a rare monogenic disease with neonatal onset, caused by mutations in the liver-specific UGT1 gene, with toxic accumulation of unconjugated bilirubin in plasma, tissues and brain. To establish an effective and long lasting cure, we applied AAV-mediated liver gene therapy to a relevant mouse model of the disease. Repeated gene transfer to adults by AAV-serotype switching, upon neonatal administration, resulted in lifelong correction of total bilirubin (TB) levels in both genders. In contrast, vector loss over time was observed after a single neonatal administration. Adult administration resulted in lifelong TB levels correction in male, but not female Ugt1 -/- mice. Our findings demonstrate that neonatal AAV-mediated gene transfer to the liver supports a second transfer of the therapeutic vector, by preventing the induction of an immune response and supporting the possibility to improve AAV-therapeutic efficacy by repeated administration.

Background: Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced neurological damage and eventually death by kernicterus. Bilirubin neurotoxicity is characterized by a wide array of neurological deficits, including irreversible abnormalities in motor, sensitive and cognitive functions, due to bilirubin accumulation in the brain. Despite the abundant literature documenting the in vitro and in vivo toxic effects of bilirubin, it is unclear which molecular and cellular events actually characterize bilirubin-induced neurodegeneration in vivo., Methods: We used a mouse model of neonatal hyperbilirubinemia to temporally and spatially define the response of the developing cerebellum to the bilirubin insult., Results: We showed that the exposure of developing cerebellum to sustained bilirubin levels induces the activation of oxidative stress, ER stress and inflammatory markers at the early stages of the disease onset. In particular, we identified TNFα and NFKβ as key mediators of bilirubin-induced inflammatory response. Moreover, we reported that M1 type microglia is increasingly activated during disease progression. Failure to counteract this overwhelming stress condition resulted in the induction of the apoptotic pathway and the generation of the glial scar. Finally, bilirubin induced the autophagy pathway in the stages preceding death of the animals., Conclusions: This study demonstrates that inflammation is a key contributor to bilirubin damage that cooperates with ER stress in the onset of neurotoxicity. Pharmacological modulation of the inflammatory pathway may be a potential intervention target to ameliorate neonatal lethality in Ugt1 -/- mice.

Moderate neonatal jaundice is the most common clinical condition during newborn life. However, a combination of factors may result in acute hyperbilirubinemia, placing infants at risk of developing bilirubin encephalopathy and death by kernicterus. While most risk factors are known, the mechanisms acting to reduce susceptibility to bilirubin neurotoxicity remain unclear. The presence of modifier genes modulating the risk of developing bilirubin-induced brain damage is increasingly being recognised. The Abcb1 and Abcc1 members of the ABC family of transporters have been suggested to have an active role in exporting unconjugated bilirubin from the central nervous system into plasma. However, their role in reducing the risk of developing neurological damage and death during neonatal development is still unknown.To this end, we mated Abcb1a/b-/- and Abcc1-/- strains with Ugt1-/- mice, which develop severe neonatal hyperbilirubinemia. While about 60% of Ugt1-/- mice survived after temporary phototherapy, all Abcb1a/b-/-/Ugt1-/- mice died before postnatal day 21, showing higher cerebellar levels of unconjugated bilirubin. Interestingly, Abcc1 role appeared to be less important.In the cerebellum of Ugt1-/- mice, hyperbilirubinemia induced the expression of Car and Pxr nuclear receptors, known regulators of genes involved in the genotoxic response.We demonstrated a critical role of Abcb1 in protecting the cerebellum from bilirubin toxicity during neonatal development, the most clinically relevant phase for human babies, providing further understanding of the mechanisms regulating bilirubin neurotoxicity in vivo. Pharmacological treatments aimed to increase Abcb1 and Abcc1 expression, could represent a therapeutic option to reduce the risk of bilirubin neurotoxicity., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The aim of our study was to assess the possible relationships among heme oxygenase (HMOX), bilirubin UDP-glucuronosyl transferase (UGT1A1) promoter gene variations, serum bilirubin levels, and Fabry disease (FD). The study included 56 patients with FD (M : F ratio = 0.65) and 185 healthy individuals. Complete standard laboratory and clinical work-up was performed on all subjects, together with the determination of total peroxyl radical-scavenging capacity. The (GT)n and (TA)n dinucleotide variations in the HMOX1 and UGT1A1 gene promoters, respectively, were determined by DNA fragment analysis. Compared to controls, patients with FD had substantially lower serum bilirubin levels (12.0 versus 8.85  μ mol/L, p = 0.003) and also total antioxidant capacity ( p < 0.05), which showed a close positive relationship with serum bilirubin levels ( p = 0.067) and the use of enzyme replacement therapy ( p = 0.036). There was no association between HMOX1 gene promoter polymorphism and manifestation of FD. However, the presence of the TA 7 allele UGT1A1 gene promoter, responsible for higher systemic bilirubin levels, was associated with a twofold lower risk of manifestation of FD (OR = 0.51, 95% CI = 0.27-0.97, p = 0.038). Markedly lower serum bilirubin levels in FD patients seem to be due to bilirubin consumption during increased oxidative stress, although UGT1A1 promoter gene polymorphism may modify the manifestation of FD as well.

