Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal pouch.

The thymus and parathyroids originate from a common primordium that develops from the third pharyngeal pouch in mice and humans. The molecular mechanism that specifies this primordium into distinct organ domains is not known. The Gcm2 and Foxn1 transcription factors are required for development of the parathyroid and thymus respectively, and are attractive candidates for this role. However, their embryonic expression patterns during pharyngeal pouch development and early thymus and parathyroid organogenesis have not been described. Here we report that Gcm2 is expressed specifically in the developing second and third pharyngeal pouches at E9.5, and is further confined to a small domain of the third pouch endoderm by E10.5. In contrast, Foxn1 is not expressed until after the common primordium is formed, beginning at E11.25. Our results show that Gcm2 and Foxn1 expression mark two complementary domains that prefigure parathyroid and thymus regions within the common primordium before morphological distinctions are present.

Johanson-Blizzard syndrome: clinical and pathological findings in 2 sibs.

Here we report on 2 sibs with the Johanson-Blizzard syndrome (JBS). The first child died in the neonatal period, the autopsy showing presence of pancreatic ducts and islets surrounded by connective tissue and a total absence of acini. Morphologic changes suggested dysplasia leading to developmental failure, but early acinar destruction could not be ruled out. The second child had a constellation of abnormalities consistent with JBS, was managed surgically, and is maintained on replacement for his pancreatic enzyme and thyroid hypofunction. At 10 years, he is in a school for the hearing-impaired and is performing appropriately for his age.

Diagnosing shoulder dislocations: time for a change of view.

Posterior shoulder dislocations are uncommon, with frequent delays in the diagnosis. Three missed posterior dislocations within our hospital caused us to review the standard radiographs taken and the knowledge of this condition. A total of 40 radiographers and 40 casualty officers were surveyed. Of the radiographers, 63% felt it unnecessary to perform two views, they complained that laterals were difficult to obtain because of patient distress. All the radiographers surveyed knew of alternative views, but would not perform them unless specifically requested. Casualty officers claimed always to request two views, but did not in 75% of cases. Only 20% were aware of alternative views, all would accept one view for exclusion of a dislocation and none were aware of the radiographic signs associated with a posterior dislocation. Increased education and a change of view would assist in decreasing the rate of missed diagnoses.

Marketing: can there ever be a real exchange of values in the Health Service?

Offers many new ideas in the field of marketing, and argues that marketing is a complex, strategic thought process, based on an exchange of customer-related values. A new model of need and want is offered, and developed into a strategic framework which considers the functional and perceptual nature of what is loosely termed "the delivery of care". Argues that, in marketing terms, strategic focus in the NHS can only be achieved by getting close to the mind of the customer.

Business planning health-check questionnaire: a survey of first, second, third and fourth wave NHS Trusts.

This paper reports the results of primary research which was carried out in July 1995 with respect to business planning within first, second, third and fourth wave National Health Service (NHS) Trusts. The purpose of the research was to examine current practice in these Trusts in three areas--namely, the levels of responsibility for business planning in general, the business planning processes applied by these Trusts, and the tools and techniques used by business planning managers in the compilation of business plans. The research, based on a 37.5% response rate, concludes that, as a general rule, business planning in first, second, third and fourth wave NHS Trusts tends to be a board-level activity, where senior managers have a job title which reflects this function. Secondly, the research shows that by far the greatest challenge for Trusts lies in the external marketplace. In areas such as patient needs forecasting, competitive (Trust) intelligence, purchaser and general practice fundholder requirements, data are difficult to acquire. Finally, the evidence suggests that there is a significant gap between what is regarded as business planning practice in the NHS and what is actually applied as best practice. The report concludes that business planning in the NHS Trusts sampled appears to be an art rather than a science, and that many assumptions made by business planning managers are founded on qualitative information rather than on specific, measurable data derived from the external and internal market.

