Committee experiences of using formal consensus in healthcare guidelines: a longitudinal qualitative study.

BACKGROUND: This feasibility study has the primary aim of capturing and comparing participant expectations and experiences of using a formal consensus method (FCM) and to explore whether these views change following participation within a guideline committee where FCM are used. METHODS: Twelve healthcare committee members and associated technical team members participated in semi-structured qualitative interviews before and after using FCM during guideline committee meetings. Interviews also focused on past experiences and expectations of informal consensus methods. RESULTS: Participants said formal consensus included a greater range of evidence. They described positive reactions and found it a useful way to encourage involvement by balancing group power dynamics. Group discussion time was identified as important to clarify ideas, supported by good group chairing. However, participants reported that undertaking FCM required additional resources and suggested targeting its use for low quality evidence, limited committee expertise, or where the evidence is controversial. CONCLUSIONS: FCM is an acceptable alternative to informal consensus methods that has qualities specifically helpful to healthcare guidelines such as encouraging participation, inclusivity of a broad range of evidence, and managing group dynamics. More research is required to better understand when using formal consensus is most appropriate and effective.

Uptake and utilization of directly acting antiviral medications for hepatitis C infection in U.S. veterans.

New drugs therapies have revolutionized the treatment of hepatitis C virus (HCV) infection. The objectives of this study were to evaluate uptake and utilization of boceprevir and telaprevir in the Department of Veterans Affairs (VA). We evaluated whether therapies conformed to response-guided protocols, whether they replaced standard interferon plus ribavirin treatment, and whether IL-28B was used to guide treatment. We performed an administrative data-based analysis of all patients receiving pharmacologic treatment for HCV in VA from October 2009 to July 2013. There were 12 737 new HCV prescriptions in VA during this time, with 5564 boceprevir or telaprevir prescriptions (44%) and 7173 prescriptions (56%) written for standard interferon plus ribavirin treatment. Prescriptions for the new treatments heavily favoured boceprevir vs telaprevir (83% vs 17%). Sixty-two percent (62%) of boceprevir-treated patients completed their minimum-specified protocol, while 69.2% of telaprevir-treated patients completed their minimum-specified protocol. From October 2010 to July 2012, 4090 patients had an IL-28B test; less than 16% of these tests guided subsequent HCV prescriptions. Uptake of boceprevir and telaprevir was rapid; the number of patients initiating treatment approximately doubled in the period after their introduction. While new prescriptions favor boceprevir or telaprevir over standard interferon plus ribavirin therapy, there appears to still be a strong role of interferon plus ribavirin in treating HCV patients. This work can inform our understanding of how other new effective HCV therapies will be used, their diffusion, and the timing of their diffusion in actual clinical practice.

Mutations in the T (brachyury) gene cause a novel syndrome consisting of sacral agenesis, abnormal ossification of the vertebral bodies and a persistent notochordal canal.

BACKGROUND: The T gene (brachyury gene) is the founding member of the T-box family of transcription factors and is vital for the formation and differentiation of the mesoderm and the axial development of all vertebrates. RESULTS: We report here on four patients from three consanguineous families exhibiting sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies, and the identification and characterisation of their underlying genetic defect. Given the consanguineous nature and the similarity of the phenotypes between the three families, we performed homozygosity mapping and identified a common 4.1 Mb homozygous region on chromosome 6q27, containing T, brachyury homologue (mouse) or T. Sequencing of T in the affected individuals led to the identification of a homozygous missense mutation, p.H171R, in the highly conserved T-box. The homozygous mutation results in diminished DNA binding, increased cell growth, and interferes with the normal expression of genes involved in ossification, notochord maintenance and axial mesoderm development. CONCLUSIONS: We have identified a shared homozygous mutation in three families in T and linked it to a novel syndrome consisting of sacral agenesis, a persistent notochordal canal and abnormal ossification of the vertebral bodies. We suggest that screening for the ossification of the vertebrae is warranted in patients with sacral agenesis to evaluate the possible causal involvement of T.

Sox4 mediates Tbx3 transcriptional regulation of the gap junction protein Cx43.

