Audit of initial use of the ultrasound-guided transversus abdominis plane block in children.

The extent of dermatomal block post transversus abdominis plane block is described in adults as T7-L1; other authors argue extent above T10 is infrequent (supra-iliac 20 ml injection). A paediatric guideline recommends this block for upper and lower abdominal surgery using 0.2 ml/kg. We aimed (through prospective audit) to document the multi-level block achieved with ultrasound-guided transversus abdominis plane block in children having abdominal surgery, during a departmental training period. Data included patient, anaesthetic and surgical details, transversus abdominis plane block characteristics (anterior supra-iliac injections) and dermatomal blockade to ice. Twenty-seven children received 38 blocks performed by 58% consultant and 42% trainee operators (90% novices): 16 unilateral/11 bilateral for umbilical (1), inguinal (13), laparoscopic (8) and laparotomy (5) surgery. Dermatomal assessment for 35 blocks (mean local anaesthetic volume 0.4 ml/kg [SD 0.2]) revealed the median blockade achieved was 3 dermatomes (interquartile range 3 to 4) involving T10 to L1 in 75% of patients. Eight blocks (six patients) also involved T8 and T9, following 0.31 to 0.81 ml/kg. One patient (3% of assessed blocks) had no block to ice at 60 minutes, but required no postoperative analgesia. Ultrasound-guided transversus abdominis plane blocks performed by supra-iliac approach and novice operators produced lower abdominal sensory blockade in children of usually 3 to 4 dermatomes, and should be offered for lower abdominal surgery only, as only 25% had upper abdominal block extension. The optimal local anaesthetic dose/volume, duration of effect and utility for these blocks in relation to peripheral and neuraxial blockade needs clarification.

Patients' attitudes towards increasing the offer of HIV testing in primary and secondary care.

The UK National Guidelines on HIV testing (2008) recommend routinely offering an HIV test to patients in certain clinical settings. We wished to investigate the acceptability of implementing these guidelines in a population with a low HIV prevalence. Patients accessing primary and secondary care were asked to circle one of the five responses to a series of statements regarding HIV testing. Of the 616 respondents, 579 (94%) stated they would be willing to be tested if presenting with a condition known to be associated with HIV. Four hundred and forty out of 616 (71%) stated they would be willing to be tested as part of their routine care, while 445/616 (72%) stated they would be willing to have the result in their main medical notes. Although the patients' responses were largely receptive to increased testing, we encountered notable negative attitudes to the project from professional and administrative staff. Resistance to increased HIV testing may be related to health-care workers rather than patients.

Evolution of the interferon alpha gene family in eutherian mammals.

Interferon alpha (IFNA) genes code for proteins with important signaling roles during the innate immune response. Phylogenetically, IFNA family members in eutherians (placental mammals) cluster together in a species-specific manner except for closely related species (i.e. Homo sapiens and Pan troglodytes) where gene-specific clustering is evident. Previous research has been unable to clarify whether gene conversion or recent gene duplication accounts for gene-specific clustering, partly because the similarity of members of the IFNA family within species has made it historically difficult to identify the exact composition of IFNA gene families. IFNA gene families were fully characterized in recently available genomes from Canis familiaris, Macaca mulatta, P. troglodytes and Rattus norvegicus, and combined with previously characterized IFNA gene families from H. sapiens and Mus musculus, for the analysis of both whole and partial gene conversion events using a variety of statistical methods. Gene conversion was inferred in every eutherian species analyzed and comparison of the IFNA gene family locus between primate species revealed independent gene duplication in M. mulatta. Thus, both gene conversion and gene duplication have shaped the evolution of the IFNA gene family in eutherian species. Scenarios may be envisaged whereby the increased production of a specific IFN-alpha protein would be beneficial against a particular pathogenic infection. Gene conversion, similar to duplication, provides a mechanism by which the protein product of a specific IFNA gene can be increased.

The impact of escalating regulatory requirements on the conduct of clinical research.

A discussion of the impact of escalating regulatory requirements is provided, from the point of view of the administration of the institutional review board of an academic institution, and from clinical investigators who conduct studies at that same institution. Current and anticipated future issues are discussed.

Mouse hepatitis virus replicase protein complexes are translocated to sites of M protein accumulation in the ERGIC at late times of infection.

The coronavirus mouse hepatitis virus (MHV) directs the synthesis of viral RNA on discrete membranous complexes that are distributed throughout the cell cytoplasm. These putative replication complexes are composed of intimately associated but biochemically distinct membrane populations, each of which contains proteins processed from the replicase (gene 1) polyprotein. Specifically, one membrane population contains the gene 1 proteins p65 and p1a-22, while the other contains the gene 1 proteins p28 and helicase, as well as the structural nucleocapsid (N) protein and newly synthesized viral RNA. In this study, immunofluorescence confocal microscopy was used to define the relationship of the membrane populations comprising the putative replication complexes at different times of infection in MHV-A59-infected delayed brain tumor cells. At 5.5 h postinfection (p.i.) the membranes containing N and helicase colocalized with the membranes containing p1a-22/p65 at foci distinct from sites of M accumulation. By 8 to 12 h p.i., however, the membranes containing helicase and N had a predominantly perinuclear distribution and colocalized with M. In contrast, the p1a-22/p65-containing membranes retained a peripheral, punctate distribution at all times of infection and did not colocalize with M. By late times of infection, helicase, N, and M each also colocalized with ERGIC p53, a specific marker for the endoplasmic reticulum-Golgi-intermediate compartment. These data demonstrated that the putative replication complexes separated into component membranes that relocalized during the course of infection. These results suggest that the membrane populations within the MHV replication complex serve distinct functions both in RNA synthesis and in delivery of replication products to sites of virus assembly.

