Impulse-powered needle-free syringe for vaccine/drug injection.

A needle-free vaccine/drug injector that works by virtue of the impulse of a moving shock wave is presented in this communication. The device can deliver controlled micro-volumes of liquid vaccines into skin and soft tissue targets in human with minimal invasion. The operation of the injector was investigated by delivering a dyed liquid into human skin samples and soft tissue models. The depth of penetration of the liquid was examined by histology of the targeted human skin samples. The delivery mechanics and the depth of penetration were analyzed theoretically with an elastic model for the skin and a viscoelastic model for the soft tissue targets, and a good agreement with experiments was observed. The current liquid vaccine/drug delivery technique can reduce pain, trauma and contamination, and can offer a cost-effective, needle-free, health-care solution.

Safety and Outcomes in 100 Consecutive Donation After Circulatory Death Liver Transplants Using a Protocol That Includes Thrombolytic Therapy.

Donation after circulatory death (DCD) liver transplantation (LT) reportedly yields inferior survival and increased complication rates compared with donation after brain death (DBD). We compare 100 consecutive DCD LT using a protocol that includes thrombolytic therapy (late DCD group) to an historical DCD group (early DCD group n = 38) and a cohort of DBD LT recipients (DBD group n = 435). Late DCD LT recipients had better 1- and 3-year graft survival rates than early DCD LT recipients (92% vs. 76.3%, p = 0.03 and 91.4% vs. 73.7%, p = 0.01). Late DCD graft survival rates were comparable to those of the DBD group (92% vs. 93.3%, p = 0.24 and 91.4% vs. 88.2%, p = 0.62). Re-transplantation occurred in 18.4% versus 1% for the early and late DCD groups, respectively (p = 0.001). Patient survival was similar in all three groups. Ischemic-type biliary lesions (ITBL) occurred in 5%, 3%, and 0.2% for early DCD, late DCD, and DBD groups, respectively, but unlike in the early DCD group, in the late DCD group ITBL was endoscopically managed and resolved in each case. Using a protocol that includes a thrombolytic therapy, DCD LT yielded patient and graft survival rates comparable to DBD LT.

Management of a pseudo-aneurysm in the hepatic artery after a laparoscopic cholecystectomy.

Introduction Symptomatic hepatic-artery pseudoaneurysm (HAP) after bile-duct injury (BDI) is a rare complication with a varied (but clinically urgent) presentation. Methods A prospectively maintained database of all patients with BDI at laparoscopic cholecystectomy (LC) referred to a tertiary specialist hepatobiliary centre between 1992 and 2011 was searched systematically to identify patients with a symptomatic HAP. Care and outcome of these patients was studied. Results Eight (6 men) of 236 patients with BDI (3.4%) with a median age of 65 (range: 54?6) years presented with symptomatic HAP. Median time of presentation of the HAP from the index LC was 31 (range: 13?16) days. Bleeding was the dominant presentation in 7 patients. One patient presented late (>2 years) with abdominal pain alone. Computed tomography angiography was the most useful investigation. Angioembolisation was successful in 7 patients. One patient died, and another patient developed liver infarction. Three patients (38%) developed biliary strictures after embolisation. Seven patients are alive and well at a median follow-up of 66 months. Conclusions Presentation of HAP is often delayed. A high index of suspicion is necessary for the diagnosis. Computed tomography angiography is the first-line investigation and selective angioembolisation can yield successful outcomes.

Spontaneous liver haemorrhage and haemobilia as initial presentation of undiagnosed polyarteritis nodosa.

Polyarteritis nodosa (PAN) is a systemic necrotising vasculitis that could result in multifocal aneurysms of visceral arteries. Isolated multiple aneurysms of the hepatic arteries in the setting of PAN is extremely rare. Patients are typically asymptomatic and, very rarely, spontaneous rupture with life threatening haemorrhage could be the initial presentation of an undiagnosed PAN. Accurate diagnosis, effective haemostasis and prompt initiation of immunosuppressive treatment with the help of a multidisciplinary team will improve the clinical outcomes.