Crigler-Najjar syndrome is a severe metabolic disease of the liver due to a reduced activity of the UDP Glucuronosyltransferase 1A1 (UGT1A1) enzyme. In an effort to translate to the clinic an adeno-associated virus vector mediated liver gene transfer approach to treat Crigler-Najjar syndrome, we developed and optimized a vector expressing the UGT1A1 transgene. For this purpose, we designed and tested in vitro and in vivo multiple codon-optimized UGT1A1 transgene cDNAs. We also optimized noncoding sequences in the transgene expression cassette. Our results indicate that transgene codon-optimization is a strategy that can improve efficacy of gene transfer but needs to be carefully tested in vitro and in vivo . Additionally, while inclusion of introns can enhance gene expression, optimization of these introns, and in particular removal of cryptic ATGs and splice sites, is an important maneuver to enhance safety and efficacy of gene transfer. Finally, using a translationally optimized adeno-associated virus vector expressing the UGT1A1 transgene, we demonstrated rescue of the phenotype of Crigler-Najjar syndrome in two animal models of the disease, Gunn rats and Ugt1a1 -/- mice. We also showed long-term (>1 year) correction of the disease in Gunn rats. These results support further translation of the approach to humans.

Objective: In vitro tests demonstrated that the new cone-bearing configuration of the Jarvik 2000 (Jarvik Heart Inc, New York, NY) left ventricular assist device exhibits better hydraulic efficiency than the previous pin-bearing design. We investigated the long-term outcomes of patients who received the Jarvik 2000 left ventricular assist device, depending on bearing design., Methods: A retrospective review of prospectively collected data from 18 centers included in the Italian Registry was performed. From May 2008 to September 2013, 99 patients with end-stage heart failure were enrolled. Patients were divided into 2 groups according to their Jarvik 2000 suspending mechanism: Group pin included patients with pin bearings (May 2008 to June 2010), and group cone included patients with newer cone bearings (July 2010 to September 2013). The 2 groups did not differ significantly in terms of baseline characteristics., Results: A total of 30 of 39 patients (group pin) and 46 of 60 patients (group cone) were discharged. During follow-up, 6 patients underwent transplantation, and in 1 patient the left ventricular assist device was explanted. The cumulative incidence competing risk of the entire cohort for noncardiovascular-related death was 28% (20%-40%); the cumulative incidence competing risk for cardiovascular-related death was 56% (42%-73%): 71% in group pin versus 26% in group cone (P = .034). The multivariate analyses confirmed that the pin-bearing design was a risk factor for cardiovascular death, along with Interagency Registry for Mechanically Assisted Circulatory Support class. Right ventricular failures and ischemic and hemorrhagic strokes were significantly higher in group pin., Conclusions: Patients with the new pump configuration showed a better freedom from cardiovascular death and lower incidence of fatal stroke and right ventricular failure. Further studies are needed to prove the favorable impact of pump-enhanced fluid dynamics on long-term results., (Copyright © 2016 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Semilunar valve regurgitation (SVR) in children on ventricular assist device (VAD) support is a difficult task. Three children with SVR prior or during VAD support, underwent uneventful semilunar valve surgery, and 2 patients were successfully bridged to heart transplantation. We conclude that surgical management of SVR during VAD support in children is feasible with low operative risk, and is mandatory to optimize VAD support while on waiting list., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Therapies