Evaluation of endothelial cell migration with a novel in vitro assay system

In this study we introduce a novel in vitro 'oil-drop' assay system for the measurement of endothelial cell (EC) migration, based on the original concept of the Teflon fence assay (Pratt et al., 1984; Am. J. Pathol. 117: 349–354). An aliquot of 15–20,000 human umbilical vein EC (HUVEC) is pipetted through a layer of mineral oil. The cells readily attach, spread and migrate on the surface of a matrix-coated tissue culture dish as a confluent circular monolayer. Migration is measured as the net increase in the total area covered at 24 hours. We have used this system to quantify EC migration on matrices composed of a mixture of type I collagen and either von Willebrand factor (vWF) or fibronectin (FN) in the presence or absence of tumor necrosis factor (TNF). Plating efficiency on both vWF/collagen and FN/collagen, measured by counting cells after attachment and spreading, is about 80%. With this method, migration on vWF/collagen was about 6.4 mm² and 5.3 mm² for TNF-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater – 6.4 mm² and 6.5 mm² with and without TNF treatment, respectively. During the 24 hour time period, HUVEC numbers increased 30–40% on vWF/collagen, and 60–80% on FN/collagen, with increased proliferation observed with TNF- treatment. EC proliferation could be completely inhibited by 2 mM hydroxyurea. This assay system has proven useful in our studies to quantify cell migration and proliferation.     

Risk management strategies in the postmarketing period : safety experience with the US and European bosentan surveillance programmes.

In view of the shortcomings of the current system for postmarketing drug surveillance that is based on voluntary spontaneous adverse drug reaction (ADR) reporting, new approaches are needed.We describe an approach involving a combination of limited distribution, patient and physician education, as well as a novel pharmaco-vigilance system that is capable of promoting the safe and adequate use of a new drug. Importantly, it provides the possibility of calculating true ADR occurrence rates, as the exposed population (denominator) and the number of patients with events (numerator) are known. These measures were taken for the oral dual endothelin ET(A)/ET(B) antagonist bosentan (Tracleer). In recent guidelines issued by the European Society of Cardiology, American College of Chest Physicians and the WHO, this drug is considered as first-line oral treatment for the treatment of pulmonary arterial hypertension, a devastating orphan disease associated with a poor prognosis. Bosentan was approved in 2001/2 on the basis of two pivotal studies that showed improved exercise capacity and haemodynamic parameters while delaying time to clinical worsening. Elevations in serum liver aminotransferase levels of >3 times the upper limit of normal were noted in 10.2% of patients (placebo-subtracted incidence). Therefore, liver function tests have to be performed on a regular basis. In addition, bosentan has potential as a teratogen.In the US, a controlled distribution network for bosentan (Tracleer) Access Program [T.A.P.]) and the development of a patient database to follow patients was set up. Accompanied by comprehensive physician and patient education programmes, T.A.P. was developed to provide a mechanism to assist with the primary risk management goals for bosentan therapy, namely pregnancy prevention and liver enzyme monitoring and prevention of hepatic injury.In Europe, the Tracleer) Excellence (TRAX PMS) database is a novel European non-interventional, prospective, internet-based surveillance system initiated by the manufacturer in cooperation with the European Medicines Agency. It collected potential safety signals associated with bosentan use including adverse events, elevations of liver aminotransferase levels, other abnormal laboratory values, death and hospitalisation. TRAX PMS has accrued 79% of all known patients in the EU and the data provide supportive 'real-life' evidence on the long-term safety of bosentan.The two different systems had similar goals and outcomes. The data received concerning thousands of patient-years of use have confirmed the clinical trial results regarding product safety and the favourable benefit/risk ratio of bosentan, especially with regard to known type A adverse events. The clinical monitoring algorithm has also been confirmed. In addition, no rare type B events were uncovered despite the increased reporting rate. These systems might serve as templates for future pharmaco-vigilance efforts regarding drugs that require particular safety attention.     

