Reprogramming human T cell function and specificity with non-viral genome targeting.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells

Reoperative Complications after Primary Orthotopic Liver Transplantation: A Contemporary Single-Center Experience in the Post–Model for End-Stage Liver Disease Era

BackgroundData on complications requiring reoperation after orthotopic liver transplantation (OLT) are limited. We sought to describe the spectrum of reoperative complications after OLT, evaluate the associations with graft and patient survival, and identify predictors of need for reoperation.Study designWe retrospectively studied adult patients who underwent primary OLT at our institution from February 2002 to July 2012. The primary outcomes included occurrence of a reoperative complication. Secondary outcomes were graft and patient survival. Multivariable logistic regression analysis was used to model the associations of recipient, donor, and operative variables with reoperation.ResultsOf 1,620 patients, 470 (29%) had complications requiring reoperation. The most common reoperative complication was bleeding (17.3%). Compared with patients not requiring reoperation, patients with reoperative complications had greater Model for End-Stage Liver Disease scores and need for pretransplantation hospitalization, mechanical ventilation, vasopressors, and renal replacement therapy; considerably longer cold and warm ischemia times and greater intraoperative blood transfusion requirements; and substantially worse 1-, 3-, and 5-year graft and patient survival rates. In multivariable analysis, predictors of reoperative complications included intraoperative transfusion of packed RBCs (odds ratio [OR] = 2.21; 95% CI, 1.91-2.56), donor length of hospitalization >8 days (OR = 1.87; 95% CI, 1.28-2.73), recipient pretransplantation mechanical ventilation (OR = 1.65; 95% CI, 1.21-2.24), cold ischemia time >9 hours (OR = 1.63; 95% CI, 1.23-2.17), warm ischemia time >55 minutes (OR = 1.58; 95% CI, 1.02-2.44), earlier major abdominal surgery (OR = 1.41; 95% CI, 1.03-1.92), and elevated donor serum sodium (OR = 1.17; 95% CI, 1.03-1.31).ConclusionsPatients who require reoperation for complications after OLT have high pretransplantation acuity and inferior post-transplantation survival. We identified factors associated with reoperative complications to guide perioperative donor-recipient matching and improve outcomes

Clinical Sequencing Exploratory Research Consortium: Accelerating Evidence-Based Practice of Genomic Medicine

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine

Acetaminophen hepatotoxicity: an updated review.

As the most common cause of acute liver failure (ALF) in the USA and UK, acetaminophen-induced hepatotoxicity remains a significant public health concern and common indication for emergent liver transplantation. This problem is largely attributable to acetaminophen combination products frequently prescribed by physicians and other healthcare professionals, with unintentional and chronic overdose accounting for over 50 % of cases of acetaminophen-related ALF. Treatment with N-acetylcysteine can effectively reduce progression to ALF if given early after an acute overdose; however, liver transplantation is the only routinely used life-saving therapy once ALF has developed. With the rapid course of acetaminophen-related ALF and limited supply of donor livers, early and accurate diagnosis of patients that will require transplantation for survival is crucial. Efforts in developing novel treatments for acetaminophen-induced ALF are directed toward bridging patients to recovery. These include auxiliary, artificial, and bioartificial support systems. This review outlines the most recent developments in diagnosis and management of acetaminophen-induced ALF

Risk factors for recurrence after repair of enterocutaneous fistula.

ObjectivesTo assess outcomes after repair of enterocutaneous fistulae (ECF) and identify factors that predict mortality and recurrence.DesignRetrospective study.SettingUniversity hospital.PatientsOne hundred thirty-five patients undergoing ECF repair between 1989 and 2005.Main outcome measuresMortality and recurrence of ECF.ResultsDefinitive operation for ECF was attempted in 135 patients. Mortality was 8%, recurrence was 17%, and 84% of patients eventually survived with a closed fistula. The primary determinant of mortality was ECF recurrence (odds ratio [OR], 6.7; 95% confidence interval [CI], 1.9-23.4). Factors independently associated with ECF recurrence by multivariate logistic regression included the presence of inflammatory bowel disease (OR, 4.9; 95% CI, 1.5-16.1), interval between diagnosis and operation of 36 weeks or longer (OR, 5.4; 95% CI, 1.8-16.4), location of fistulae in the small intestine (OR, 9.8; 95% CI, 1.7-57.6), and resection with stapled anastomosis (OR, 4.1; 95% CI, 1.3-13.2). Recurrence of ECF was 35% with resection and stapled anastomosis, 22% with simple oversew, and 11% with resection and hand-sewn anastomosis. Recurrence of ECF was 12% when operation was performed prior to 36 weeks from diagnosis, compared with 36% if performed at or beyond 36 weeks.ConclusionsThe primary determinant of mortality after ECF repair is a failed operation leading to recurrence of the fistula. Risk factors for ECF recurrence include inflammatory bowel disease, fistula located in the small intestine, an interval of 36 weeks or longer between diagnosis and operation, and resection with stapled anastomosis

Risk factors for recurrence after repair of enterocutaneous fistula.

ObjectivesTo assess outcomes after repair of enterocutaneous fistulae (ECF) and identify factors that predict mortality and recurrence.DesignRetrospective study.SettingUniversity hospital.PatientsOne hundred thirty-five patients undergoing ECF repair between 1989 and 2005.Main outcome measuresMortality and recurrence of ECF.ResultsDefinitive operation for ECF was attempted in 135 patients. Mortality was 8%, recurrence was 17%, and 84% of patients eventually survived with a closed fistula. The primary determinant of mortality was ECF recurrence (odds ratio [OR], 6.7; 95% confidence interval [CI], 1.9-23.4). Factors independently associated with ECF recurrence by multivariate logistic regression included the presence of inflammatory bowel disease (OR, 4.9; 95% CI, 1.5-16.1), interval between diagnosis and operation of 36 weeks or longer (OR, 5.4; 95% CI, 1.8-16.4), location of fistulae in the small intestine (OR, 9.8; 95% CI, 1.7-57.6), and resection with stapled anastomosis (OR, 4.1; 95% CI, 1.3-13.2). Recurrence of ECF was 35% with resection and stapled anastomosis, 22% with simple oversew, and 11% with resection and hand-sewn anastomosis. Recurrence of ECF was 12% when operation was performed prior to 36 weeks from diagnosis, compared with 36% if performed at or beyond 36 weeks.ConclusionsThe primary determinant of mortality after ECF repair is a failed operation leading to recurrence of the fistula. Risk factors for ECF recurrence include inflammatory bowel disease, fistula located in the small intestine, an interval of 36 weeks or longer between diagnosis and operation, and resection with stapled anastomosis