to prevent severe neonatal unconjugated hyperbilirubinemia and kernicterus are phototherapy and, in unresponsive cases, exchange transfusion, which has significant morbidity and mortality risks. Neurotoxicity is caused by the fraction of unconjugated bilirubin not bound to albumin (free bilirubin, Bf). Human serum albumin (HSA) administration was suggested to increase plasma bilirubin-binding capacity. However, its clinical use is infrequent due to difficulties to address its potential preventive and curative benefits, and to the absence of reliable markers to monitor bilirubin neurotoxicity risk. We used a genetic mouse model of unconjugated hyperbilirubinemia showing severe neurological impairment and neonatal lethality. We treated mutant pups with repeated HSA administration since birth, without phototherapy application. Daily intraperitoneal HSA administration completely rescued neurological damage and lethality, depending on dosage and administration frequency. Albumin infusion increased plasma bilirubin-binding capacity, mobilizing bilirubin from tissues to plasma. This resulted in reduced plasma Bf, forebrain and cerebellum bilirubin levels. We showed that, in our experimental model, Bf is the best marker to determine the risk of developing neurological damage. These results support the potential use of albumin administration in severe acute hyperbilirubinemia conditions to prevent or treat bilirubin neurotoxicity in situations in which exchange transfusion may be required.

We describe a modified implantation technique for the HeartWare ventricular assist device. We access the apex through a left minithoracotomy. The outflow graft is tunneled through a small incision in the fourth intercostal space and then subcutaneously to the subclavian region. After division of the left axillary artery, an end-to-end anastomosis is performed to the proximal part, and the distal vessel is connected end-to-side through a fenestration in the outflow graft. We believe that this technique, particularly suitable for redo scenarios or severely calcified aorta, achieves a more direct blood flow into the aorta and reduces cerebrovascular events while avoiding excessive flow to the arm., (Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Aim: To investigate the contribution of anti-platelet therapy and derangements of pre-operative classical coagulation and thromboelastometry parameters to major bleeding post-coronary artery bypass grafting (CABG)., Methods: Two groups of CABG patients were studied: Group A, treated with aspirin alone (n = 50), and Group B treated with aspirin and clopidogrel (n = 50). Both had similar preoperative, clinical, biologic characteristics and operative management. Classic coagulation parameters and rotational thromboelastometry (ROTEM) profiles were determined preoperatively for both groups and the same heparin treatment was administered. ROTEM profiles (INTEM and EXTEM assays) were analyzed, both for traditional parameters, and thrombin generation potential, expressed by area-under-curve (AUC)., Results: There was no significant difference between rates of major bleeding between patients treated with aspirin alone, compared with those treated with aspirin and clopidogrel (12% vs 16%, P = 0.77). In the 14 cases of major bleeding, pre-operative classic coagulation and traditional ROTEM parameters were comparable. Conversely we observed that the AUC in the EXTEM test was significantly lower in bleeders (5030 ± 1115 Ohm*min) than non-bleeders (6568 ± 548 Ohm*min) (P < 0.0001)., Conclusion: We observed that patients with a low AUC value were at a significantly higher risk of bleeding compared to patients with higher AUC, regardless of antiplatelet treatment. This suggests that thrombin generation potential, irrespective of the degree of platelet inhibition, correlates with surgical bleeding.