Early laparoscopic cholecystectomy service provision is feasible and safe in the current UK National Health Service.

INTRODUCTION: Despite increasing evidence of the benefits and safety of early laparoscopic cholecystectomy (LC) in acute gallstone disease, it is not widely practised in England. The Royal College of Surgeons of England support the separation of emergency and elective surgical care. The aim of this prospective study was to examine the impact of the implementation of 'Surgeon of the Week (SoW)' model on the number of early LCs performed and the efficiency of the emergency theatre activity in our hospital. This study also looked into its implications on specialist registrar training for early LC, and the financial impact to the hospital. PATIENTS AND METHODS: Between January 2007 and May 2008, demographic data, admission and discharge dates, complications, conversions to an open operation and deaths were collected for all patients who underwent early laparoscopic cholecystectomies. For ease of comparison, patients were divided into Group A representing before introduction of SoW (1 January 2007 to 30 August 2007) and Group B representing after introduction of SoW (1 October 2007 to 31 May 2008). The total numbers of operations performed in the emergency theatre list in the two groups were also calculated. RESULTS: A total of 1361 emergency operations were performed on the emergency theatre list in Group A, of which 951 were general surgical procedures. In Group B, the numbers of emergency procedures were 1537, of which 1138 were general surgical operations. There was a significant increase in the number of general surgical operations after introduction of SoW (P = 0.013). Before introduction of the SoW rota, 45 early LCs were performed. This increased to 118 after SoW which was significant (P < 0.001). In Group A, the number of early LCs performed by surgical trainees was 10 (22%). In Group B, the number of LCs performed by surgical trainees was 35 (30%; not significant). CONCLUSIONS: This study has demonstrated an increase in the efficiency of the emergency theatre with an increase in the number of early LCs on their index admission without extra morbidity following implementation of the SOW model in our hospital. We recommend the introduction of a suitable emergency surgical consultant on-call model separating emergency and elective surgical care depending on local circumstances. This can lead to significant cost savings and reduce re-admissions with gallstone-related complications.

Auxiliary liver transplantation for propionic acidemia: a 10-year follow-up.

Orthotopic liver transplantation (OLT) is an established treatment for patients with liver-based metabolic disorders that produce structural and functional impairment. Auxiliary liver transplantation (ALT) has been proposed as an alternative approach due to the potential advantage of preserving the native liver that could be used for future gene therapy and also serves as a back-up should the graft fail. The aim of our study was to determine if ALT has the long-term potential to correct the underlying abnormality in propionic acidemia (PA). A retrospective analysis was performed on graft function, metabolic parameters and effects on development in a child who underwent ALT for PA at our institute. The clinical and biochemical parameters are near normal with no diet restrictions and with good graft survival. A normal growth and an acceptable neurological and psychomotor development were achieved in the child. ALT is feasible and provides adequate liver mass to prevent metabolic decompensation in PA.

Leg wound infections following cardiac surgery: a scoring system for assessment and management.

OBJECTIVE: Well-defined criteria are needed to provide guidance for the appropriate management of leg wounds following saphenous vein harvest in coronary artery bypass graft surgery (CABG). METHOD: A score named DISINFECT was devised to carefully define the variables to be considered for assessing saphenous vein harvest wounds. RESULTS: This preliminary study included 100 consecutive patients undergoing first-time isolated CABG requiring the saphenous vein as a conduit. Wounds were assessed and the points combined to create a daily score (D) according to the presence of increased C-reactive protein/white blood cells (I), surrounding tissue (S), quality of the incision (I), new skin (N), foreign material (F), exudate (E), positive cultures (C) and temperature (T). CONCLUSION: Taking into account the stages of wound healing, severity of infection and appropriate use of antibiotics, this method of wound management would improve the consistency with which leg wounds are managed, reduce hospital stays and increase resistance to hospital-acquired infection.

The use of the stroke risk index to predict neurological complications following coronary revascularisation on cardiopulmonary bypass.

