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This experiment tested the hypothesis that relocating cattle is detrimental to their growth. The study examined the effect of having relocated cattle mixed with, or segregated from, the local acclimatised cattle at the destination property. Bos indicus cross steers (120) were allocated to three groups and were relocated, in two separate cohorts, 980 km from northern Queensland to improved pastures in central Queensland. At the start of Phase 1, the control group (C) was moved 3 months before the other two groups. The remaining two groups grazed native pastures; one group was supplemented (SR) to increase growth rate similar to that expected from improved pasture in central Queensland and the other was not supplemented (R). At the end of Phase 1, C was significantly (P < 0.05) heavier than SR, which was significantly (P < 0.05) heavier than R. At the start of Phase 2, the SR and R groups were relocated and after transportation the R and SR groups lost 12 kg or 4.4% of liveweight and 18 kg or 5.7% of liveweight, respectively; this weight loss was recovered after 5 days. All steers were reallocated to segregated (SEG) or mixed (MIX) treatment groups forming six treatments (SEG.C, SEG.R and SEG.SR and MIX.C, MIX.R and MIX.SR). There were no significant differences in liveweights within the SEG treatments by 57 days or within the MIX treatments by 106 days after relocation. There were few if any significant differences in the plasma constituents and differential leucocyte counts of the steers and most results were within physiologically normal ranges. We conclude on the basis of these results and of other experiments that the anecdotal poor performance of cattle after relocation appears to be unfounded. [ABSTRACT FROM AUTHOR]

Purpose: Management of renal cell carcinoma (RCC) with inferior vena cava (IVC) extension is one of the greatest challenges in urology. The gold standard treatment includes extracorporeal circulation and deep hypothermic circulatory arrest (DHCA). However, this surgical treatment has an impact on survival and prognosis. We aim to present a less invasive surgical approach for the treatment of level III and IV IVC thrombi in RCC., Methods: We identified all patients undergoing radical nephrectomy and thrombectomy at our Institution between 2016 and 2020. Patients with level IV thrombi were treated with a combined thoracic and abdominal procedure: beating heart surgery with normothermic cardiopulmonary bypass (CPB) was used for thrombus retrieval. Level III thrombi were approached exclusively through an abdominal access., Results: We identified 12 patients treated with radical nephrectomy and thrombectomy among 243 patients undergoing radical nephrectomy for RCC. Mean age was 69.3 years, most patients were in T3c clinical stage, without lymph-node involvement (75%) and no metastases (66.7%); 58.3% of patients had Mayo III thrombus classification. Median intensive care unit stay was 2 days and total in-hospital stay 10.5 days. In-hospital mortality was 0%, with a 66.6% survival rate at 2 years., Conclusions: Beating heart surgery with normothermic CPB can be considered a less invasive method for radical resection of Mayo level IV IVC thrombi. This surgical approach is safe and feasible, with good in-hospital and short-term outcomes., Competing Interests: Declarations. Research involving Human Participants and/or Animals: Data collection adhere to the principles of the Declaration of Helsinki. All patients signed an informed consent agreeing to share their own anonymous information for future studies Informed consent: All patients signed an informed consent agreeing to share their own anonymous information for future studies. Conflicts of interest: None., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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).)

Background: Patients with the Crigler-Najjar syndrome lack the enzyme uridine diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1), the absence of which leads to severe unconjugated hyperbilirubinemia that can cause irreversible neurologic injury and death. Prolonged, daily phototherapy partially controls the jaundice, but the only definitive cure is liver transplantation., Methods: We report the results of the dose-escalation portion of a phase 1-2 study evaluating the safety and efficacy of a single intravenous infusion of an adeno-associated virus serotype 8 vector encoding UGT1A1 in patients with the Crigler-Najjar syndrome that was being treated with phototherapy. Five patients received a single infusion of the gene construct (GNT0003): two received 2×10 12 vector genomes (vg) per kilogram of body weight, and three received 5×10 12 vg per kilogram. The primary end points were measures of safety and efficacy; efficacy was defined as a serum bilirubin level of 300 μmol per liter or lower measured at 17 weeks, 1 week after discontinuation of phototherapy., Results: No serious adverse events were reported. The most common adverse events were headache and alterations in liver-enzyme levels. Alanine aminotransferase increased to levels above the upper limit of the normal range in four patients, a finding potentially related to an immune response against the infused vector; these patients were treated with a course of glucocorticoids. By week 16, serum bilirubin levels in patients who received the lower dose of GNT0003 exceeded 300 μmol per liter. The patients who received the higher dose had bilirubin levels below 300 μmol per liter in the absence of phototherapy at the end of follow-up (mean [±SD] baseline bilirubin level, 351±56 μmol per liter; mean level at the final follow-up visit [week 78 in two patients and week 80 in the other], 149±33 μmol per liter)., Conclusions: No serious adverse events were reported in patients treated with the gene-therapy vector GNT0003 in this small study. Patients who received the higher dose had a decrease in bilirubin levels and were not receiving phototherapy at least 78 weeks after vector administration. (Funded by Genethon and others; ClinicalTrials.gov number, NCT03466463.)., (Copyright © 2023 Massachusetts Medical Society.)