Tbx3, a T-box transcription factor, regulates key steps in development of the heart and other organ systems. Here, we identify Sox4 as an interacting partner of Tbx3. Pull-down and nuclear retention assays verify this interaction and in situ hybridization reveals Tbx3 and Sox4 to co-localize extensively in the embryo including the atrioventricular and outflow tract cushion mesenchyme and a small area of interventricular myocardium. Tbx3, SOX4, and SOX2 ChIP data, identify a region in intron 1 of Gja1 bound by all tree proteins and subsequent ChIP experiments verify that this sequence is bound, in vivo, in the developing heart. In a luciferase reporter assay, this element displays a synergistic antagonistic response to co-transfection of Tbx3 and Sox4 and in vivo, in zebrafish, drives expression of a reporter in the heart, confirming its function as a cardiac enhancer. Mechanistically, we postulate that Sox4 is a mediator of Tbx3 transcriptional activity.

Toward a global foot and mouth disease vaccine bank network.

A network of foot and mouth (FMD) vaccine banks has been initiated with the support of vaccine bank managers and technical advisors that participated in a workshop held at the Institute for Animal Health, Pirbright, in the United Kingdom in April 2006. Terms of Reference that provide guidance for coordinated activities are under consultation. Practical and economic benefits can be realised from collaboration, which will be achieved through mutually acceptable mechanisms for the exchange of information and materials relevant to vaccine banks and their management. If administrative and technical hurdles can be overcome, the network has the potential to contribute significantly to the improved control of FMD worldwide. A 'global' and interactive vaccine bank association could be created by agreeing a system of resource sharing that could orchestrate additional emergency cover with vaccine or antigen from the reserves of network members.

Nuclear magnetic resonance imaging of the vitreous body.

Imaging with proton nuclear magnetic resonance is a valuable new tool for studying the vitreous body of the eye. It is particularly suited for the detection of vitreal liquefaction and intraocular hemorrhage because of the dependence of the signal on the physical environment of water. Conversely, the vitreous body provides a new model for studying changes in proton relaxation times of protein solutions in biological systems.

Evaluation of a monoclonal antibody-based approach for the selection of foot-and-mouth disease (FMD) vaccine strains.

Foot-and-mouth disease (FMD) virus exists as seven serotypes within which are numerous variants necessitating careful selection of vaccine strains. Currently, a serological assay system based on the use of polyclonal vaccine antisera is widely used for this selection. However, inherent variability in the matching antisera used makes the tests poorly reproducible and difficult to interpret. In this study, we have explored the possibility of replacing or supplementing the polyclonal antibody (PAb)-based method with one based on use of monoclonal antibodies (MAb). Panels of MAbs raised against two serotype O vaccine strains were examined for reactivity with 22 field viruses, isolated over a 10-year period between 1991 and 2001. Antigenic site 2 was found to comprise more than one epitope. The sequence variation in capsid protein VP2 harbouring antigenic site 2 was analysed and the amino acid residues at positions 79 and 134 appeared to greatly influence the binding of site 2 MAbs. Prediction of antigenic match based on MAb reactivity did not correlate closely with the results of a PAb-based "gold-standard" method and it was concluded that a wider panel of MAbs are needed that recognise all protective epitopes present on the surface of FMD virus together with a better understanding of those epitopes which are important in conferring protection.

Osmotic blood-brain barrier disruption. Computerized tomographic monitoring of chemotherapeutic agent delivery.

The present study describes a canine model of transient reversible blood-brain barrier disruption with hyperosmolar mannitol infusion into the internal carotid artery. Studies in this model show that osmotic blood-brain barrier disruption before intracarotid infusion of methotrexate results in markedly elevated (therapeutic) levels of drug in the ipsilateral cerebral hemisphere. Levels in the cerebrospinal fluid correlate poorly and inconsistently with brain levels. Computerized tomograms in this canine model provide a noninvasive monitor of the degree, time-course, and localization of osmotic blood-brain barrier disruption.

Saccharomyces cerevisiae PTS1 receptor Pex5p interacts with the SH3 domain of the peroxisomal membrane protein Pex13p in an unconventional, non-PXXP-related manner.

