Sphingosine-1-phosphate induces pro-remodelling response in airway smooth muscle cells.

BACKGROUND: Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. OBJECTIVE: To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. METHODS: Airway smooth muscle cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and Western blotting. Receptor signalling and function were determined by mRNA knockdown and intracellular calcium mobilization experiments. RESULTS: S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P2 and S1P3 receptors activated intracellular calcium mobilization and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. CONCLUSION: S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma.

Gene expression profiling of endobronchial ultrasound (EBUS)-derived cytological fine needle aspirates from hilar and mediastinal lymph nodes in non-small cell lung cancer.

OBJECTIVE: Endobronchial ultrasound (EBUS) allows minimally invasive sampling of hilar and mediastinal lymph nodes and has an established role in non-small cell lung cancer (NSCLC) diagnosis and staging. Molecular biomarkers are being explored increasingly in lung cancer research. Gene expression profiling (GEP) is a microarray-based technology that comprehensively assesses genome-wide changes in gene expression that can provide tumour-specific molecular signatures with the potential to predict prognosis and treatment responsiveness. We assessed the feasibility of using EBUS-derived aspirates from benign and tumour-infiltrated lymph nodes for GEP. METHODS: RNA was extracted from EBUS-directed transbronchial fine needle aspiration samples in routine clinical practice. GEP was subsequently performed in six patients with NSCLC, three of whom had tumour-infiltrated nodes and three who had benign lymph nodes; the differences in gene expression were then compared. RESULTS: RNA was successfully extracted in 29 of 32 patients, 12 of whom were diagnosed with NSCLC. RNA yield (median, 12.1 µg) and RNA integrity (median, 6.3) were sufficient after amplification for GEP. Benign and malignant nodes in adenocarcinoma were discriminated by principal component analysis and hierarchical clustering with different expression patterns between malignant and benign nodes. CONCLUSION: We have demonstrated the feasibility of RNA extraction and GEP on EBUS-derived transbronchial fine needle aspirates from benign and tumour-infiltrated lymph nodes in patients with known NSCLC in routine clinical practice. Further studies on larger patient cohorts are required to identify expression profiles that robustly differentiate benign from malignant lymph nodes in NSCLC.

NHS Diabetes guideline for the perioperative management of the adult patient with diabetes.

These Joint British Diabetes Societies guidelines, commissioned by NHS Diabetes, for the perioperative management of the adult patient undergoing surgery are available in full in the Supporting Information. This document goes through the seven stages of the patient journey when having surgery. These are: primary care referral; surgical outpatients; preoperative assessment; hospital admission; surgery; post-operative care; discharge. Each stage is given its own considerations, outlining the roles and responsibilities of each group of healthcare professionals. The evidence base for the recommendations made at each stage, discussion of controversial areas and references are provided in the report. This document has two key recommendations. Firstly, that the management of the elective adult surgery patients should be with modification to their usual diabetes treatment if the fasting is minimized because the routine use of a variable rate intravenous insulin infusion is not recommended. Secondly, that poor preoperative glycaemic control leads to post-outcomes and thus, where appropriate, needs to be addressed prior to referral for surgery.

White matter lesions in euthymic patients with bipolar disorder.

OBJECTIVE: We aimed to quantify both load and regional distributions of hyperintensities on magnetic resonance imaging (MRI) in prospectively verified euthymic bipolar patients and matched controls. METHOD: Cerebral hyperintensities on T2, proton density and fluid-attenuated inversion recovery (FLAIR) MRI were compared between 48 bipolar and 47 control subjects using semi-quantitative rating scales. RESULTS: Bipolar subjects had more severe frontal deep white matter lesions (DWML). Hyperintensity load was independent of age in bipolar patients but increased with age in controls. Global prevalence and severity of hyperintensities did not differ between groups. Exploratory analysis showed DWML in excess in the left hemisphere in bipolar subjects but not in controls. CONCLUSION: Findings are consistent with clinical, particularly some neurocognitive, features of bipolar disorder and implicate fronto-subcortical circuits in its neurobiology. They more probably reflect a trait abnormality or illness scar rather than a mood state-dependent finding. Processes other than ageing and vascular factors may underlie their development.

How to improve the efficiency of veterinary laboratories.

Veterinary laboratories around the globe are currently facing a number of important challenges. These challenges include an ageing workforce, lack of trained veterinary specialists, increased stringency for test validation, increasing emphasis on quality management, safety standards and certified accreditation processes, increasing regulation and audit processes and high costs of replacement infrastructure. Importantly, increased collaboration and linkages are high on the agenda in most countries to enhance efficiency within the sector. In Australia, mechanisms are in place to deal with some of these issues, and it is likely similar mechanisms are being used in other countries. The issues and some possible solutions are discussed.