Background: The role of extracorporeal life support (ECLS) in primary cardiogenic shock (PCS) is well established. In this study, we evaluated the impact of etiology on outcomes., Methods: Between January 2009 and March 2013, we implanted a total of 249 patients with ECLS; we focused on 64 patients for whom peripheral ECLS was the treatment for PCS. Of these, 37 cases (58%) were "acute" (mostly acute myocardial infarction: 39%); 27 (42%) had an exacerbation of "chronic" heart failure (dilated cardiomyopathy: 30%; post-ischemic cardiomyopathy: 9%; and congenital: 3%)., Results: In the group with chronic etiology, 23 patients were bridged to a left ventricular assist device (52%) or heart transplantation (33%). In the group with acute etiology, ECLS was used as a bridge-to-transplantation in 3 patients (8%), a bridge-to-bridge in 9 (24%), and a bridge-to-recovery in 18 (49%). One patient in each group was bridged to conventional surgery. Recovery of cardiac function was achieved in only the group with acute primary cardiogenic shock (18 vs 0 patients, P = .0001). A mean flow during support of ≤60% of the theoretic flow (body surface area × 2.4) was a predictor of successful weaning (P = .02). Median duration of ECLS support was 7 days (range: 2-11.5 days). Nine patients (14%) died during support; 30-day overall survival was 80% (51 of 64 patients); and 59% of patients were discharged, in whom survival at 48 months was 90%. Thirty-day survival was correlated with duration of ECLS support., Conclusions: In "chronic" heart failure, ECLS represents a bridge to a ventricular assist device or heart transplantation, whereas in "acute" settings, it offers a considerable chance of recovery, and is often the only required therapy., (Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Severe hyperbilirubinemia is toxic during central nervous system development. Prolonged and uncontrolled high levels of unconjugated bilirubin lead to bilirubin-induced encephalopathy and eventually death by kernicterus. Despite extensive studies, the molecular and cellular mechanisms of bilirubin toxicity are still poorly defined. To fill this gap, we investigated the molecular processes underlying neuronal injury in a mouse model of severe neonatal jaundice, which develops hyperbilirubinemia as a consequence of a null mutation in the Ugt1 gene. These mutant mice show cerebellar abnormalities and hypoplasia, neuronal cell death and die shortly after birth because of bilirubin neurotoxicity. To identify protein changes associated with bilirubin-induced cell death, we performed proteomic analysis of cerebella from Ugt1 mutant and wild-type mice. Proteomic data pointed-out to oxidoreductase activities or antioxidant processes as important intracellular mechanisms altered during bilirubin-induced neurotoxicity. In particular, they revealed that down-representation of DJ-1, superoxide dismutase, peroxiredoxins 2 and 6 was associated with hyperbilirubinemia in the cerebellum of mutant mice. Interestingly, the reduction in protein levels seems to result from post-translational mechanisms because we did not detect significant quantitative differences in the corresponding mRNAs. We also observed an increase in neuro-specific enolase 2 both in the cerebellum and in the serum of mutant mice, supporting its potential use as a biomarker of bilirubin-induced neurological damage. In conclusion, our data show that different protective mechanisms fail to contrast oxidative burst in bilirubin-affected brain regions, ultimately leading to neurodegeneration.

Background: Much of the morbidity and mortality associated with ventricular assist devices (VADs) is due to haemorrhagic and thrombotic complications. To manage antithrombotic therapy, interactions between the patient and pump should be better understood., Methods: We have compared the Jarvik 2000, an axial flow left ventricular assist device (LVAD), with the HeartWare ventricular assist device (HVAD) centrifugal pump, regarding conventional laboratory findings, thromboelastometric and aggregometric tests., Results: Patients with the Jarvik 2000 experienced a significant reduction in platelet count following implantation, a phenomenon not seen with the HeartWare model. Conversely, we observed that levels of platelet activation, as assessed by a platelet function analyzer, and activation of the coagulation system, as assessed by thromboelastometry, were significantly greater in the HeartWare group., Conclusions: It seems that axial flow pumps, being more destructive on blood cells, tend to reduce platelet numbers. On the other hand, centrifugal flow is associated with a hypercoagulable state, possibly resulting from the activation of the coagulation system in the absence of platelet destruction.