National toxicovigilance for pesticide exposures resulting in health care contact - An example from the UK's National Poisons Information Service.

BACKGROUND: Although there are extensive systems in place for pharmacovigilance, similar systems for detecting adverse health effects relating to pesticide exposure are rare. In 2004, the National Poisons Information Service (NPIS) pesticide surveillance study was implemented to identify cases requiring health care contact in the UK. This report describes the epidemiology of pesticide exposures reported to poison centres in the UK over a 9-year period. METHODS: Data on exposures were gathered through monitoring access to the NPIS's online clinical toxicology database TOXBASE(®) and through monitoring calls to the four NPIS units (Edinburgh, Cardiff, Newcastle and Birmingham). Severity was judged by both caller and NPIS staff. RESULTS: During the 9 years, 34,092 enquiries concerning pesticides were recorded; 7,804 cases of pesticide exposure were derived from these enquiries. Exposures were predominantly unintentional and acute (6,789; 87.0%); 217 (2.8%) and 755 (9.7%) were chronic unintentional and acute deliberate self-harm exposures, respectively. The majority of cases occurred in children, especially the 0-4 year age group The minimum incidence of pesticide exposure requiring health care contact was 2.0 cases/100,000 population per year. Reported numbers were 6- to 25-fold greater than those picked up through other UK pesticide toxicovigilance schemes. There were 81 cases of severe toxicity and 38 cases of fatal exposure. Deliberate self-harm accounted for 62.3% of severe cases and 79% of deaths. Aluminium phosphide, paraquat, diquat and glyphosate were responsible for most severe and fatal cases. CONCLUSIONS: The data gathered from this pesticide surveillance study indicate that poison centre resources can usefully monitor pesticide exposures resulting in health care contact in the UK. The NPIS may usefully be one component of the UK's response to European legislation requiring surveillance of complications resulting from pesticide use.

Business planning: can the health service move from strategy into action?

Advances the case for the use of one particular business planning technique within a National Health Service Trust. At the present time, NHS trusts are required to write strategic direction statements. Evidence suggests that these documents provide an accurate account of past performance and present position of the trust, but do not express the future position intended to be achieved. These documents also tend to be lengthy and lack strategic focus, which means that they are not helpful to managers who want clear organizational goals and objectives to which to work. Attempts to address the difficulties associated with determining how existing skills and resources can be used as the platform for future growth strategies by using the Ansoff Matrix and SWOT Analysis planning tools, given the external changes in the marketplace. Also attempts to shed light on some of the important links between busines strategy and management development by extending planning theory into practice.

Measurement of nitric oxide concentration in human skin in vivo using dermal microdialysis.

Using microdialysis, we measured nitric oxide (NO) levels in healthy human skin, in vivo, before and during the local inflammatory response to histamine. Basal dialysate NO concentration, assayed using an amperometric technique, was 0.49+/-0.06 microM (mean+/-S.E.M., 21 probes, 14 subjects). Histamine injection produced transient increases in NO concentration within both the weal and flare which was blocked by the NO synthase inhibitor, NG-nitro-L-arginine-methyl ester (L-NAME). Dialysate NO concentration also increased following transdermal delivery of the nitrosovasodilator, glyceryl trinitrate. Thus, using microdialysis, it is possible to quantify NO production in human skin in vivo and study its modulation during the acute inflammatory response.

Effects of H1 antagonists on the cutaneous vascular response to histamine and bradykinin: a study using scanning laser Doppler imaging.