Redefinition of the cleavage sites of DNase I on the nucleosome core particle.

DNase I has been widely used for the footprinting of DNA-protein interactions including analyses of nucleosome core particle (NCP) structure. Our understanding of the relationship between the footprint and the structure of the nucleosome complex comes mainly from digestion studies of NCPs, since they have a well-defined quasi-symmetrical structure and have been widely investigated. However, several recent results suggest that the established consensus of opinion regarding the mode of digestion of NCPs by DNase I may be based on erroneous interpretation of results concerning the relationship between the NCP ends and the dyad axis. Here, we have used reconstituted NCPs with defined ends, bulk NCPs prepared with micrococcal nuclease and molecular modelling to reassess the mode of DNase I digestion. Our results indicate that DNase I cuts the two strands of the nucleosomal DNA independently with an average stagger of 4 nt with the 3'-ends protruding. The previously accepted value of 2 nt stagger is explained by the finding that micrococcal nuclease produces NCPs not with flush ends, but with approximately 1 nt 5'-recessed ends. Furthermore we explain why the DNA stagger is an even and not an odd number of nucleotides. These results are important for studies using DNase I to probe nucleosome structure in complex with other proteins or any DNA-protein complex containing B-form DNA. We also determine the origin of the 10n +/- 5 nt periodicity found in the internucleosomal ladder of DNase I digests of chromatin from various species. The explanation of the 10n +/- 5 nt ladder may have implications for the structure of the 30 nm fibre.

A review of tests available for use in the diagnosis of tuberculosis in non-bovine species.

Bovine tuberculosis is an important disease that has impacts on regional and international trade. The disease can affect both social and economic stability and have a deleterious affect on species diversity. The intradermal tuberculin test has been in use for almost a century and, despite the technological advances of the last two decades, is still the only prescribed test for the diagnosis of tuberculosis in cattle. Many other species of animal, including humans, can be infected with Mycobacterium bovis. This paper reviews the various tests that have been used by researchers for detecting infection with M. bovis in a variety of animal species, and attempts to prioritise or comment on the importance of having appropriately validated diagnostics for the different species. The difficulties of test validation using small numbers of animals, especially when tuberculosis occurs in only a few instances or the species of animal affected is rare and/or valuable, are discussed.

White matter lesions and season of birth of patients with bipolar affective disorder.

OBJECTIVE: It is established that patients with bipolar disorder have an excess of births in winter or early spring. The authors investigated a link between season of birth and white matter lesions with magnetic resonance imaging (MRI). METHOD: T(2)-weighted and proton density MRI scans were examined for 79 patients with bipolar disorder (DSM-IV) for the presence of deep subcortical and periventricular white matter lesions. The birth seasons of patients with white matter lesions were compared with those of the general population. RESULTS: Thirteen subjects exhibited deep subcortical white matter lesions, of whom nine (69.2%) were born in the winter months (January to March). Seven of these patients remained symptomatic, despite adequate treatment for more than 2 years. CONCLUSIONS: Birth season, illness outcome, and deep subcortical white matter lesions appear to be closely linked. Deep subcortical white matter lesions may be a marker of a toxic or infective insult in utero.

Australia's campaign to eradicate bovine tuberculosis: the battle for freedom and beyond.

In 1970, voluntary State-based TB control programs in Australia were replaced by a coordinated national campaign to eliminate both brucellosis and tuberculosis from the cattle population. The campaign was funded and managed under tripartite agreement by State/Territory and Commonwealth governments and Industry. The tuberculosis component of the campaign relied on test and slaughter with surveillance for the disease in abattoirs and trace-back to property of origin an essential component. Because of the moderate sensitivity of the skin test ( approximately 70%), testing was repeated at prescribed intervals over a number of years. In the more hostile environment of northern Australia, novel strategies were developed to maximize musters and remove 'at risk' animals. Australia is fortunate it did not have a feral host for M. bovis (apart from buffalo, which were included in the campaign) to complicate eradication. A national granuloma submission program was implemented in 1992 to increase the intensity of abattoir monitoring. Selective or total depopulation was used in some herds to achieve the requirements of the national Standard Definitions and Rules of the Campaign and achieve the status of 'TB Free Area' in December 1997. Monitoring for tuberculosis has continued under the 5-year Tuberculosis Freedom Assurance Program and measures to further reduce the risk of new cases have been implemented.

Genetic differentiation of Australian isolates of Mycobacterium tuberculosis by pulsed-field gel electrophoresis.