Neonatal jaundice is caused by high levels of unconjugated bilirubin. It is usually a temporary condition caused by delayed induction of UGT1A1, which conjugates bilirubin in the liver. To reduce bilirubin levels, affected babies are exposed to phototherapy (PT), which converts toxic bilirubin into water-soluble photoisomers that are readily excreted out. However, in some cases uncontrolled hyperbilirubinemia leads to neurotoxicity. To study the mechanisms of bilirubin-induced neurological damage (BIND) in vivo, we generated a mouse model lacking the Ugt1a1 protein and, consequently, mutant mice developed jaundice as early as 36 hours after birth. The mutation was transferred into two genetic backgrounds (C57BL/6 and FVB/NJ). We exposed mutant mice to PT for different periods and analyzed the resulting phenotypes from the molecular, histological and behavioral points of view. Severity of BIND was associated with genetic background, with 50% survival of C57BL/6‑Ugt1(-/-) mutant mice at postnatal day 5 (P5), and of FVB/NJ-Ugt1(-/-) mice at P11. Life-long exposure to PT prevented cerebellar architecture alterations and rescued neuronal damage in FVB/NJ-Ugt1(-/-) but not in C57BL/6-Ugt1(-/-) mice. Survival of FVB/NJ-Ugt1(-/-) mice was directly related to the extent of PT treatment. PT treatment of FVB/NJ-Ugt1(-/-) mice from P0 to P8 did not prevent bilirubin-induced reduction in dendritic arborization and spine density of Purkinje cells. Moreover, PT treatment from P8 to P20 did not rescue BIND accumulated up to P8. However, PT treatment administered in the time-window P0-P15 was sufficient to obtain full rescue of cerebellar damage and motor impairment in FVB/NJ-Ugt1(-/-) mice. The possibility to modulate the severity of the phenotype by PT makes FVB/NJ-Ugt1(-/-) mice an excellent and versatile model to study bilirubin neurotoxicity, the role of modifier genes, alternative therapies and cerebellar development during high bilirubin conditions., (© 2014. Published by The Company of Biologists Ltd.)

Null mutations in the UGT1A1 gene result in Crigler-Najjar syndrome type I (CNSI), characterized by severe hyperbilirubinemia and constant risk of developing neurological damage. Phototherapy treatment lowers plasma bilirubin levels, but its efficacy is limited and liver transplantation is required. To find alternative therapies, we applied AAV liver-specific gene therapy to a lethal mouse model of CNSI. We demonstrated that a single neonatal hUGT1A1 gene transfer was successful and the therapeutic effect lasted up to 17 months postinjection. The therapeutic effect was mediated by the presence of transcriptionally active double-stranded episomes. We also compared the efficacy of two different gene therapy approaches: liver versus skeletal muscle transgene expression. We observed that 5-8% of normal liver expression and activity levels were sufficient to significantly reduce bilirubin levels and maintain lifelong low plasma bilirubin concentration (3.1±1.5 mg/dl). In contrast, skeletal muscle was not able to efficiently lower bilirubin (6.4±2.0 mg/dl), despite 20-30% of hUgt1a1 expression levels, compared with normal liver. We propose that this remarkable difference in gene therapy efficacy could be related to the absence of the Mrp2 and Mrp3 transporters of conjugated bilirubin in muscle. Taken together, our data support the concept that liver is the best organ for efficient and long-term CNSI gene therapy, and suggest that the use of extra-hepatic tissues should be coupled to the presence of bilirubin transporters.

The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It is characterized by unconjugated hyperbilirubinemia that may result in severe neurologic damage and death if untreated. To date, liver transplantation is the only curative treatment. With the aim of generating mutant cell lines of the Ugt1 gene, we utilized the TALEN technology to introduce site-specific mutations in Ugt1 exon 4. We report a fast and efficient method to perform gene knockout in tissue culture cells, based on the use of TALEN pairs targeting restriction enzyme (RE) sites in the region of interest. This strategy overcame the presence of allele-specific single nucleotide polymorphisms (SNPs) and pseudogenes, conditions that limit INDELs' detection by Surveyor. We obtained liver-derived murine N-Muli cell clones having INDELs with efficiency close to 40%, depending on the TALEN pair and RE target site. Sequencing of the target locus and WB analysis of the isolated cell clones showed a high proportion of biallelic mutations in cells treated with the most efficient TALEN pair. Ugt glucuronidation activity was reduced basal levels in the biallelic mutant clones. These mutant liver-derived cell lines could be a very useful tool to study biochemical aspects of Ugt1 enzyme activity in a more natural context, such as substrate specificity, requirement of specific co-factors, the study of inhibitors and other pharmacological aspects, and to correlate enzyme activity to the presence of specific mutations in the gene, by adding back to the mutant cell clones specific variants of the Ugt1 gene. In addition, since genome editing has recently emerged as a potential therapeutic approach to cure genetic diseases, the definition of the most efficient TALEN pair could be an important step towards setting up a platform to perform genome editing in CNSI.