We aimed to identify prognostic laboratory markers during extracorporeal life support (ECLS) in patients with primary refractory cardiogenic shock (RCS) and to create a preliminary specific mortality score. All 208 consecutive subjects admitted for primary RCS and treated with ECLS between January-2009 and December-2018 were retrospectively analyzed. Multivariate regression analysis on laboratory markers during the first nine days of ECLS was used to develop a "Refractory End-stage Shock CUred with Ecls" (RESCUE) score. Serum creatinine (OR = 3.72, 95%CI: 2.01-6.88), direct bilirubin (OR = 1.40, 95%CI: 1.05-1.8), and platelet count (OR = 0.62, 95%CI: 0.42-0.94) were independent predictors of in-hospital mortality and were included in the score. The mean AUC was 0.763 (95%CI: 0.698-0.828) in the development cohort and 0.729 (95%CI: 0.664-0.794) in the bootstrap internal validation cohort. The RESCUE score represents a novel promising instrument to predict early mortality during the first critical days of ECLS and to help in properly guiding the therapeutic decision-making process., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: We investigated the synergistic effect of the new cone-bearing design of Jarvik 2000 (Jarvik Heart Inc., NY) together with a minimally-invasive approach to outcomes of LVAD patients., Methods: We retrospectively reviewed all patients from 5 institutions involved in the Jarvik 2000 Italian Registry, from October 2008 to October 2016. Patients were divided into three groups according to pump design and implantation technique: pin-bearing design and conventional approach (Group 1); cone-bearing and conventional approach (Group 2); cone-bearing and minimally-invasive implantation (Group 3)., Results: A total of 150 adult patients with end-stage heart failure were enrolled: 26 subjects in Group 1, 74 in Group 2, and 50 in Group 3. Nineteen patients (73%) in Group 1, 51 (69%) in Group 2, and 36 (72%) in Group 3 were discharged. During follow-up, 22 patients underwent transplantation, while in 3 patients the LVAD was explanted. The overall 1-year survival was 58 ± 10%, 64 ± 6%, and 74% ± 7% in Groups 1, 2, and 3, respectively (p = 0.034). The competing-risks-adjusted cumulative incidence rate for adverse events was 42.1 [27-62.7] per 100 patient-years in Group 1, 35.4 [25.3-48.2] in Group 2, and 22.1 [12.4-36.4] in Group 3 (p = 0.046 for Group 1 vs. 3)., Conclusions: The association of the modern cone-bearing configuration of Jarvik 2000 and minimally invasive surgery improved survival and minimized the risk for cardiovascular events, as a result of combining technology and technique., (© 2022 International Center for Artificial Organs and Transplantation and Wiley Periodicals LLC.)

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.

Continuous flow left ventricular assist devices (LVADs) have become a valuable therapy for end-stage heart failure. In vitro research highlighted a role of outflow cannula position on the pattern of blood flow in the aorta. However, the clinical effects of the alterations of flow remain unclear. We investigate short- and long-term outcomes of patients implanted with Jarvik 2000 LVAD, according to the ascending (Group 1) versus descending (Group 2) outflow graft connection to the aorta in a multicenter study. From May 2008 to October 2014, 140 consecutive end-stage heart failure patients underwent Jarvik 2000 LVAD implantation in 17 Italian centers. According with a preliminary multivariate analysis, we selected the 90 patients implanted in the four high-volume centers to avoid bias (Group 1 n = 39, Group 2 n = 51). Among the groups, no differences were recorded in the hospital mortality and the main complications occurring after LVAD implantation were similar. In multivariable analysis, the ascending aorta outflow cannula position and higher creatinine at discharge were significant predictors for long-term survival. Postimplant hemolysis was more pronounced in descending aorta outflow graft anastomosis. Outflow graft anastomosis to the ascending aorta is associated with better long-term survival, independent of age and perfusion techniques, reflecting the previous in vitro results., (© 2020 International Center for Artificial Organs and Transplantation and Wiley Periodicals LLC.)

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.

All pre-term newborns and a high proportion of term newborns develop neonatal jaundice. Neonatal jaundice is usually a benign condition and self-resolves within few days after birth. However, a combination of unfavorable complications may lead to acute hyperbilirubinemia. Excessive hyperbilirubinemia may be toxic for the developing nervous system leading to severe neurological damage and death by kernicterus. Survivors show irreversible neurological deficits such as motor, sensitive and cognitive abnormalities. Current therapies rely on the use of phototherapy and, in unresponsive cases, exchange transfusion, which is performed only in specialized centers. During bilirubin-induced neurotoxicity different molecular pathways are activated, ranging from oxidative stress to endoplasmic reticulum (ER) stress response and inflammation, but the contribution of each pathway in the development of the disease still requires further investigation. Thus, to increase our understanding of the pathophysiology of bilirubin neurotoxicity, encephalopathy and kernicterus, we pharmacologically modulated neurodegeneration and neuroinflammation in a lethal mouse model of neonatal hyperbilirubinemia. Treatment of mutant mice with minocycline, a second-generation tetracycline with anti-inflammatory and neuroprotective properties, resulted in a dose-dependent rescue of lethality, due to reduction of neurodegeneration and neuroinflammation, without affecting plasma bilirubin levels. In particular, rescued mice showed normal motor-coordination capabilities and behavior, as determined by the accelerating rotarod and open field tests, respectively. From the molecular point of view, rescued mice showed a dose-dependent reduction in apoptosis of cerebellar neurons and improvement of dendritic arborization of Purkinje cells. Moreover, we observed a decrease of bilirubin-induced M1 microglia activation at the sites of damage with a reduction in oxidative and ER stress markers in these cells. Collectively, these data indicate that neurodegeneration and neuro-inflammation are key factors of bilirubin-induced neonatal lethality and neuro-behavioral abnormalities. We propose that the application of pharmacological treatments having anti-inflammatory and neuroprotective effects, to be used in combination with the current treatments, may significantly improve the management of acute neonatal hyperbilirubinemia, protecting from bilirubin-induced neurological damage and death., (Copyright © 2018 Elsevier Inc. All rights reserved.)