A number of peroxisome-associated proteins have been described that are involved in the import of proteins into peroxisomes, among which is the receptor for peroxisomal targeting signal 1 (PTS1) proteins Pex5p, the integral membrane protein Pex13p, which contains an Src homology 3 (SH3) domain, and the peripheral membrane protein Pex14p. In the yeast Saccharomyces cerevisiae, both Pex5p and Pex14p are able to bind Pex13p via its SH3 domain. Pex14p contains the classical SH3 binding motif PXXP, whereas this sequence is absent in Pex5p. Mutation of the conserved tryptophan in the PXXP binding pocket of Pex13-SH3 abolished interaction with Pex14p, but did not affect interaction with Pex5p, suggesting that Pex14p is the classical SH3 domain ligand and that Pex5p binds the SH3 domain in an alternative way. To identify the SH3 binding site in Pex5p, we screened a randomly mutagenized PEX5 library for loss of interaction with Pex13-SH3. Such mutations were all located in a small region in the N-terminal half of Pex5p. One of the altered residues (F208) was part of the sequence W(204)XXQF(208), that is conserved between Pex5 proteins of different species. Site-directed mutagenesis of Trp204 confirmed the essential role of this motif in recognition of the SH3 domain. The Pex5p mutants could only partially restore PTS1-protein import in pex5Delta cells in vivo. In vitro binding studies showed that these Pex5p mutants failed to interact with Pex13-SH3 in the absence of Pex14p, but regained their ability to bind in the presence of Pex14p, suggesting the formation of a heterotrimeric complex consisting of Pex5p, Pex14p, and Pex13-SH3. In vivo, these Pex5p mutants, like wild-type Pex5p, were still found to be associated with peroxisomes. Taken together, this indicates that in the absence of Pex13-SH3 interaction, other protein(s) is able to bind Pex5p at the peroxisome; Pex14p is a likely candidate for this function.

Porcine gammadelta T cells: possible roles on the innate and adaptive immune responses following virus infection.

gammadelta T cells recognise different types of antigen in alternative ways to alphabeta T cells, and thus appear to play a complementary role in the immune response. However, unlike alphabeta T cells, the role or function of gammadelta T cells is still unclear. As pigs possess a high proportion of circulating gammadelta T cells, they are suitable large animal model to study gammadelta T cell functions. This as yet has not been fully exploited, leaving porcine gammadelta T cell biology and its role in immunity in its infancy. Foot-and-mouth disease (FMD) high potency "emergency" vaccines are able to induce early protection from challenge and it has been suggested that, in part, there is some involvement of innate immune responses. The antigen component of the vaccine is able to stimulate purified naive pig gammadelta T cells and induce the mRNA of various cytokines and chemokines. This observation suggests that gammadelta T cells probably contribute to the early phase of the immune responses to FMD vaccination, and perhaps infection. A subset of these circulating gammadelta T cells display a phenotype similar to professional antigen presenting cells and are able to take up and present soluble antigen to CD4(+) T cells in a direct cell-cell interaction via MHC class II. This direct interaction between gammadelta T cells and CD4(+) T cells is likely to have a significant influence on the out come of the adaptive immune response.

Pharmacology and neurotoxicity of cis-diamminedichloroplatinum, bleomycin, 5-fluorouracil, and cyclophosphamide administration following osmotic blood-brain barrier modification.

cis-Platinum, bleomycin, 5-fluorouracil, and cyclophosphamide were administered to rodent and canine models of osmotic blood-brain barrier modification to evaluate the relationship between tissue drug concentration in brain and the physiological and neuropathological effects of the drug. The toxicity studies were carried out in the canine, and the pharmacological studies were carried out in the rodent. Even without the osmotic procedure, intracarotid cis-platinum, in contrast to the other drugs, produced modification of the blood-brain barrier with resultant severe neurotoxicity. Osmotic blood-brain barrier modification was used to increase delivery of bleomycin and 5-fluorouracil to the ipsilateral brain region, but the increased delivery was associated with evident neurotoxicity. Cyclophosphamide administration in association with osmotic blood-brain barrier opening did not cause significant neural toxicity. These studies indicate that some chemotherapeutic agents given in association with osmotic blood-brain barrier opening can result in neurotoxicity. The corollary of the known limited neurotoxicity when these chemotherapeutic agents are used in a conventional manner appears to be due to the presence of the blood-brain barrier.

Pharmacology and toxicity of intracarotid adriamycin administration following osmotic blood-brain barrier modification.