Histamine plays an important part in the cutaneous weal and flare response which underlies many allergic skin conditions. It has a direct effect on the local vasculature to promote vasodilatation and increase microvascular permeability and may also initiate the more widely spread neurogenic flare. Quantification of these responses and studies of the mediator mechanisms underlying them have been limited by the lack of appropriate techniques to investigate them. To address this we have used two relatively new techniques, scanning laser Doppler imaging (LDI) and dermal microdialysis to measure changes in skin blood flow and the release of histamine within the weal and flare, following intradermal injection of histamine or bradykinin. These measurements have been made both in the absence and presence of the H1 receptor blockers cetirizine and loratadine. Scanning LDI of the inflammatory response revealed marked differences in both the development and steady state responses to the intradermal injection of histamine (1-3 mumol/L) and bradykinin (1 mumol/L). The development of the flare and the weal response to both histamine and bradykinin was significantly reduced by cetirizine but not by loratadine. The histamine-induced flare area fell by 57 +/- 4% (mean +/- SEM, n = 10, P < 0.001) after cetirizine and the area of the weal fell by 73 +/- 11% (P < 0.009). Bradykinin-induced inflammatory responses were similarly reduced by cetirizine, the weal by 60 +/- 16% (P < 0.02) and the flare by 61 +/- 4% (P < 0.005). Measurement of histamine concentration in skin using microdialysis, in six subjects, confirmed that histamine levels rose in the dialysate collected from the weal to 310 +/- 16 nmol/L following injection of histamine. Histamine levels also rose following bradykinin injection in some subjects (mean 147 +/- 46 nmol/L, range 18-336). Little increase in histamine concentration was seen in the dialysate from the flare following injection of either histamine or bradykinin. The histamine concentration in dialysate from unprovoked skin was 4.19 +/- 0.75 nmol/L. These data reveal differences in the dermal responses to different mediators when assessed using scanning LDI. They confirm that histamine is released within the weal but not the flare response to the intradermal injection of both histamine and bradykinin and that its effects on the local vasculature to cause the oedematous weal and the axon reflex-mediated flare are significantly attenuated by the H1 antagonist cetirizine and to a lesser extent by the H1 antagonist loratadine.

The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures.

The use of immunomagnetic separation (IMS) techniques has been reported to reduce the total test time, and improve the sensitivity, of microbiological tests done on foods. This approach is being adopted in epidemiological investigations into suspected foodborne outbreaks of Escherichia coli O157 infection and has gained acceptance by public health laboratories and the food industry. This study demonstrated the ability of a commercially available IMS procedure, Dynabeads anti-E. coli O157, to enable detection of a few cells of E. coli O157 in 25 g of inoculated minced beef, giving results 1 d earlier than a cultural analysis of similar sensitivity. With correct choice of enrichment broths, IMS may increase isolation rate of E. coli O157 compared to that obtained using conventional cultural methods. It is suggested that this may be due to an increase in relative concentration of E. coli O157 compared with the background microflora present in minced beef, which may reduce reliability of non-IMS detection procedures by masking or mimicking target cells on selective/differential solid media. The use of an immunoassay incorporating an IMS step, EHEC-Tek (Organon-Teknika), enabled detection of a few cells of E. coli O157 in 25 g of minced beef. Comparison of the IMS-ELISA with a standard ELISA procedure (Tecra) indicated the sensitivity of the latter system to be greater, perhaps resulting in the higher isolation rate. The use of a method to reliability isolate and detect extremely low levels of E. coli O157 in a food is necessary to aid reduction in the incidence of this most serious of foodborne pathogens.

Evaluation of methods for the isolation and detection of Escherichia coli O157 in minced beef.

This study has evaluated enrichment and detection procedures for the isolation and detection of Escherichia coli O157 inoculated into minced beef. The use of a 24 h enrichment in modified EC broth containing novobiocin allowed low numbers of contaminating cells to multiply to levels detectable on culture media and by ELISA test kits. Total analysis time was reduced by the use of the Dynabead immunomagnetic separation system. The use of the Petrifilm Test Kit-HEC for E. coli O157:H7 and Organon Teknika EHEC-TEK system detected low numbers of contaminating cells following enrichment and reduced analysis time by 1 d. The incorporation of cefixime and tellurite into Sorbitol MacConkey Agar increased the rate and ease of isolation of E. coli O157 and its use is therefore recommended.

