Christopher Burns

Generation of a human embryonic stem cell line encoding the cystic fibrosis mutation ΔF508, using preimplantation genetic diagnosis

Human embryonic stem (hES) cells are pluripotent cells isolated from early human embryos. They can be grown in vitro and made to differentiate into many different cell types. These properties have suggested that they may be useful in cell replacement therapy for many degenerative diseases. However, if hES cells could also be manufactured with mutations significant in human disease, they could provide a powerful in-vitro tool for modelling disease processes and progression in a number of different cell types, as well as providing an ideal system for studying in-vitro toxicity and efficacy of drugs and other therapeutic systems such as gene therapy. Embryos with such mutations are generated as part of routine genetic testing during preimplantation genetic diagnosis, providing the opportunity to generate cell lines with significant mutations. A human embryonic stem cell line homozygous for the most common mutation leading to cystic fibrosis in humans (delta F508) has been generated and characterized. This cell line has the same morphology and expresses proteins typical of other unaffected hES cell lines. This cell line represents an important in-vitro tool for understanding the pathophysiology of cystic fibrosis, and presents exciting opportunities to test the efficacy and toxicity of new therapies relevant to CF.

Preimplantation genetic diagnosis as a novel source of embryos for stem cell research.

The generation of human embryonic stem (hES) cells has captured the public and professional imagination, largely due their potential as a means of overcoming many debilitating and degenerative diseases by cell replacement therapy. Despite this potential, few well-characterized hES cell lines have been derived. Indeed, in the UK, despite several centres having been active in this area for more than 2 years, there are as yet no published reports of human embryonic stem cells having been generated. Part of the reason for this lack of progress may relate to the quality of embryos available for research. Embryos surplus to therapeutic requirements following routine assisted reproduction treatment are often of poor quality and a large proportion may be aneuploid. This study reports a new approach to hES cell derivation. Embryos surplus to therapeutic requirements following preimplantation genetic diagnosis were used. Although unsuitable for embryo transfer due to the high risk of genetic disease, these embryos are from fertile couples and thus may be of better quality than fresh embryos surplus to assisted reproduction treatment cycles. Embryos donated after cryopreservation were also used, and putative hES lines were derived from both sources of embryos. The cell lines described here are thought to be the first reported hES cell lines to have been derived in the UK.

Prehospital Interventions Performed in a Combat Zone: A Prospective Multicenter Study of 1,003 Combat Wounded

BACKGROUND Battlefield care given to a casualty before hospital arrival impacts clinical outcomes. To date, the published data regarding care given in the prehospital setting of a combat zone are limited. The purpose of this study was to describe the incidence and efficacy of specific prehospital lifesaving interventions (LSIs; interventions that could affect the outcome of the casualty), consistent with the Tactical Combat Casualty Care paradigm, performed during the resuscitation of casualties in a combat zone. METHODS We performed a prospective observational study between November 2009 and November 2011. Casualties were enrolled as they were treated at six US surgical facilities in Afghanistan. Descriptive data were collected on a standardized data collection form and included mechanism of injury, airway management, chest and hemorrhage interventions, vascular access, type of fluid administered, and hypothermia prevention. On arrival to the military hospital, the treating physician determined whether an intervention was performed correctly and whether an intervention was not performed that should have been performed (missed LSI). RESULTS A total of 1,003 patients met the inclusion criteria. Their mean (SD) age was 25 (8.5) years and 97% were male. The mechanism of injury was explosion in 60% of patients, penetrating in 24% of patients, blunt in 15% of patients, and burn in 0.8% of patients. The most commonly performed LSIs included hemorrhage control (n = 599), hypothermia prevention (n = 429), and vascular access (n = 388). Of the missed LSIs, 252 were identified with the highest percentage of missed opportunities being composed of endotracheal intubation, chest needle decompression, and hypotensive resuscitation. In contrast, tourniquet application had the lowest percentage of missed opportunities. CONCLUSIONS In our prospective study of prehospital LSIs performed in a combat zone, we observed a higher rate of incorrectly performed and missed LSIs in airway and chest (breathing) interventions than hemorrhage control interventions. The most commonly performed LSIs had lower incorrect and missed LSI rates. LEVEL OF EVIDENCE Prognostic study, level III.