As part of an epidemiological study of tuberculosis in Australia, 84 isolates of Mycobacterium tuberculosis from patients were analysed by pulsed-field gel electrophoresis (PFGE). The isolates were genetically heterogeneous, with 66 different DNA banding patterns obtained following digestion of genomic DNA with Dra1 and 53 patterns with Xba1. When the results were compared with those previously obtained in restriction fragment length polymorphism analysis (RFLP), in 87% of cases the results with Dra1 were consistent with those obtained with insertion sequence IS6110 as a probe in RFLP. However, PFGE was able to differentiate four of eight isolates which were identical with IS6110 typing. The high polymorphism amongst strains and the high average age of the patients (51 years) suggested that most organisms were cultured from patients who had reactivation of existing infections. Isolates with identical DNA patterns were found in different states of Australia, but no one strain predominated in any area. This suggests that tuberculosis has been introduced into Australia from various sources.

Developing a uniform reporting system for preventable adverse drug events.

The United States Pharmacopeia (USP) Practitioners' Reporting Network utilized nearly three decades of experience to design and develop a nationwide, anonymous, Internet-accessible service for hospitals to report and benchmark medication errors (also known as "preventable adverse drug events"). As nationwide experiential reporting enters the 21st century, the features of MedMARx will provide facilities with the unprecedented ability to conduct daily continuous quality improvement that encompasses medication error risk analysis and prevention based on the experiences of other facilities. Data elements were developed based on review by expert panels and USP's experience with the Medication Errors Reporting Program. The success of this model could indicate its usefulness in other practice settings and for broader applications in health care.

Tuberculosis due to Mycobacterium bovis in the Australian population: cases recorded during 1970-1994.

OBJECTIVE: To determine the contribution of Mycobacterium bovis to active tuberculosis in the Australian population during 1970-1994, and to collate and analyse demographic data from bacteriologically proven cases. DESIGN: Summary data for tuberculosis cases notified by Australian public health agencies during 1970-1985 and 1991-1994 were obtained from the database of notifiable diseases maintained by the Department of Health and Family Services. More detailed demographic data for cases confirmed by bacteriology during 1970-1994 were supplied by the Australian Mycobacterium Reference Laboratory Network. RESULTS: At least 236 cases of bovine tuberculosis (TB) occurred in the Australian population during 1970-1994 (mean 9.4 cases; range 3-22 cases annually). The bovine strain has accounted for around 1% of Australian cases of TB during this period. Laboratory sources provided demographic data for 150 cases with positive bacteriology. For this group, the mean age was 54 years (range 22-86), and the male:female ratio was 2.4:1. The majority of cases (74%) involved pulmonary disease. Australian-born persons accounted for 68% of the total cases and typically had histories of employment in meat and/or livestock industries. CONCLUSION: M. bovis was responsible for less than 1.5% of cases of TB in the Australian population during 1970-1994. Most cases were apparently due to reactivation of infection acquired through occupational exposure. Thus, although virtual eradication of M. bovis from Australia's cattle herds has now reduced the risk of exposure, it can be expected that human cases of bovine TB will continue to be detected for years to come. The bovine strain should be considered as the possible agent of TB in foreign-born Australians.

Tuberculosis due to Mycobacterium bovis in the Australian population: DNA typing of isolates, 1970-1994.

SETTING: Bacteriologically confirmed cases of Mycobacterium bovis in the Australian population. OBJECTIVE: To evaluate the DNA fingerprinting techniques commonly used for M. bovis on isolates from humans and determine whether they were useful for determining the origin of human infection. DESIGN: M. bovis strains isolated between 1970 and 1994 were obtained from five Australian Reference Laboratories. Four DNA fingerprinting techniques, comprising Southern hybridisation with three different probes (the insertion sequence [IS]6110, the polymorphic guanine-cytosine-rich sequence [PGRS] and the direct repeat [DR]) and a PCR-based method (spoligotyping) were used. RESULTS: The PGRS, DR and IS6110 RFLP methods identified 32, 22 and 14 different types respectively from the 45 isolates available. Spoligotyping identified 18 different types. When all methods were combined 41 different strains were identified. Clear differences were found between many isolates from Australian-born patients and those from patients born overseas. CONCLUSIONS: The PGRS RFLP method was the most effective method for typing the human strains, but a combination of methods is recommended for maximum sensitivity. Most Australian-born patients that had worked in the meat and livestock industries were infected with strains similar to those that are commonly found in Australian cattle, confirming the occupational risk in these industries. Patients born overseas were typically infected with strains genetically different from those of patients born in Australia. This suggests that patients born overseas identified with M. bovis were presenting with reactivation of infection acquired outside Australia.

