Comparison of the hybrid capture tube test and PCR for detection of human papillomavirus DNA in cervical specimens.

The strong association of human papillomavirus (HPV) and cervical cancer makes it important to study HPV detection methods that may play a role in cervical cancer screening. We compared two DNA methods that are commonly used for HPV research in the United States: the MY09/MY11 L1 consensus primer PCR-based test and the first-generation Hybrid Capture tube method (HCT). Laboratory assays by each method were performed with 596 cervicovaginal specimens collected from participants in a large cohort study conducted in Portland, Oreg. Included were 499 specimens from women whose cytology was normal and 97 specimens from women with squamous intraepithelial lesions (SILs). The overall HPV DNA positivity for known types was 22.5% by PCR compared to 13.6% by HCT. When the analysis was restricted to the 14 HPV types detectable by both methods, the sensitivity of HCT, with PCR used as the standard for HPV status, was higher for specimens from women with concurrent SILs (81.0%) than for specimens from women with normal cytology (46.7%). Among specimens testing positive by both methods, 97.2% of the time the two methods agreed on whether specimens were positive for cancer-associated HPV types. Both of these HPV test methods provide information that supplements the information provided by the Pap smear. The PCR method has higher analytic sensitivity than HCT in detecting HPV, but HCT may be helpful in identifying women with concurrent SILs.     

Experimental analysis of the annotation of promoters in the public database

The ability to identify and examine promoter elements is important to researchers who wish to understand how gene expression is regulated in normal and pathological states. Unfortunately, the number of human promoters that have been directly experimentally defined is small. In order to determine if promoter sequences can be identified by simply aligning mRNA and genomic sequences, we have used a reporter gene assay to assess the promoter activity of the immediate 5' region flanking 38 mRNAs mapping to chromosome 21. For comparison, we have measured the activities of 19 sequences not thought to be promoters and 39 sequences taken from the Eukaryotic Promoter Database. Our results suggest that alignment of reference mRNAs to genomic sequence allows promoters to be identified for at least 75% of genes. These data provide the first empirical evidence that the current state of annotation of the genome is sufficient to allow molecular geneticists to correctly identify promoter sequences for most genes for which reference mRNA and genomic sequences are available.     

Bronchiectasis and deteriorating lung function in agammaglobulinaemia despite immunoglobulin replacement therapy

Immunoglobulin replacement therapy enhances survival and reduces infection risk in patients with agammaglobulinaemia. We hypothesized that despite regular immunoglobulin therapy, some patients will experience ongoing respiratory infections and develop progressive bronchiectasis with deteriorating lung function. One hundred and thirty‐nine (70%) of 199 patients aged 1–80 years from nine cities in the United Kingdom with agammaglobulinaemia currently listed on the UK Primary Immune Deficiency (UKPID) registry were recruited into this retrospective case study and their clinical and laboratory features analysed; 94% were male, 78% of whom had Bruton tyrosine kinase (BTK) gene mutations. All patients were on immunoglobulin replacement therapy and 52% had commenced therapy by the time they were 2 years old. Sixty per cent were also taking prophylactic oral antibiotics; 56% of patients had radiological evidence of bronchiectasis, which developed between the ages of 7 and 45 years. Multivariate analysis showed that three factors were associated significantly with bronchiectasis: reaching 18 years old [relative risk (RR) = 14·2, 95% confidence interval (CI) = 2·7–74·6], history of pneumonia (RR = 3·9, 95% CI = 1·1–13·8) and intravenous immunoglobulin (IVIG) rather than subcutaneous immunoglobulin (SCIG) = (RR = 3·5, 95% CI = 1·2–10·1), while starting immunoglobulin replacement after reaching 2 years of age, gender and recent serum IgG concentration were not associated significantly. Independent of age, patients with bronchiectasis had significantly poorer lung function [predicted forced expiratory volume in 1 s 74% (50–91)] than those without this complication [92% (84–101)] (P < 0·001). We conclude that despite immunoglobulin replacement therapy, many patients with agammaglobulinaemia can develop chronic lung disease and progressive impairment of lung function.     

The active metabolite of prasugrel effectively blocks the platelet P2Y12 receptor and inhibits procoagulant and pro-inflammatory platelet responses

The aim of these studies was to investigate the extent of platelet P2Y(12) receptor inhibition by the thienopyridine active metabolite of prasugrel, R-138727. Blood was taken from healthy volunteers and pre-incubated with R-138727 or cangrelor (AR-C66931MX). Platelet aggregation was assessed in platelet rich plasma (PRP) and whole blood (WB). Vasodilator stimulated phosphoprotein (VASP) phosphorylation, platelet procoagulant activity (annexin V binding and microparticle formation) and calcium mobilisation were measured by flow cytometry. Platelet-leukocyte co-aggregate formation and sCD40L release, both pro-inflammatory responses of platelets, were measured by flow cytometry and ELISA, respectively. P2Y(12) receptor antagonism was determined using a radioligand binding assay ((33)P 2-MeSADP) in resting and stimulated platelets and the effects of clopidogrel administration were also assessed. R-138727 yielded concentration-dependent inhibition of platelet aggregation, VASP phosphorylation inhibition, procoagulant activity and pro-inflammatory responses. In the presence of R-138727 or cangrelor there was increased calcium reuptake following agonist stimulation. R-138727 30 micromol/L and cangrelor 1 micromol/L completely inhibited (33)P 2-MeSADP binding, compared to partial inhibition following clopidogrel administration. Platelet activation and granule secretion did not expose an additional pool of P2Y(12) receptors. Prasugrel's active metabolite effectively blocks the P2Y(12) receptor with the highest concentrations tested yielding complete inhibition of P2Y(12)-mediated amplification of several important platelet responses.     

Isolation and characterisation of Mauritius lowland day gecko Phelsuma guimbeaui microsatellite loci

We isolated 315 sequences from a Phelsuma guimbeaui microsatellite-enriched genomic library. Primer sets were designed for 44 loci and used to genotype 29 unrelated individuals belonging to a population in the west of Mauritius. All the loci were polymorphic and the number of alleles ranged from 6 to 34. Mean observed and expected heterozygosity varied from 0.29 to 1.00 and 0.48 to 0.97, respectively. Thirteen loci displayed evidence of deviation from Hardy–Weinberg equilibrium, of which 11 also had an estimated null allele frequency ≥10 %. The microsatellite loci will be used to evaluate the population structure and genetic diversity of P. guimbeaui in Mauritius.