The effect of reversible blood-brain barrier modification on the delivery of Adriamycin to the brain was studied in a rodent and canine model. Pharmacokinetic and physiological studies were done in these animals after a wide range of doses of Adriamycin (0.1 to 1.0 mg/kg) were administered into the carotid artery following osmotic barrier modification with mannitol. In the absence of barrier modification, no immunoreactive Adriamycin was detected in the cerebrum; whereas, following barrier modification, up to 4.5 micrograms of drug and/or metabolites per g of brain were found. Optimum tissue levels of Adriamycin and metabolites were achieved following barrier modification when the drug was administered by either bolus or slow continuous (15-min) infusion. Immunoreactive drug was identified in brain for up to 6 hr after administration. Significant functional neurotoxicity occurred at all dose levels, even at 0.1 mg/kg, a level at which Adriamycin concentration in the brain was below the level of detectability. Neuropathological examination revealed the presence of necrosis and hemorrhagic infarcts. Thus, these pharmacological and toxicity studies suggest that Adriamycin (or its metabolites) may produce significant clinical neurotoxicity when even small amounts penetrate the blood-brain barrier.

Delivery of melanoma-associated immunoglobulin monoclonal antibody and Fab fragments to normal brain utilizing osmotic blood-brain barrier disruption.

Iodinated monoclonal antibodies (IgG 96.5 and two monomeric Fab fragments 96.5 and 48.7) to melanoma-associated antigens were administered after osmotic blood-brain barrier (BBB) opening in normal rats. Osmotic BBB disruption significantly (P less than 0.0001) increased monoclonal antibody delivery to the brain. Following BBB opening and intracarotid administration, there was no difference in the disrupted brain concentration integral area under the curve between Fab and IgG over the 72-h experimental period. However, Fab concentration in the disrupted brain was initially higher than IgG, and the clearance was more rapid (P less than 0.0001), decreasing 50% by approximately 4.5 h compared to 25.5 h for IgG. Plasma clearance was also more rapid for the Fab than IgG. The levels decreased 50% by 1.5 h for Fab and 15 h for IgG. The route and timing of antibody infusion had a significant effect on delivery to the disrupted brain with the Fab fragments but not with the intact IgG. Antibody recovered from disrupted brain retained its immunological reactivity as measured by a cell binding assay for at least 24 h. IgG and Fab delivery to the ipsilateral brain after BBB disruption increased (P less than 0.001) with increasing dose over a more than 3-log dose range. These data provide information applicable to the therapeutic use of monoclonal antibodies in brain tumor treatment.

Growth of human lung tumor in the brain of the nude rat as a model to evaluate antitumor agent delivery across the blood-brain barrier.

We have developed a brain tumor model in the nude rat utilizing NCI-N417D human small cell carcinoma of the lung grown both intracerebrally and s.c. The median latency period from the time of intracerebral tumor inoculation to the onset of neurological symptoms is 13 days with an intracerebral tumor take rate of 91% (29 of 32). The median survival is 13 days, and all animals were dead by Day 26. The tumor is discrete, well circumscribed, with occasional leptomeningeal spread and with minimal evidence of surrounding cerebral edema. Intracerebrally, this tumor is usually impermeable to Evan's blue:albumin (Mr 68,500) but not fluorescein (Mr 376). Although variable, the intracerebral tumor is less permeable to methotrexate than is the same tumor grown s.c. in the same animal (P less than 0.005). The intraarterial and i.v. routes of methotrexate administration in the presence and absence of blood-brain barrier opening were evaluated. Drug delivery to the intracerebral tumor and ipsilateral brain was significantly (P less than 0.025) greater when the methotrexate was given intraarterially and was significantly (P less than 0.0025) increased after osmotic blood-brain barrier opening. After barrier opening, methotrexate concentration was enhanced 3- to 4-fold in tumor and 10- to 20-fold in brain around tumor. Thus, the nude rat provides a model to investigate the biology and therapeutic responsiveness of human small cell carcinoma of the lung grown intracerebrally where it develops a blood-tumor barrier similar to that seen in humans. This model further provides the unique opportunity to investigate the role of osmotic blood-brain barrier opening in the treatment of a tumor which is sensitive to chemotherapeutic agents and for which tumor-specific monoclonal antibodies are available.

Professionalism of physicians at a major teaching hospital during the Fukushima nuclear disaster.

It poses a serious problem if physicians leave a hospital without having a replacement or without permission. A huge earthquake followed by a devastating tsunami seriously damaged the Fukushima-Daiichi nuclear power plant. This disaster overwhelmed a major teaching hospital in the local area and many hospital employees, including some resident physicians, left the premises. Since the threat of severe radiation exposure poses a potentially greater lifetime risk to younger individuals, letting the young resident physicians leave the hospital was not only allowed, it was actually recommended by many attending physicians and hospital administrators. The hospital administrator was required to make the difficult decision of whether to make all efforts to provide the highest level of medical care, including keeping all of the physicians on the premises, or to evacuate the resident physicians in order to preserve their health and their potential future contributions to healthcare. Consideration and compassion needed to be provided to all people, regardless of the reason they wanted to leave. From an ethical perspective, the roles of performance under these complex circumstances should be understood and embraced by us as individuals, professionals, supervisors and society as a whole.

