Comparative expression profiling in primary and immortalized endothelial cells: changes in gene expression in response to hydroxy methylglutaryl-coenzyme A reductase inhibition.

Immortalized cell lines offer significant logistical advantages over primary cells when used for in-vitro studies. Immortalized cells may, however, exhibit important differences relative to their primary cell counterparts. In this study, microarrays were used to make a genome-wide comparison between primary human umbilical vein endothelial cells (HUVECs) and EA.hy926, an immortalized HUVEC cell line, in their baseline properties and in their response to inhibition of the mevalonate pathway with an inhibitor of hydroxy methylglutaryl-coenzyme A reductase (statin). HUVECs and EA.hy926 were incubated with control medium, atorvastatin, mevalonate, or a combination of atorvastatin and mevalonate for 24 h. Gene expression profiles were obtained in duplicates using Affymetrix Human Genome U133A 2.0 arrays (Santa Clara, California, USA). Probe-sets were selected according to the following criteria: a twofold or greater increase/decrease in atorvastatin-treated cells compared with untreated cells; a twofold or greater reversal of the effect of atorvastatin by combined treatment with atorvastatin and mevalonate; no significant change in gene expression in cells treated with mevalonate alone compared with untreated cells. Most genes that were expressed by untreated HUVECs, were also expressed by untreated EA.hy926 cells. EA.hy926 cells, however, constitutively expressed a large number of additional genes, many of which were related to cell cycle control and apoptosis. Atorvastatin induced differential expression (> or = twofold) of 103 genes in HUVECs (10 up, 93 down) and 466 genes in EA.hy926 cells (198 up, 268 down). Applying the above selection criteria, thrombomodulin and tissue plasminogen activator were up-regulated in both cell types, whereas, connective tissue growth factor, thrombospondin-1, and cysteine-rich angiogenic inducer 61 were down-regulated. In conclusion, EA.hy926 cells retain most of the characteristics of endothelial cells under baseline conditions as well as after treatment with atorvastatin. It is necessary, however, to carefully select and validate changes in genes that are the focus of studies when using EA.hy926 cells. While this cell line is highly useful in studies on some genes, including genes encoding molecules involved in regulating thrombohemorrhagic homeostasis, they appear to be less suited for studies focused on other genes, particularly those involved in the regulation of cell proliferation and apoptosis.     

Chronic obstructive pulmonary disease in Brazilian primary care: Diagnostic competence and case-finding.

AIMS: The developing world is particularly at risk of an increasing health burden due to an increased prevalence of Chronic Obstructive Pulmonary Disease (COPD) secondary to increasing tobacco consumption. However, research is scarce. The objectives of this study were to assess the current competence for diagnosing COPD in primary care in a resource-limited setting in Brazil, and to develop a local patient profile for case-finding. METHODS: 34 general practitioners (GPs) in five areas of northern Brazil recruited adult patients with principal complaints of cough and/or shortness of breath who then had spirometry (n = 142). RESULTS: For the dichotomous variable 'COPD' the degree of agreement between GP diagnosis (n = 64, 18.3%) and spirometric outcome (n = 36, 25.4%) was poor, with Kappa = 0.055 (SE 0.087) and DOR = 1.35. False-positive and false-negative diagnosis proportions were 19.8% and 75%, respectively. Independent risk factors were 'smoking history of more than five pack years' and 'presence of both dyspnoea and cough'. It requires the testing of 2.2 smokers with more than five pack years to detect one patient at risk. CONCLUSIONS: COPD is a common yet underdiagnosed disease in Brazilian primary care. Spirometry improves diagnostic competence and case-finding substantially. If applied in a pre-selected high-risk population, we believe spirometry can be a cost-effective diagnostic tool for case-finding in the resource-limited setting. This study provides important baseline information for effective guideline implementation.     

