Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.     

Improving the quality of reports of meta-analyses of randomized controlled trials: the QUOROM Statement

BACKGROUND: The Quality of Reporting of Meta-analyses (QUOROM) Conference was convened to address standards for improving the quality of reporting of meta-analyses of clinical randomised controlled trials (RCTs). METHODS: The QUOROM group consists of 30 clinical epidemiologists, clinicians, statisticians, editors, and researchers. In conference, the group was asked to identify items they thought should be included in a checklist of standards. Whenever possible, checklist items were guided by research evidence suggesting that failure to adhere to the item proposed could lead to biased results. A modified Delphi technique was used in assessing candidate items. RESULTS: The conference resulted in the QUOROM statement, a checklist, and a flow diagram. The checklist describes our preferred way to present the abstract, introduction, methods, results, and discussion sections of a report of a meta-analysis. it is organized into 21 headings and subheadings regarding searches, selection, validity assessment, data abstraction, study characteristics, and quantitative data synthesis, and in the results with 'trial flow', study characteristics, and quantitative data synthesis; research documentation was identified for eight of the 18 items. The flow diagram provides information about both the numbers of RCTs identified, included, and excluded and the reasons for exclusion of trials. INTERPRETATION: We hope this report will generate further thought about ways to improve the quality of reports of meta-analyses of RCTs and that interested readers, reviewers, researchers, and editors will use the QUOROM statement and generate ideas for its improvement.     

The proteomics of ageing

Proteomics provides an extremely powerful tool for the study of variations in protein expression between individuals, different disease states and different conditions. One of the major challenges facing the medical profession in the forthcoming decades is to understand the changes that occur in individuals as they become older and to attempt to develop means to improve the quality of life for the otherwise healthy ageing population. The present study describes the first phase of such an investigation in which the andaged protein composition of human skeletal muscle sample from young and aged subjects are compared. These results provide the beginning of a Human Aged Skeletal Muscle Profile reference map which is an essential first step to further investigations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Alpha2 macroglobulin and the risk of Alzheimer's disease

BACKGROUND: alpha2 Macroglobulin is a panproteinase inhibitor that is found immunohistochemically in neuritic plaques, a requisite neuropathologic feature of AD. Recently, a pentanucleotide deletion near the 5' end of the "bait region" of the alpha2 macroglobulin (A2M) gene was reported to be associated with AD in a large cohort of sibpairs, in which the mutation conferred a similar odds ratio with AD as the APOE-epsilon4 allele for carriers of at least one copy of the A2M gene (Mantel-Haenszel odds ratio, 3.56). METHODS: We studied three independent association samples of AD patients (n = 309) with an age range of 50 to 94 years and representative controls (n = 281) to characterize the allele frequency of the pentanucleotide deletion in this cohort. We detected the mutation near the 5' splice site of exon 18 using standard PCR and restriction fragment length polymorphism methods. The results were adjusted for age, gender, education, and APOE polymorphism. RESULTS: We found that the A2M gene polymorphism conferred an increased risk for AD, with an estimated Mantel-Haenszel ratio of 1.5 (95% CI 1.1 to 2.2; p = 0.025). There was no age- or gender-dependent increase in A2M gene allele frequencies in AD patients compared with controls. The combined sample showed the expected association between AD and APOE-epsilon 4. In one of our three samples there was an interaction between the A2M and APOE-epsilon4 genes, but the other two samples showed no interaction between the two risk factors. CONCLUSIONS: Our data support an association between the A2M gene and AD. This association is less pronounced, however, in our cohort than in the previously reported sample of sibpairs.     

The operative management of displaced intra-articular fractures of the calcaneum: a two-centre study using a defined protocol

The management of intra-articular calcaneal fractures remains controversial with strong arguments supporting both conservative and operative management. This study assesses the results of open reduction and internal fixation (ORIF) of displaced intra-articular fractures of the calcaneum in two independent centres where the indications for operative management had been strictly defined.Forty-seven patients (51 fractures) who had sustained such injuries underwent an ORIF performed by one of the senior surgeons using the technique described by Eastwood et al. [JBJS 75-B(1993)189] All of the fractures were assessed by plain X-ray and CT scan and graded according to the Sanders classification [Clin. Orthop. 290(1993)97]. Patients were assessed at a minimum of 2 years post-surgery both clinically and with a standardised questionnaire based on that described by Kerr et al. [Injury 27(1996)35].The mean age at operation was 42 and 50 years for the two centres, and the mean follow-up was 44 months. Seventy-six per cent of the patients were male. Eighty-eight per cent of the injuries were due to a fall from a height. Fifteen patients had contralateral foot/ankle injuries. Thirty-four of 46 patients were in employment at the time of the injury, 24 of these were in physical jobs and 20 sustained their injury whilst at work.Overall, the satisfaction rate was 90%. Ninety-four per cent of patients in work returned to work at a median of 6 months. Bilateral injuries were associated with a significantly poorer outcome as were those with heel pad pain. Delay to operation greater than 14 days was associated with a higher infection rate. Smoking was not related to infection rate.     

Ketoacidosis at the diagnosis of type 1 (insulin dependent) diabetes mellitus is related to poor residual beta cell function. Childhood Diabetes in Finland Study Group

The determinants of the degree of metabolic decompensation at the diagnosis of type 1 (insulin dependent) diabetes mellitus (IDDM) and the possible role of diabetic ketoacidosis in the preservation and recovery of residual beta cell function were examined in 745 Finnish children and adolescents. Children younger than 2 years or older than 10 years of age were found to be more susceptible to diabetic ketoacidosis than children between 2 and 10 years of age (< 2 years: 53.3%; 2-10 years: 16.9%; > 10 years: 33.3%). Children from families with poor parental educational level had ketoacidosis more often than those from families with high parental educational level (24.4% v 16.9%). A serum C peptide concentration of 0.10 nmol/l or more was associated with a favourable metabolic situation. Low serum C peptide concentrations, high requirement of exogenous insulin, low prevalence of remission, and high glycated haemoglobin concentrations were observed during the follow up in the group of probands having diabetic ketoacidosis at the diagnosis of IDDM. Thus diabetic ketoacidosis at diagnosis is related to a decreased capacity for beta cell recovery after the clinical manifestation of IDDM in children.     

Aortic aneurysm complicating bacterial endocarditis in childhood

Bacterial endocarditis is an uncommon diagnosis in childhood with significant morbidity and mortality. Aortic aneurysm as a complication is well described in adults but there are few reports in the paediatric literature. Two children with bacterial endocarditis are described, whose illnesses were complicated by aortic aneurysm formation requiring surgical intervention.