The cell biology of coronavirus infection.

The ability to obtain entire volume data on infected cells will allow us to define much more accurately the interactions of viral proteins with host cell structures such as ER, Golgi, and cytoskeletal elements. In addition, the demonstrated ability to express viral proteins fused to fluorescent markers in in live cells will allow us to follow specific proteins or complexes during the course of infection and to determine if exogenously expressed proteins are able to target to sites of active viral replication. This in turn will allow new approaches to the study of viral and cellular protein-protein interactions, as methods to study the biology and pathogenesis of MHV infection at a cellular level. Finally, the approaches described here will allow us to define protein complementation of defective viruses at a cellular level, rather than being dependent on population measurements of RNA, protein, or progeny virus. By combining these approaches with available biochemical and molecular biological approaches and the emerging reverse genetic and recombinant genetic approaches, rapid progress in understanding the details of coronavirus-cell interactions should be possible.

Extracorporeal liver perfusion using human and pig livers for acute liver failure.

BACKGROUND: Patients with fulminant hepatic failure (FHF) often die awaiting liver transplantation. Extracorporeal liver perfusion (ECLP) has been proposed as a method of "bridging" such patients to transplantation. We report the largest experience to date of ECLP using human and porcine livers in patients with acute liver failure. METHODS: Patients with FHF unlikely to survive without liver transplantation were identified. ECLP was performed with human or porcine livers. Patients underwent continuous perfusion until liver transplantation or withdrawal of support. Two perfusion circuits were used: direct perfusion of patient blood through the extracorporeal liver and indirect perfusion with a plasma filter between the patient and the liver. FINDINGS: Fourteen patients were treated with 16 livers in 18 perfusion circuits. Nine patients were successfully "bridged" to transplantation. ECLP stabilized intracranial pressure (ICP) and cerebral perfusion pressure (CPP). Arterial ammonia levels fell from a median of 146 to 83 micromol/liter within 12 hr and this reduction was maintained at least 48 hr. Pig and human ECLP lowered ammonia levels equally. Serum bilirubin levels also fell from a median of 385 to 198 micromol/liter over the first 12 hr but the response was not sustained as well with porcine livers. There was no immunological benefit to using the the filtered perfusion circuit. INTERPRETATION: These data demonstrate that ECLP is safe and can provide metabolic support for comatose patients with fulminant hepatic failure for up to 5 days. While labor and resource intensive, this technology is available to centers caring for patients with acute liver failure and deserves wider evaluation and application.

Protection of human subjects in the United States: a short history.

The purpose of this article is to describe a few pivotal events that led to the development of federal regulations that now govern human subjects research in the United States.

The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis.

The coronavirus mouse hepatitis virus (MHV) translates its replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed to be involved in viral RNA synthesis. Antibodies directed against polypeptides encoded by open reading frame 1b were used to characterize the expression and processing of the MHV helicase and to define the relationship of helicase to the viral nucleocapsid protein (N) and to sites of viral RNA synthesis in MHV-infected cells. The antihelicase antibodies detected a 67-kDa protein in MHV-infected cells that was translated and processed throughout the virus life cycle. Processing of the 67-kDa helicase from polyprotein 1ab was abolished by E64d, a known inhibitor of the MHV 3C-like proteinase. When infected cells were probed for helicase by immunofluorescence laser confocal microscopy, the protein was detected in patterns that varied from punctate perinuclear complexes to large structures that occupied much of the cell cytoplasm. Dual-labeling studies of infected cells for helicase and bromo-UTP-labeled RNA demonstrated that the vast majority of helicase-containing complexes were active in viral RNA synthesis. Dual-labeling studies for helicase and the MHV N protein showed that the two proteins almost completely colocalized, indicating that N was associated with the helicase-containing complexes. This study demonstrates that the putative RNA helicase is closely associated with MHV RNA synthesis and suggests that complexes containing helicase, N, and new viral RNA are the viral replication complexes.

Determining when a clinical activity should be classified as research requiring Institutional Review Board Review.

The boundary between therapy and research may at times be difficult to distinguish, and it is, therefore, important for health care professionals to recognize when a clinical activity should be properly classified as research. Research may be subject to federal regulations which require advance review and approval by an Institutional Review Board (IRB) in order to protect the rights and welfare of patients who serve as human subjects. This paper will discuss the criteria health care professionals can use to distinguish between therapy, innovative therapy, and therapeutic or clinical research.

Research involving fetal drug therapy: ethical, legal, and practical considerations.

Fetal drug therapies have emerged as a promising avenue for the prevention or correction of disease during fetal or immediate postnatal life. Despite slow progress, several medications have been developed for in utero therapy of disorders which relate to fetal and neonatal pulmonary, cardiac, neurologic, and growth disorders. However, ethical and regulatory constraints require protection of the mother and fetus while causing no more than necessary additional risk. Appreciating these constraints will lead to the identification of pragmatic questions which should be answered before evaluating the efficacy and safety of a particular treatment or research proposal.