The aim of this study was to investigate the validity of the stroke risk index (SRI) in identifying patients who develop a stroke following coronary artery bypass grafting on cardiopulmonary bypass. Retrospective data were analysed from 6846 patients who underwent adult coronary artery surgical procedures on cardiopulmonary bypass between 1997 and 2003. Patients were risk stratified pre-operatively using the SRI assessment model into low (Group A < or = 50), medium (Group B, 51-100) and high risk (Group C, > 100). A total of 217 patients (3.2%) with a mean age 65.9 +/- 11.7 years developed adverse neurological events following surgery. The CNS injury risk was 4% in Group A, 23% in Group B, and 8% in Group C. Pre-operatively, patients in Group B were older (p < 0.05), had a greater proportion of redo operations (OR 3.02; p < 0.001), diabetes mellitus (OR 2.51; p < 0.05), hypertension (OR 1.64; p < 0.01), myocardial infarction (OR 3.79; p < 0.05), ejection fraction < 30% (OR 1.46; p < 0.01) and absence of sinus rhythm (OR 2.52; p < 0.05) when compared with their counterparts. CNS events increased the patients' hospital stay by 40% in Groups A and C (p = 0.04) and 72% in Group B (p < 0.001). Only 31% returned home, compared with 85% of patients without cerebral complications (p < 0.001). These findings demonstrate that factors such as a pre-operative history of redo procedures, myocardial infarction, ejection fraction < 30% and absence of sinus rhythm play an important role in identifying the at-risk population. We conclude, therefore, that a further refinement and validation of the SRI is necessary.

Metabolic activation of 2-acetylaminofluorene is required for induction of multidrug resistance gene expression in rat liver cells.

P-Glycoprotein the multidrug resistance (mdr) efflux transporter is encoded by class 1 mdr genes (mdr1) in humans and rodent species. In rat liver and in rat hepatocytes in primary culture, expression of mdr1 genes can be induced with the carcinogenic aromatic amine 2-acetylaminofluorene (2-AAF). As a consequence, increased P-glycoprotein levels led to an accelerated efflux of vinblastine from the hepatocytes and to resistance towards vinblastine-mediated cytotoxicity. N-Hydroxylation, an obligatory initial step in the activation of 2-AAF into electrophilic DNA-binding metabolites is catalyzed predominantly by cytochrome P450 (CYP)1A2, an isozyme present in normal rat liver. In rat hepatocytes in primary culture, mdr1 induction with 2-AAF could be inhibited by addition of the CYP1A-inhibitor alpha-naphthoflavone, indicating the requirement for metabolic conversion of 2-AAF to act as an inducer of mdr1 gene expression. Both N-hydroxy-2-AAF and the mutagenic 2-AAF derivative N-acetoxy-2-AAF (AAAF) were more potent than 2-AAF as mdr1 inducers. mdr1 induction also decreased when deacetylation of AAAF, which strongly accelerates its conversion into a mutagen, was inhibited with paraoxon. Furthermore, rat liver epithelial cells stably transfected with mouse CYP1A2 showed inducibility of mdr1 gene expression with 2-AAF, whereas the parental cell line, which is devoid of CYP1A2 activity, did not. These findings indicate that electrophilic metabolites formed during 2-AAF or AAAF metabolism are responsible for mdr1 induction in rat hepatocytes. The increased mdr1 gene expression may reflect an adaptive cellular response to electrophiles which includes enhanced synthesis of P-glycoprotein aimed to protect the cell from further damage.

Recombinant mouse cytochromes P1-450 and P3-450: enzymatic characterization of the hemoprotein expressed in human cells infected with recombinant vaccinia virus.