Objectives: Sternal wound dehiscence (SWD) after cardiac surgery is a rare but serious condition associated with considerable costs and morbidity. We sought to evaluate the results of the introduction of vacuum-assisted closure (VAC) therapy in the management of sternal wound dehiscence, compared with those of previous conventional treatments., Methods: We retrospectively collected 7148 patients who underwent cardiac surgery at our institution between January 2002 and June 2012. A total of 152 (2.1%) patients had a sternal wound dehiscence: 107 were treated with conventional treatments (Group A) and 45 were managed with VAC therapy (Group B). Patients were stratified according to preoperative risk factors and type of sternal wound dehiscence (superficial or deep; infected or not) and compared by means of a propensity-matched analysis. A cost analysis was also performed., Results: Forty-five patients of each group matched for all preoperative risk factors and type of sternal wound dehiscence. SWD-related mortality rate was significantly lower in Group B (11 vs 0%; P = 0.05). Incidence of mediastinitis (P < 0.0001), sepsis (P = 0.04), delayed SWD infection (P = 0.05), other complication (P = 0.05), surgical sternal revision (P = 0.04) and surgical superficial revision (P < 0.0001) were all significantly lower in Group B. Mean patient cost was 31 106€ in Group A and 24 383€ in Group B, thus achieving a mean saving of 6723€ per patient., Conclusions: In our experience, the use of VAC therapy for the management of SWD was considerably effective in decreasing mortality (SWD related), incidence of complications and need for surgical procedures; thus, leading to a significant reduction of costs., (© The Author 2014. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.)

Astrocytomas and their most malignant variant glioblastoma multiforme (GBM) represent the vast majority of primary brain tumors. Despite the current progress in neurosurgery, radiation therapy and chemotherapy, most astrocytomas remain fatal disorders. Although brain tumor biology is a matter of intense research, the cell-of-origin and the complete astrocytoma-inducing signaling pathway remain unknown. To further identify the mechanisms leading to gliomagenesis, we transduced primary astrocytes on a p53(-/-) background with c-Myc, constitutively active myr-Akt or both, myr-Akt and c-Myc. Transduced astrocytes showed oncogene-specific alterations of morphology, proliferation and differentiation. Following prolonged periods of cultivation, oncogene-transduced astrocytes expressed several stem-cell markers. Furthermore, astrocytes coexpressing c-Myc and Akt were tumorigenic when implanted into the brain of immunocompetent C57BL/6 mice. Our results reveal that the loss of p53 combined with oncogene overexpression in mature astrocytes simulates pivotal features of glioma pathogenesis, providing a good model for assessing the development of secondary glioblastomas.

The aim of the present study was to detect differentially expressed genes in the human osteoblast-like osteosarcoma cell line SaOS-2 using non-radioactive RNA fingerprinting (RNA arbitrarily primed polymerase chain reaction, RAP-PCR). RNA was isolated at different time points from SaOS-2 cells grown with and without dexamethasone (DEX). By RAP-PCR we detected changes in band patterns of cells treated with DEX compared with untreated cells. PCR fragments further characterized and sequences from three of these gave perfect matches to the coding sequences of the human nucleophosmin gene B23, cDNA clone 4&#95;c6 from P1 H25 and the human TRA1 gene, respectively. differential regulation of these genes in DEX-stimulated SaOS-2 cells could be demonstrated by RT-PCR.