Diabetes Mellitus: A Potential Target for Stem Cell Therapy

Type 1 diabetes mellitus has received much attention recently as a potential target for the emerging science of stem cell medicine. In this autoimmune disease, the insulin-secreting beta-cells of the pancreas are selectively and irreversibly destroyed by autoimmune assault. Advances in islet transplantation procedures now mean that patients with the disease can be cured by transplantation of primary human islets of Langerhans. A major drawback in this therapy is the availability of donor islets, and the search for substitute transplant tissues has intensified in the last few years. This review will describe the essential requirements of a material designed as a replacement beta-cell and will look at the potential sources of such replacements. These include embryonic stem (ES) cells and multipotent adult stem/progenitor cells from a range of tissues including the pancreas, intestine, liver, bone marrow and brain. These stem cell populations will be evaluated and the different experimental approaches that have been employed to derive functional insulin-expressing cells will be discussed. The review will also look at the capability of human ES (hES) cells generated by somatic cell nuclear transfer and some adult stem cell populations such as bone marrow-derived stem cells, to offer autologous transplant material that would remove the need for immunosuppression. In patients with Type 1 diabetes, auto-reactive T-cells are programmed to recognise the insulin-producing beta-cells. As a result, for therapeutic replacement tissues, it may be more sensible to derive cells that behave like beta-cells but are immunologically distinct. Thus, the potential of cells derived from non-beta-cell origin to avoid the autoimmune response will also be discussed. Finally, the review will summarise the future prospects for stem cell therapies for diabetes and will highlight some of the problems that may be faced by researchers working in this area, such as malignancy, irreproducible differentiation strategies, immune-system rejection and social and ethical concerns over the use of hES cells.

Transfusion-associated microchimerism in combat casualties.

BACKGROUND Fresh whole blood (FrWB) is routinely used in the resuscitation of combat casualties in Operation Iraqi Freedom and Operation Enduring Freedom. However, studies have shown high rates (20%-40%) of transfusion-associated microchimerism (TA-MC) in civilian trauma patients receiving allogenic red blood cell (RBC) transfusions. We explored the incidence of TA-MC in combat casualties receiving FrWB compared with patients receiving standard stored RBC transfusions. METHODS Prospective data on TA-MC at >or=14 days posttransfusion were collected on 26 severely injured combat casualties admitted to the National Naval Medical Center between December 2006 and March 2007. Demographic variables included age, sex, Injury Severity Score, and transfusion history. Data are expressed as mean +/- SD. RESULTS The mean age of the study cohort was 24 +/- 7; mean Injury Severity Score was 17 +/- 12. All were men and suffered penetrating injury. Average hospital length of stay was 46 +/- 35 days. TA-MC was present in 45% (10 of 22) patients who were transfused at least 1 unit of blood. The four nontransfused patients all tested negative for TA-MC. Among six patients who received 4 to 43 units of FrWB, five also received RBCs and one aphaeresis platelets. The remaining 16 transfused patients who received RBCs (no FrWB) included seven who also received platelets in theater. The prevalence of TA-MC was 50% (3 of 6) in FrWB patients, 50% in patients given platelets (4 of 8), and 38% (3 of 8) in those given only RBCs as a cellular component (p = 0.61). CONCLUSIONS Although these preliminary data do not demonstrate a significantly increased rate of TA-MC in FrWB or apheresis platelets recipients compared with RBC recipients, the overall 45% (10 of 22) rate of TA-MC in transfused soldiers warrants further study to ascertain possible clinical consequences such as graft-versus-host or autoimmune disease syndromes.

A Laparoscopic Swine Model of Noncompressible Torso Hemorrhage

BACKGROUND Hemorrhage persists as the leading cause of potentially preventable civilian and military death. Noncompressible torso hemorrhage (NCTH) is a particularly lethal injury complex, with few contemporary prehospital interventions available. Various porcine models of hemorrhage have been developed for civilian and military trauma research. However, the predominant contemporary models lack key physiologic characteristics including the natural tamponade provided by an intact abdominal wall. To improve physiologic and clinical relevance, we developed a laparoscopic model of NCTH. This approach maintains both the integrity of the peritoneum and the natural tamponade effect of an intact abdominal wall while preserving the intrinsic physiologic responses to hemorrhage. Furthermore, we present data quantifying the contribution of the swine contractile spleen in the context of uncontrolled hemorrhage. METHODS Anesthetized adult male Yorkshire swine underwent a laparoscopic Grade V liver injury, with or without open preinjury splenectomy. Animals were observed without intervention for a total of 120 minutes after injury to simulate point of injury, transport time, and arrival at hospital. RESULTS Shed blood–to–body weight ratio did not differ among groups; however, mortality was higher in splenectomized animals (67% vs. 33%). Cox regression modeling demonstrated a critical time point of 45 minutes and blood pressure as significant predictors of mortality. CONCLUSION This study describes a model of NCTH that reflects clinically relevant physiology in trauma and uncontrolled hemorrhage. In addition, it quantitatively assesses the role of the swine contractile spleen in the described model.