Towards a standardized approach to DNA fingerprinting of Mycobacterium bovis. International Union Against Tuberculosis and Lung Disease, Tuberculosis in Animals Subsection.

The Tuberculosis in Animals Subsection of the International Union Against Tuberculosis and Lung Disease (IUATLD) recently identified a need to standardize the deoxyribonucleic acid (DNA) strain typing of Mycobacterium bovis. The standard method for strain typing of M. tuberculosis isolates cannot be directly extrapolated to M. bovis due to the low copy number of IS6110 identified in the majority of M. bovis strains, particularly from cattle. To improve the resolution of M. bovis strains, alternative methods and additional DNA probes have been investigated. In combination with studies of published literature, laboratories performing M. bovis DNA fingerprinting were surveyed. Results of these surveys allowed us to reach consensus and to make recommendations for DNA typing of M. bovis isolates, which hopefully will lead towards a standardized approach to the DNA fingerprinting of this organism. This approach, in conjunction with conventional epidemiological traceback approaches, should facilitate more accurate and effective investigations into the epidemiology, maintenance and transmission of M. bovis within and between man and domesticated, feral and wild animals, both at a local and a global level.

Spacer oligonucleotide typing of bacteria of the Mycobacterium tuberculosis complex: recommendations for standardised nomenclature.

Spacer oligonucleotide typing (spoligotyping) is widely used for differentiation of bacteria of the Mycobacterium tuberculosis complex. However, the absence of any standardised method for concise description of spoligotypes makes it difficult to compare the results from different laboratories. This paper describes unambiguous, interconvertible systems for the designation of spoligotype patterns, the adoption of which will be beneficial to mycobacterial research.

Rapid identification of Haemophilus somnus, Histophilus ovis and Actinobacillus seminis using the API ZYM system.

The API ZYM system, a commercially-available technique that measures bacterial enzyme activity was used to test 43 isolates identified as H. somnus, H. ovis or A. seminis and 19 from related genera. The enzyme patterns resulting from the API ZYM differentiated H. somnus and H. ovis from A. seminis and related genera but not from each other. An identification scheme based on 9 of the enzymes in the API ZYM and a few simple biochemical tests is proposed for the rapid and reliable identification of these bacteria in a diagnostic bacteriology laboratory.

Preparation, assessment and preservation of antigen from Brucella abortus strain 45/20 for use in the rough antigen complement fixation test.

Two methods of extraction were used to prepare antigens from Brucella abortus rough strain 45/20. The antigens were assessed for use in the complement fixation test. A suitable antigen was prepared using the saline extraction method of Miller et al. (1976) and used extensively in CF tests. Four methods of preservation were compared; -20 degrees C, -196 degrees C, 0.5% phenol at 4 degrees C, and lyophilisation. The antigen could be stored at -20 degrees C or -196 degrees C for up to 2 years.

The use of the enzyme-linked immunosorbent assay (ELISA) to detect the IgM and IgG antibody response to Leptospira interrogans serovars hardjo, pomona and tarassovi in cattle.

Leptospira interrogans serovars pomona, hardjo and tarassovi were each used to inoculate 6 cattle. Three-hundred and ninety-nine sera collected from the inoculated animals and from a control group over a 3-month period were tested using the microscopic agglutination test (MAT) and the enzyme-linked immunosorbent assay (ELISA). Leptospiruria was monitored by microscopic examination and culture. The ELISA detected specific IgM antibody against the serovars in all infected cattle 1 week after inoculation. This IgM antibody persisted in most of the animals for 3-5 weeks. Specific IgG antibody appeared at the same time or just after IgM, but persisted for much longer. Levels of antibody detected by the ELISA and the MAT did not correlate with each other, nor with the periods of leptospiruria found in the infected cattle.

Use of DNA amplification for the rapid identification of Mycobacterium bovis.

DNA amplification using the polymerase chain reaction technique was evaluated for rapid identification of Mycobacterium bovis. Two oligonucleotide primers of 20 bases in length were constructed to target a region of the gene encoding the M. bovis secretory protein, MPB70. The amplification reaction produced a single product 372 bp in size which was readily detected by agarose gel electrophoresis. All 84 strains of M. bovis tested produced a positive signal in the amplification reaction. In addition all isolates fro the M. tuberculosis complex tested, with the exception of M. microti, gave a single band at 372 bp. No amplified product was detected when 24 other species of mycobacteria and species from four other genera were tested. The sensitivity of the test was such that a single viable cell could be detected in the reaction. This technique provides a simple and extremely sensitive method of identifying isolates of M. bovis and other pathogenic M. tuberculosis complex organisms.