Molecular characterization of human factor XSan Antonio

Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.     

Estrogen-induced vitellogenin mRNA and protein in sheepshead minnow (Cyprinodon variegatus)

Many environmentally persistent xenobiotic chemicals appear to disrupt normal endocrine function by acting as ligands for endogenous steroid receptors, including the estrogen receptor. Xenobiotics that bind to the estrogen receptor may elicit several effects, one of which is activating estrogen-responsive genes, such as vitellogenin (Vtg). Primers to vitellogenin mRNA have been used to amplify a portion of the coding sequence in sheepshead minnow (SHM) (Cyprinodon variegatus). Two Vtg cDNA fragments from SHM were isolated exhibiting 72% sequence homology and corresponding to the two Vtg genes identified in the mummichog, Fundulus heteroclitus. Using these Vtg cDNA fragments as sensitive genetic probes, we evaluated the initial estrogenic response of fish exposed to natural or anthropogenic chemicals. These probes were used to study in vivo gene induction in SHM exposed to 17beta-estradiol (E(2)) and ethinylestradiol (EE(2)) under controlled laboratory conditions. Hepatic Vtg mRNA was upregulated and plasma Vtg synthesis in estrogen-induced SHM was assessed. Two in vivo time-course experiments were conducted; a single injection of E(2) followed over 72 h and a double E(2) injection examined for 12 days. These two protocols provided evidence for differential hepatic Vtg mRNA regulation resulting from a single or a double injection. In a separate experiment using an aqueous flowthrough system, constant exposures to low doses of E(2) (200 ng/L) and EE(2) (100 ng/L) induced hepatic Vtg mRNA and plasma Vtg to levels comparable with the E(2) injections. Larger aqueous exposure doses (2000 ng/L E(2) or 1000 ng/L EE(2)) in the flowthrough experiment resulted in greater responses of hepatic Vtg mRNA and plasma Vtg at 7 days. Constant aqueous exposure to E(2) (2000 ng/L) or EE(2) (1000 ng/L) may thus be more effective than a single large-dose injection (5 mg/kg) to stimulate Vtg gene activation and synthesis.     

Right arm ischemia following intentional stent-graft coverage of an anomalous right subclavian artery.

PURPOSE: To report thoracic aortic stent-graft repair in a patient with abnormal aortic arch anatomy. CASE REPORT: An anomalous right subclavian artery was covered with a stent-graft in a 38-year-old woman being treated for a false aneurysm after coarctation repair. The right arm became relatively ischemic, but was viable and managed conservatively. CONCLUSIONS: Aneurysms close to left or aberrant right subclavian arteries can be safely and effectively treated by endoluminal repair without the need for revascularization procedures; ischemic symptoms that develop are often mild and transient.     

A radioreceptor assay for quantitating plasma factor VIII/von Willebrand's protein

A sensitive and precise radioreceptor assay for determining plasma levels of human factor VIII/von Willebrand's factor (FVIII/vWF) has been developed by taking advantage of the FVIII/vWF receptor sites on human platelets. Paraformaldehyde-fixed platelets, which were processed and then stored, retained FVIII/vWF binding activity and therefore could be used as a convenient source of receptors. The human plasma samples to be tested were initially filtered on 4% agarose columns to concentrate the FVIII/vWF protein in the void volume and to remove the factor(s) that interferes with the assay. The percent recovery of FVIII/vWF in the pooled eluent was measured by the recovery of added trace 125I-FVIII/vWF. The coefficients of intra- and interassay variation were 6% and 10%, respectively. The plasma FVIII/vWF concentrations determined by the assay for pooled normal plasma, hemophilia A plasma, and plasmas from two patients with von Willebrand's disease were 16.3 +/- 0.5, 52.6 +/- 1.5, 6.8 +/- 0.8, and 3.2 +/- 0.2 microgram/ml, respectively. The range of plasma FVIII/vWF concentrations varied between 8.3 microgram/ml and 24.9 microgram/ml for 10 normal adults. The plasma FVIII/vWF concentrations determined by the radioreceptor assay correlated well with levels measured by the ristocetin-induced platelet aggregation method, thus demonstrating the functional relevancy of the radioreceptor assay for plasma FVIII/vWF.     

Identification and structural analysis of residues in the V1 region of CD4 involved in interaction with human immunodeficiency virus envelope glycoprotein gp120 and class II major histocompatibility complex molecules.