Use of pyrrolidonyl peptidase to distinguish Citrobacter from Salmonella.

In the routine testing of foods for Salmonella, Citrobacter and other members of the Enterobacteriaceae often produce colonies which are almost indistinguishable from Salmonella on commonly used selective agars. Biochemical confirmation of such colonies can be expensive and time-consuming. It has been suggested that the enzyme pyrrolidonyl peptidase (PYRase) could be used as a rapid test to distinguish Citrobacter colonies (PYRase-positive) from Salmonella (PYRase-negative). Pure cultures of Salmonella, Citrobacter and other Enterobacteriaceae were tested for PYRase activity; all strains of Salmonella tested were PYRase-negative, and all Citrobacter tested were PYRase-positive. Inoculated and naturally contaminated food samples were tested for the presence of Salmonella by a standard cultural method. A PYR test was used to test Salmonella-like colonies isolated on selective agar and potentially, eliminate PYR-positive isolates from further biochemical testing. The test was able to screen out 6% of colonies selected from samples inoculated with Salmonella, and 43% of colonies selected from uninoculated samples.

Rapid and definitive detection of Salmonella in foods by PCR.

The BAX system for screening Salmonella is one of the first commercial PCR-based systems for the detection of food-borne pathogens. It is able to give a confirmed result within 28 h. There was 98.6% and 95.8% agreement between the BAX system and conventional cultural analysis for the detection of Salmonella in artificially inoculated and uninoculated food samples, respectively. In both cases, the BAX system generated more positive detections than cultural analysis. The speed of assay, case of use and high specificity and sensitivity of the BAX system for the detection of food-borne Salmonella make it an attractive method for routine food microbiology laboratories.

The use of bacteriophage-based systems for the separation and concentration of Salmonella.

Techniques for the separation/concentration of micro-organisms from background food matrices can be applied to increase the speed of analysis and ease of isolation and detection of target micro-organisms. One recent example of such a technique is the immunomagnetic separation (IMS) procedure that has been used for the separation of specific micro-organisms from foods. This paper describes the use of a novel biosorbent consisting of a Salmonella-specific bacteriophage (phage) immobilized to a solid phase that was used for the separation and concentration of Salmonella from food materials. This work has shown that a Salmonella-specific phage-based biosorbent could remove Salmonella from culture fluid and separate Salmonella from suspensions of other Enterobacteriaceae. The ease of production of phage, high affinity of phage-cell interaction and the ability of phage to infect host cells in heterogeneous environments indicates the potential of such a biosorbent as the basis for a reliable separation system in food microbiological analysis.

Production and characterization of monoclonal antibodies specific for human mast cell tryptase.

Human mast cell tryptase was purified from lung tissue by high salt extraction, ammonium sulphate precipitation, octyl Sepharose and heparin-agarose chromatography. The tryptase isolated was a tetramer with a molecular weight of 132 kD on gel filtration, and on SDS-polyacrylamide gel electrophoresis was reduced to a single diffuse band with a mean molecular weight of 32.5 kD. Purified tryptase catalysed the cleavage of the tryptic substrates tosyl L-arginine methyl ester and benzoyl DL-arginine p-nitroanilide; enzymatic activity was enhanced in the presence of heparin but markedly decreased in the presence of 2 M sodium chloride. Rabbit antisera and three new monoclonal antibodies (AA1, AA3 and AA5) were produced which were specific for tryptase in indirect ELISAs, immunoenzymatic overlay in crossed immunoelectrophoresis and by Western blotting. Additive and competitive ELISA experiments suggested that the three monoclonal antibodies all recognized epitopes within a single highly immunogenic area of the tryptase molecule, and enzyme assays indicated that this site was distant from the active site. Binding of monoclonal antibodies to tryptase was not affected by the presence of heparin, or by periodate treatment of the antigen suggesting that carbohydrate epitopes were not recognized. Western blotting indicated that some heterogeneity in molecular weight for monomeric tryptase was not reflected in antigenic differences. An immunofluorescence procedure with cytocentrifuge preparations of enzymatically dispersed lung, colon and skin revealed highly specific localization of tryptase to the granules of all mast cells, but there was no binding to other cells in these preparations, to cultured keratinocytes, to basophils or to any other blood leucocyte.