Decision trees based on automatic learning and their use in cardiology

Computerized information systems, especially decision support systems, have become an increasingly important role in medical applications, particularly in those where important decision must be made effectively and reliably. But the possibility of using computers in medical decision making is limited by many difficulties, including the complexity of conventional computer languages, methodologies and tools. Thus a conceptual simple decision making model with the possibility of automating learning should be used. In this paper we introduce a cardiological knowledge-based system based on the decision tree approach supporting the mitral valve prolapse determination. Prolapse is defined as the displacement of a bodily part from its normal position. The term mitral valve prolaps (PMV), therefore, implies that the mitral leaflets are displaced relative to some structure, generally taken to be the mitral annulus. The implications of the PMV are the following: disturbed normal laminar blood flow, turbulence of the blood flow, injury of the chordae tendinae, the possibility of thrombus's composition, bacterial endocarditis, and finally hemodynamic changes defined as mitral insufficiency and mitral regurgitation. Uncertainty persists about how it should be diagnosed and about its clinical importance. It is our deep belief that the echocardiography enables properly trained experts armed with proper criteria to evaluate PMV almost 100%. But unfortunately, there are some problems concerned with the use of echocardiography. In that manner we have decided to start a research project aimed at finding new criteria and enabling the general practitioner to evaluate PMV using conventional methods and to select potential patients from the general population. To empower one to perform needed activities we have developed a computer tool called ROSE (computeRised prOlaps Syndrom dEtermination) based on algorithms of automatic learning. This tool supports the definition of new criteria and the selection of potential PMV-patients.     

Health status,work limitations,and return-to-work trajectories in injured workers with musculoskeletal disorders

BACKGROUND: The purpose of this study was to describe the health status and work limitations in injured workers with musculoskeletal disorders at 1 month post-injury, stratified by return-to-work status, and to document their return-to-work trajectories 6 months post-injury. METHODS: A sample of 632 workers with a back or upper extremity musculoskeletal disorder, who filed a Workplace Safety and Insurance Board lost-time claim injury, participated in this prospective study. Participants were assessed at baseline (1 month post-injury) and at 6 months follow-up. RESULTS: One month post-injury, poor physical health, high levels of depressive symptoms and high work limitations are prevalent in workers, including in those with a sustained first return to work. Workers with a sustained first return to work report a better health status and fewer work limitations than those who experienced a recurrence of work absence or who never returned to work. Six months post-injury, the rate of recurrence of work absence in the trajectories of injured workers who have made at least one return to work attempt is high (38%), including the rate for workers with an initial sustained first return to work (27%). CONCLUSIONS: There are return-to-work status specific health outcomes in injured workers. A sustained first return to work is not equivalent to a complete recovery from musculoskeletal disorders.     

Assessment of hand-arm vibration exposure among traffic police motorcyclists

 The aims of this study were (1) to evaluate subjective symptoms in the hand-arm system of all traffic police motorcyclists of a city located in the central part of Japan and (2) to assess their hand-arm vibration exposure associated with traffic police motorcycle riding. The study population consisted of 119 motorcycling traffic policemen and 49 male controls. By means of a questionnaire, information on the occupational history and the presence of subjective symptoms in the hand-arm system of all subjects was obtained. Vibration was measured on the handlebars of the representative motorcycles and on the hands of the riders. The 4- and 8-h energy-equivalent frequency-weighted acceleration as well as the lifetime vibration dose were calculated for all police motorcyclists. The prevalence of finger blanching in the traffic police motorcyclists was 4.2%, but none of the controls had this symptom. The rates of finger numbness (19.3%), finger stiffness (16.0%), shoulder pain (13.4%), and shoulder stiffness (45.4%) were significantly higher among police motorcyclists as compared with controls. The root-mean-square (rms) frequency-weighted acceleration on the handlebars of police motorcycles was in the range of 2.2–4.9 m/s² rms. The computed 4- and 8-h energy-equivalent frequency-weighted acceleration values were 2.8– 4.5 and 2.0 –3.2 m/s² rms, respectively. A pattern of increasing percentage prevalence with increasing cumulative vibration dose was noticed. The subjects with a lifetime vibration dose of more than 20.1 m² h ³ s^-4 (ln scale) showed significantly higher prevalence rates for symptoms in the fingers and shoulders as compared with the control group. As occupational vibration exposure of traffic police motorcyclists might be considered a risk factor for the development of symptoms in the hand-arm system of the riders, its evaluation and control is needed for prevention methodology evolution. Received: 15 April 1996/Accepted: 8 November 1996     

Silencing the major apple allergen Mal d 1 by using the RNA interference approach

BACKGROUND: Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. OBJECTIVE: The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. METHODS: In vitro -grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1-specific inverted repeat sequence separated by a Mal d 1-specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1-reactive mAb and with IgE antibodies against Mal d 1. RESULTS: After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P < .05) higher allergenicity than 5 of the transformants. Reduction of expression of Mal d 1 was confirmed by immunoblotting. In wild-type and unsuccessful transformants, a strong band was detected with Mal d 1-reactive mAb 5H8 at the expected apparent M r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. CONCLUSIONS: Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.     