We expressed mouse cytochrome P1-450 and P3-450 using recombinant vaccinia virus gene expression system in HeLa cells that were devoid of significant basal levels of P-450. HeLa cells were infected with the recombinant vaccinia virus containing either mouse cytochrome P1-450 or P3-450 cDNA, and the cell lysates were analyzed for the kinetics of P-450 enzyme activity and protein expression at the same time. 7-Ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities were measured as an expression of the cytochrome P-450 enzyme activities. Both cell lines began to express these enzyme activities as early as 12h after infection. The activities increased linearly up to the 24 h time point, and were kept for 36 h. Western immunoblot analysis showed that these cytochrome P-450 proteins were detected at 16 h and reached maximum quantity at 24 h after infection. These data showed a good correlation between cytochrome P-450 enzyme activity and protein concentration throughout the process of P-450 gene expression by vaccinia virus vector, suggesting a complete formation of cytochrome P-450 holoenzyme from the early stage of the protein expression.

Biotransformation of methylxanthines in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms. Allocation of metabolic pathways to isoforms and inhibitory effects of quinolones.

V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2, P450IIB1, human P450IA2, and rat liver epithelial cells expressing murine P450IA2 were used to allocate metabolic pathways of methylxanthines to specific isoforms and to test the suitability of such cell lines for investigations on drug interactions occurring at the cytochrome expressed. The cell lines were exposed to caffeine and/or theophylline and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. The results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity. Addition of the quinolone antibiotic agents pipemidic acid or pefloxacin, both known to inhibit caffeine metabolism in vivo and in human liver microsomes, reduced formation rates of all metabolites of caffeine in cells expressing rat and human P450IA2. Theophylline was mainly metabolized via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1.

Retrovirus-mediated stable expression of human CYP2A6 in mammalian cells.

To study the pharmacological and toxicological significance of the human cytochrome P450 isoform CYP2A6, we expressed it in mammalian cells by retrovirus-mediated gene transfer. The LXSN vector and PA317 packaging cells were used to create amphotropic recombinant retroviruses containing CYP2A6 cDNA. NIH 3T3 and HeLa cells were infected with these retroviruses and cell clones expressing CYP2A6 were selected. The integration of the CYP2A6 construct was verified by PCR analysis and northern blot analysis showed that a 5 kb mRNA containing the CYP2A6 was present in the cells. The integrated cDNA directed the expression of catalytically active CYP2A6 enzyme which has remained stable over numerous cell passages. No oxidation of several other P450 substrates was detected. The promutagen aflatoxin B1 was metabolized to intermediates binding to the host cell genomic DNA by the 3T3 2A6 cells. These cell lines are thus well suited for the study of the catalytic profile and the biological consequences of promutagen activation by the human CYP2A6 isoform.

Stable expression of human cytochrome P450IIE1 in mammalian cells: metabolic activation of nitrosodimethylamine and formation of adducts with cellular DNA.

To introduce cytochrome P450IIE1 DNA stably into the chromosomal DNA of mammalian cells, we constructed recombinant retroviruses containing the full-length complementary DNA for human cytochrome P450IIE1 and a selectable neo gene. Rat and mouse cells were infected with these viruses, and clones expressing the neo marker gene product were selected in G418-containing medium. Analysis of the DNA of the clones by Southern blotting showed that the viral DNA was integrated into the cellular DNA. Enzymatic analysis of the clones showed that the transduced DNA directed the expression of enzymatically active cytochrome P450IIE1. Treatment of the cells with the carcinogen [14C]-nitrosodimethylamine and analysis of the cellular DNA by CsCl equilibrium density gradients showed incorporation of the label into DNA, indicating the formation of covalent adducts with the cell DNA. Construction of recombinant cell lines constitutively expressing cytochrome P450IIE1 provides a permanent source for this enzyme and can aid in the analysis of its catalytic properties, such as the metabolic activation of chemical mutagens/carcinogens, and the consequent cytotoxic and genotoxic effects of these compounds.

Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms.

Primary steps in the metabolism of caffeine and theophylline are cleavage of methyl groups and/or hydroxylation at position 8, mediated by cytochromes P450. V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2 and P450IIBI and human P450IA2 and rat liver epithelial cells expressing murine P450IA2 were used to overcome problems arising in the proper allocation of metabolic pathways to specific isoforms by conventional techniques. These cell lines were exposed to caffeine and/or theophylline, and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P450IA2, resulting in the formation of four primary demethylated and hydroxylated metabolites. However, there were differences in the relative amounts of the metabolites. The human and the mouse P450IA2 isoforms predominantly mediated 3-demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3-demethylation and 1-demethylation of caffeine to a similar extent. Theophylline was metabolized mainly via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or human P450IA2, or rat P450IA1. These results support the hypothesis that caffeine plasma clearance is a specific in vivo probe for determining human P450IA2 activity.