Mast cell tryptase and histamine concentrations in bronchoalveolar lavage fluid from patients with interstitial lung disease.

1. Mast cell activation in the lung was investigated by measuring concentrations of mast cell tryptase and histamine in the bronchoalveolar lavage fluid from patients with bronchial carcinoma, sarcoidosis, extrinsic allergic alveolitis or cryptogenic fibrosing alveolitis and from normal subjects. 2. Histamine concentrations in bronchoalveolar lavage fluid supernatants were elevated in the bronchial carcinoma and cryptogenic fibrosing alveolitis groups, and were correlated with the histamine content of the cells recovered. 3. An avidin-biotin-enhanced antigen-capture e.l.i.s.a., using polyclonal rabbit antibody specific for tryptase, and mouse monoclonal antibody AA5, allowed the quantification of tryptase in all samples of bronchoalveolar lavage fluid. Tryptase concentrations were increased in the bronchial carcinoma and extrinsic allergic alveolitis groups and in some of the patients with sarcoidosis, and the levels correlated with mast cell numbers and also with concentrations of albumin. 4. There was no significant correlation between levels of tryptase and histamine, suggesting differences in the rates of metabolism or different cellular sources. 5. The tryptase and histamine concentrations measured suggest that there is continuous degranulation of mast cells within the normal lung, but that this process is more pronounced in patients with bronchial carcinoma or interstitial lung disease.

Application of oligo-(14-amino-3,6,9,12-tetraoxatetradecanoic acid) lipid conjugates as steric barrier molecules in liposomal formulations.

Lipid conjugates of oligo-(14-amino-3,6,9,12-tetraoxatetradecanoic acid) (ATTAn) were synthesized as monodisperse analogues of poly(ethylene glycol) (PEG) derivatives used in liposomal drug delivery systems. The new lipids were shown to be at least equivalent to MePEGA-2000-DSPE in assays designed to evaluate the effectiveness of polymers as steric barrier molecules in liposomes. Liposomes containing 1-5% of ATTA8-DSPE (octamer) showed comparable long circulation behavior relative to PEG-2000-DSPE analogues. Surprisingly, the shorter ATTA4-DSPE (tetramer) appeared to be quite effective in reducing clearance. Liver enzyme levels and systemic single dose tolerability of ATTA8-DSPE liposomes were comparable to controls, suggesting that the new materials are nontoxic. Prolonged exposure of ATTA8-DSPE liposomes to splenocytes in vitro showed no evidence of mitogenicity relative to controls or MePEGA-2000-DSPE liposomes. ATTA8-DSPE was as effective as MePEGC-2000-DSPE in preventing complement activation by cationic liposome systems. Repeat dosage in vivo regimes in ICR mice using DSPC/cholesterol liposomes, with and without 5% ATTA8-DSPE and MePEGC-2000-DSPE, showed no evidence of enhanced clearance on successive doses. Splenocytes recovered after repeat doses showed no significant evidence of mitogenicity on restimulation with liposomes. Cellular differentiation and activation marker levels in splenocytes recovered after the fourth in vivo administration were at normal levels. These results suggest that ATTAn oligomers do not induce an immune response in isolation. It was demonstrated that ATTA8-DSPE could be used to replace PEG-lipids in the formulation of doxorubicin, plasmid DNA and oligonucleotides using a variety of formulation techniques. The study demonstrates that ATTAn oligomers can be safely and effectively used in place of poly(ethylene glycol) as well-defined biomaterials in liposomal applications where reproducible behavior is critical.