Wound healing trajectories in burn patients and their impact on mortality

The rate of wound healing and its effect on mortality has not been well described. The objective of this article is to report wound healing trajectories in burn patients and analyze their effects on in-hospital mortality. The authors used software (WoundFlow) to depict burn wounds, surgical results, and healing progression at multiple time points throughout admission. Data for all patients admitted to the intensive care unit with ≥ 20% TBSA burned were collected retrospectively. The open wound size (OWS), which includes both unhealed burns and unhealed donor sites, was measured. We calculated the rate of wound closure (healing rate), which we defined as the change in OWS/time. We also determined the time delay (DAYS) from day of burn until day on which there was a reduction in OWS < 10%. Data are medians [interquartile range]. There were 38 patients with complete data; 25 had documentation of successful healing (H), and 13 did not (NH). H differed from NH on age (38 years [32–57] vs 63 [51–74]), body mass index (27 [21–28] vs 32 [19–52]), 24-hour fluid resuscitation (12 L [10–16] vs 18 [15–20]), pressors during first 48 hours (72% vs 100%), use of renal replacement therapy (32% vs 92%), and mortality (4% vs 100%). Repeated measures analysis of covariance showed a significant difference between survivors and nonsurvivors on OWS as a function of time (P<.001). Patients with a positive healing rate (+2%/day) after postburn day 20 had 100% survival whereas those with a negative healing rate (−2%/day) had 100% mortality. For H patients, median DAYS was 41 (28–54); median DAYS/TBSA was 1.3 (1.0–1.9). Survivors had a 0.62% drop in OWS/day, or 4.3%/week. In this cohort of patients with ≥ 20% TBSA, there was a difference in mortality after postburn day 20, between patients with a positive healing rate (+2%/day, 100% survival) and those with a negative healing rate (−2%/day, 100% mortality, P < .05).

Nonoperative closure of persistent gastrocutaneous fistulas in children with 2-octylcyanoacrylate.

UNLABELLED A persistent gastrocutaneous fistula (pGCF) is an all-too-common complication following removal of a gastrostomy tube (GT) in a child and is associated with significant morbidity. The most common initial methods to manage pGCF include local would care and occlusion techniques. Failure of this approach is followed by surgical excision of the fistula tract and closure of the gastrostomy under general anesthesia. We report the first use of a tissue adhesive, 2-octylcyanoacrylate (2OC) (Dermabond; Ethicon, Sommerville, NJ) as a non-surgical method to close pGCF in children. METHODS The families of children presenting to the pediatric surgical division for management of a pGCF were offered the option of 2OC closure. Children not receiving or who failed to achieve closure with 2OC therapy underwent surgery for excision of the pGCF with primary closure of the stomach and soft tissues. RESULTS Seven children underwent 2OC therapy. 57% (4 of 7) of the children had complete closure of the pGCF with 2OC therapy. Three children underwent operative closure without complication. CONCLUSIONS The tissue adhesive 2OC can successfully close a pGCF in children after GT removal. This therapy is cost-effective, non-invasive, does not require general anesthesia, and can be performed in an outpatient setting.

Venous Thromboembolic Disease in Colorectal Patients

Venous thromboembolic disease, which includes deep vein thromboses as well as pulmonary emboli, can be a significant complication in the postoperative patient. In particular, colorectal patients often carry a higher risk for venous thromboembolism when compared with patients undergoing other operative procedures. Features unique to colorectal patients are the high incidence of inflammatory bowel disease or malignancy. Typically, these patients will undergo lengthy pelvic procedures, which also contribute to a cumulative risk of venous thrombosis. It is critical that all patients and the proposed operative procedure are appropriately risk stratified. Risk stratification allows for easier implementation of an appropriate prophylactic strategy. There are a wide range of safe and effective mechanical and pharmacologic measures available. The authors provide very specific recommendations, but note that clinical judgment plays a significant role.

Prevalence and Impact of Late Defecation in the Critically Ill, Thermally Injured Adult Patient

The aim of this study was to determine the prevalence of late defecation (absence of laxation for more than 6 days after admission) as an indicator of lower-gastrointestinal (GI) tract dysfunction in burn patients. In addition, the authors wanted to determine whether the addition of polyethylene glycol 3350 to the standard bowel regimen led to improvement in markers of lower-GI function and outcomes. The authors conducted a retrospective chart review of patients admitted to the burn intensive care unit during a 26-month period. Inclusion criteria were 20% or more TBSA burn, requirement for mechanical ventilation, and age over 18 years. Of 83 patients included, the prevalence of late defecation was 36.1% (n = 30). There was no association between late defecation and mortality. Patients with late defecation had more frequent episodes of constipation after first defecation (P =.03), of feeding intolerance (P =.007), and received total parenteral nutrition more frequently (P =.005). The addition of polyethylene glycol to the standard bowel regimen did not affect markers of lower-GI function. Late defecation occurs in more than one third of critically ill burn patients. Late defecation was associated with ongoing lower-GI dysfunction, feeding intolerance, and the use of total parenteral nutrition. The causal relationship between these problems has not been determined. A prospective study at the authors’ institution is currently planned to attempt to validate late defecation as a marker of lower-GI tract dysfunction, determine its relationship to various outcomes, and determine risk factors for its development.