The human CD4 molecule binds both human immunodeficiency virus envelope protein gp120 and class II major histocompatibility complex (MHC) molecules. We have studied a series of mutants in the region of amino acids 42-49 of CD4 for their ability to bind gp120, to interact with class II MHC, to enhance T-cell activation, and to bind a panel of anti-CD4 antibodies. The mutation Q40P (Gln40----Pro) and the deletion d42-49 were found to disrupt most antibody epitopes in the V1 domain of CD4, suggesting major conformational changes, whereas mutants F43L, G47R, and P48S retained the binding of most of the anti-CD4 antibodies tested. The mutants d42-49, Q40P, F43L, and G47R lost both gp120 and class II MHC binding as well as the ability to enhance T-cell activation. In contrast, the mutation P48S affected neither gp120 binding, nor class II MHC binding, nor T-cell activation. We conclude that within this region the binding sites for gp120 and for class II MHC molecules overlap and that amino acids Phe43 and Gly47 comprise an intimate part of both binding sites. These observations are consistent with a three-dimensional model of the V1 domain of CD4 that was developed in order to understand the structural basis for binding to CD4.     

High-frequency oscillatory ventilation in adults: the Toronto experience

STUDY OBJECTIVES: To review the clinical experience with high-frequency oscillatory ventilation (HFOV) in three medical-surgical ICUs in Toronto, ON, Canada, and to describe patient characteristics, HFOV strategies, and outcomes. DESIGN AND PATIENTS: Retrospective chart review of all patients treated with HFOV at three academic university-affiliated ICUs since 1998. The data extracted included patient demographics, etiology of respiratory failure, ventilator settings, and gas exchange and cardiovascular data from baseline to 72 h of treatment, as well as at the transition from HFOV to conventional ventilation (CV). Heart rate and BP were recorded at regular intervals in all patients, and hemodynamic data were recorded in 32 patients who had pulmonary artery catheters in place. Cointerventions and ICU mortality were also recorded. MEASUREMENTS AND RESULTS: A total of 156 adults (67 women and 89 men; mean [+/- SD] age, 48 +/- 18 years; mean acute physiology and chronic health evaluation [APACHE] II score, 23.8 +/- 7.5) with severe ARDS (ie, mean Pao(2)/fraction of inspired oxygen [Fio(2)] ratio, 91 +/- 48 mm Hg; mean oxygenation index [OI], 31 +/- 14) who had received CV for a duration of 5.6 +/- 7.6 days underwent 171 trials of HFOV. HFOV was discontinued within 4 h in 19 patients (12%) because of difficulties with oxygenation, ventilation, or hemodynamics. Pao(2)/Fio(2) ratios and OI ([Fio(2) x mean airway pressure x 100]/Pao(2)) improved significantly with the application of HFOV, and this benefit persisted for the 72-h study duration. Significant changes in hemodynamics following HFOV initiation included an increase in central venous pressure and a reduction in cardiac output (throughout the 72 h), and an increase in pulmonary artery occlusion pressure (at 3 and 6 h). Patients were treated with HFOV for 5.1 +/- 6.3 days. The 30-day mortality rate was 61.7%. Pneumothorax occurred in 21.8% of patients, 43.6% of patients were treated with inhaled nitric oxide, and 37.2% of patients were treated with steroids. Independent predictors of mortality on multivariate analysis were older age, higher APACHE II score, lower pH at the initiation of HFOV, and a greater number of days receiving CV prior to HFOV. CONCLUSIONS: HFOV has beneficial effects on Pao(2)/Fio(2) ratios and OI, and may be an effective rescue therapy for adults with severe oxygenation failure. The early institution of HFOV may be advantageous.     

Implementing clinical guidelines for chronic obstructive pulmonary disease: barriers and solutions

Chronic obstructive pulmonary disease (COPD) is a complex chronic lung disease characterised by progressive fixed airflow limitation and acute exacerbations that frequently require hospitalisation. Evidence-based clinical guidelines for the diagnosis and management of COPD are now widely available. However, the uptake of these COPD guidelines in clinical practice is highly variable, as is the case for many other chronic disease guidelines. Studies have identified many barriers to implementation of COPD and other guidelines, including factors such as lack of familiarity with guidelines amongst clinicians and inadequate implementation programs. Several methods for enhancing adherence to clinical practice guidelines have been evaluated, including distribution methods, professional education sessions, electronic health records (EHR), point of care reminders and computer decision support systems (CDSS). Results of these studies are mixed to date, and the most effective ways to implement clinical practice guidelines remain unclear. Given the significant resources dedicated to evidence-based medicine, effective dissemination and implementation of best practice at the patient level is an important final step in the process of guideline development. Future efforts should focus on identifying optimal methods for translating the evidence into everyday clinical practice to ensure that patients receive the best care.