Identification and susceptibility testing of Enterobacteriaceae and Pseudomonas aeruginosa by direct inoculation from positive BACTEC blood culture bottles into Vitek 2

Inoculation of an automated system for rapid identification (ID) and antimicrobial susceptibility testing (AST) directly from positive blood culture bottles will reduce the turnaround time of laboratory diagnosis of septicemic patients, which benefits clinical outcome and decreases patient costs. Direct test results, however, must always be confirmed by testing a pure overnight culture, which is the "gold standard." We studied the accuracy of direct testing versus repeat testing in order to investigate the possibility of refraining from repeat testing. We also assessed the clinical risk of reporting results based on direct testing only. We inoculated Vitek 2 (bioMérieux) directly from 410 positive BACTEC 9240 (BD) blood culture bottles containing gram-negative rods and studied the ID and AST results. In a comparison of direct inoculation with the standard method, a total of 344 isolates of Enterobacteriaceae and Pseudomonas aeruginosa were tested, and 93.0% were correctly identified. Of the 39 (10.2%) samples that contained bacilli not identifiable by Vitek 2, only 1 gave a conclusive, correct result. The overall MIC agreement among 312 isolates was 99.2%, with 0.8% very major and 0.02% major error rates. Of only three (polymicrobial) samples, the direct susceptibility pattern would be reported to the clinician as too sensitive. Vitek 2 results obtained from direct inoculation of blood culture bottles containing gram-negative bacilli are safe enough for immediate reporting, provided that ID and AST are consistent. Repeat testing is not necessary, unless Gram stain or overnight subculture results raise doubt about the purity of the culture.     

The apolipoprotein C-II variant apoC-IILys19→Thr is not associated with dyslipidemia in an affected kindred

The rare apolipoprotein C-II (apoC-II) mutation, apoC-II_Lys19→Thr, also known as apoC-II-v, has been found previously in association with hyperlipoproteinemia. From a lipid clinic screening we identified three unrelated individuals who had the apoC-II_Lys19→Thr mutation. Among eight family members of one proband, we have found another four who were affected. None of the inviduals in this kindred is dyslipidemic and there is no difference in lipid levels between affected and unaffected family members. Therefore, we conclude that the presence of this apolipoprotein variant by itself has no effect on lipoprotein levels. In addition, the apolipoprotein E (apoE) isoform, apoE4 does not have a synergistic effect on lipoprotein levels in this kindred, in contrast to observations on the interaction of apoE4 with another apoC-II mutant (apoC-II_Toronto). The single nucleotide substitution that causes the apoC-II_Lys19→Thr variant introduces a previously unrecognized restriction site (for Mae III), that provides for easy screening.     

A new CARD15 mutation in Blau syndrome

The caspase recruitment domain gene CARD15/NOD2, encoding a cellular receptor involved in an NF-kappaB-mediated pathway of innate immunity, was first identified as a major susceptibility gene for Crohn's disease (CD), and more recently, as responsible for Blau syndrome (BS), a rare autosomal-dominant trait characterized by arthritis, uveitis, skin rash and granulomatous inflammation. While CARD15 variants associated with CD are located within or near the C-terminal leucine-rich repeat domain and cause decreased NF-kappaB activation, BS mutations affect the central nucleotide-binding NACHT domain and result in increased NF-kappaB activation. In an Italian family with BS, we detected a novel mutation E383K, whose pathogenicity is strongly supported by cosegregation with the disease in the family and absence in controls, and by the evolutionary conservation and structural role of the affected glutamate close to the Walker B motif of the nucleotide-binding site in the NACHT domain. Interestingly, substitutions at corresponding positions in another NACHT family member cause similar autoinflammatory phenotypes.