Rat hypoxanthine phosphoribosyltransferase cDNA cloning and sequence analysis.

We have determined the nucleotide sequence of the rat hprt (hypoxanthine phosphoribosyltransferase; EC 2.4.2.8.) mRNA coding region and of adjacent, untranslated 5' and 3' mRNA, and we have designed an oligonucleotide primer pair for efficient PCR amplification of the rat hprt coding region. These sequence data and rat-specific primer pair will aid workers interested in coupling well-developed rat toxicologic and carcinogenicity bioassays with quantitative and molecular analyses of somatic mutation induction in rat cells in vivo and in vitro.

Cytochrome P4501A2 constitutively expressed from transduced DNA mediates metabolic activation and DNA-adduct formation of aromatic amine carcinogens in NIH 3T3 cells.

We transduced mouse cytochrome P4501A2 DNA into NIH 3T3 cells by retrovirus-mediated gene transfer. The capacity of the transduced cytochrome P4501A2 for metabolic activation and DNA-carcinogen adduct formation of aromatic amine carcinogens was investigated. Clones of NIH 3T3 cells that constitutively express cytochrome P4501A2 and controls were exposed to a prototype food-derived carcinogenic heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and an aromatic amine, 2-acetylaminofluorene (AAF), and their genomic DNAs were analyzed for adducts by 32P-postlabeling assays. Kinetic analysis of DNA-carcinogen adducts indicated that adduct formation was dependent on the level of the enzyme, the dose of carcinogen, and the duration of exposure. Addition of 7,8-benzoflavone, an inhibitor of P4501A2, blocked both the enzyme activity and DNA-adduct formation, indicating the specific role of P4501A2 in metabolic activation and adduct formation. Three specific IQ-DNA adducts were detected in cells expressing P4501A2. Fingerprints of the in situ DNA adducts were similar to those of the in vivo adducts in rodent hepatic DNA after the administration of IQ. A single AAF-DNA adduct was observed in cells exposed to AAF, but other minor adducts were also detected in vivo. These results show that cells expressing constitutive levels of single cytochrome P450s provide an excellent in situ model system for analyzing the catalytic specificity, metabolic activation, and genotoxicity of putative toxic, mutagenic, and carcinogenic substances.

Transduction of cytochrome P3-450 by retroviruses: constitutive expression of enzymatically active microsomal hemoprotein in animal cells.

Cell lines were established which produce replication-defective ecotropic and amphotropic host range recombinant retroviruses containing the cDNA for mouse cytochrome P3-450 as well as the bacterial Neo gene for G418 resistance. The G418-resistant clones derived from virus-infected cultures were analyzed for the expression, subcellular localization, and catalytic activities of the cytochrome P3-450. Southern blot analysis of the genomic DNAs indicates that the viral DNA was stably integrated into the cellular DNA. Western blot analysis of the proteins showed that the size of the constitutively expressed product was Mr 54,000, indistinguishable from the cytochrome P3-450 found in mouse liver microsomes. Spectral characterization of the P3-450 proteins indicates that the newly synthesized apoprotein incorporated heme and integrated into the microsomes. Enzymatic analysis of the cell homogenates in vitro and of the dividing cells in situ showed very high acetanilide hydroxylase activity and very low aryl hydrocarbon hydroxylase activity, a diagnostic feature of the cytochrome P3-450. The precise transmission of the recombinant retroviral sequences into the target cells and the exceptional fidelity of expression of the enzyme in cells will allow the analysis of an increasing number of cloned genes of cytochrome P-450s by defining the individual enzyme specificities, their physiological role in cells, and consequences of their functional expression, such as in toxicity, mutagenesis, and carcinogenesis.