Functional identity of catalytic subunits of acetylcholinesterase.

11 S acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the electric eel Electrophorus electricus essentially consists of four catalytic subunits which appear to be identical structurally but to be assembled with slight asymmetry. During isolation and storage of the enzyme, proteolysis cleaves a portion of the subunits into major fragments containing the active site and minor fragments containing no active sites without change in the enzyme molecular weight. A previous report (Gentinetta, R. and Brodbeck, U. (1976) Biochim. Biophys. Acta 438 437--448) indicated that the intact and the fragmented subunits reacted with diisopropylfluorophosphate at different rates and that the reaction rate in the presence of excess phosphorylating agent was not strictly first order. Those findings could not be reproduced in this report. Intact and fragmented subunits were observed to react at the same rate with diisopropylfluorophosphate. In addition, the overall reaction kinetics both of 11 S and 18 S plus 14 S acetylcholinesterase were found to be strictly first order in the presence of an excess of diisopropylfluorophosphate throughout the course of reaction. These results are consistent with several previous reports that only one type of active site can be detected in acetylcholinesterase. The proteolysis which fragments a portion of the catalytic subunit has no apparent effect on the catalytic properties of the enzyme.

The regulation of the vanadium chloroperoxidase from Curvularia inaequalis.

The effects of carbon and nitrogen source on the regulation of the vanadium chloroperoxidase secreted by the fungus Curnularia inaequalis were investigated. The addition of glucose showed a repressing effect on both the observed messenger RNA level and the measured enzyme activities, whereas the addition of glutamate as nitrogen source and the addition of both glutamate and glycerol had no effect. Addition of vanadate had no effect on the level of mRNA. Eight hundred base pairs of the upstream promoter region of vCPO were sequenced and various features of interest are highlighted. Closer inspection of the mycelium revealed that once secreted, vCPO probably remains tightly associated with the hyphae in two forms, one of which may be a proform of the enzyme. A possible cleavage event at the C-terminus may lower its potential for hyphal association and permit its disassociation into the growth medium. A putative role for the vanadium chloroperoxidase is put forward.

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.

The effect of oxidation on sorbitol pathway kinetics.

The rapid conversion of glucose to sorbitol by aldose reductase and the consequent hyperosmolarity of the cytoplasm has been shown to be the primary cause of the so-called "sugar" or "osmotic" cataract in many animal lenses. It is not as clear, however, that hyperosmolarity is the principal factor in the etiology of cataracts in human diabetic subjects. In fact, the comparatively low activity of aldose reductase in the human lens as compared with several animal lenses, and the osmotically insignificant levels of sorbitol pathway products (sorbitol and fructose), suggest that hyperosmolarity, per se, may not be as important a factor in human cataract formation as it is in animals. We present evidence that the flux of glucose and sorbitol through the rat lens is markedly reduced by oxidative stress (0.1 mM H2O2). Sorbitol accumulation is reduced by 114%, sorbitol turnover is reduced by 78%, sorbitol production is reduced by 90%, fructose accumulation is reduced by 60%, and fructose turnover is reduced by 76% in the presence of 36 mM glucose. H2O2 does not affect glucose turnover, the glucose rate constant, or the ATP level significantly at 36 mM glucose, but at 5.5 mM glucose, 0.2 mM H2O2 leads to a rapid loss of ATP that can be prevented by 0.04 mM sorbinil, an aldose reductase inhibitor. These results suggest that inhibition of aldose reductase by sorbinil renders rat lenses better able to cope with oxidative stress. In the absence of an aldose reductase inhibitor, elevating ambient glucose may render a lens less able to scavenge oxidants by diverting NADPH into sorbitol production.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct measurement of polyol pathway activity in the ocular lens.

A method to measure the polyol pathway metabolic flux in the intact rabbit lens by 13C nuclear magnetic resonance spectroscopy is described. In the lens exposed to 35.5 mM glucose, the polyol pathway accounts for 1/3 of the total glucose turnover. The high metabolic activity of the pathway suggests a significant alteration in the reduced to oxidized pyridine nucleotide ratio in the lens exposed to high glucose.