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination,accessory, and epigenetic profiles

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity.Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a serious infectious disease of humans and animals and a leading cause of community-acquired sepsis and pneumonia in endemic regions (Currie et al. 2010). Initially thought to be confined to Southeast Asia and Northern Australia, the prevalence of Bp appears to be spreading (Wiersinga et al. 2012), and Bp has been designated a biothreat select agent in the United States. Bp can persist in extreme environmental conditions and can infect several plant and animal hosts, including birds, dolphins, and humans (Wuthiekanun et al. 1995; Howard and Inglis 2003; Sprague and Neubauer 2004; Larsen et al. 2013). Treatment of clinical melioidosis is challenging because the bacterium is inherently resistant to many antibiotics, and Bp infections can persist in humans for more than a decade (Hayden et al. 2012; Wiersinga et al. 2012).The Bp genome comprises one of the largest and most complex bacterial genomes sequenced to date. Consisting of two large circular replicons (chromosomes) with a combined 7.2-Mb genome size (Holden et al. 2004), it contains a rich arsenal of genes related to virulence (e.g., Type III and Type VI secretion systems, polysaccharide biosynthesis clusters), metabolic pathways, and environmental adaptation (Wiersinga et al. 2012). Besides conserved regions, accessory genes on mobile elements and genomic islands may also contribute to phenotypic and clinical differences in microbial behavior (Currie et al. 2000; Sim et al. 2008). Analysis of the Bp genome has revealed previously unknown toxins and mechanisms of antibiotic resistance (Chantratita et al. 2011; Cruz-Migoni et al. 2011).Most large-scale studies of Bp genetic diversity to date have analyzed strains using multilocus sequence typing (MLST). These studies have suggested a high degree of genetic variability between Bp strains and related Burkholderia species (Cheng et al. 2008), and have shown that Bp strains belonging to different sequence types (STs) can often coexist in the same locale and sometimes even within the same sample (Pitt et al. 2007; Wuthiekanun et al. 2009). However, due to the limited number of genes analyzed by MLST, these studies cannot comment on the global proportion of genetic material shared between strains of different STs nor on the relative contribution of recombination, mutation, and horizontal gene transfer on intraspecies genetic diversity. Moreover, although previous studies have applied whole-genome sequencing (WGS) to study global patterns of Bp genetic heterogeneity and evolution, earlier Bp WGS reports have been confined to a limited number of isolates (10–12) derived from diverse geographical regions (Nandi et al. 2010), where geophysical barriers likely limit the propensity of the analyzed strains to exchange genetic material. To achieve a comprehensive understanding of genetic variation among closely related Bp strains, WGS analysis of much larger strain panels, ideally performed on strains isolated from a common region and belonging to the same (or closely related) ST groups, is required.In this study, we attempted to fill this important knowledge gap by performing WGS on 106 Bp strains drawn from a restricted Asian locale (Singapore and Malaysia). The WGS data, exceeding previous Bp WGS studies by 10-fold, enabled us to identify specific genomic clades of Bp, molecular features of Bp recombination at the whole-genome level, and accessory genome features contributing to recombination and horizontal gene transfer. We found a consistent pattern of genetic separation correlating with MLST, recombination haplotypes, shared accessory genes, and restriction modification (RM) systems. We provide evidence that restriction modification, beyond its role as defense against foreign DNA invasion, may have also partitioned the Bp species by restricting gene flow, resulting in the other observed correlations. Because RM systems are widely dispersed through the bacterial kingdom, it is possible that similar principles may apply to other bacterial species, implicating a potential role for epigenetic barriers as a driver of early incipient speciation.     

Juvenile and adenomatous gastrointestinal polyposis

Summary This is the fourth report of a case showing an association between juvenile and adenomatous polyposis. Starting at age 14, this patient underwent multiple polypectomies and gastrointestinal resections over a 15-year period. Although initial biopsies were diagnosed as juvenile polyps, later biopsies showed both adenomatous polyps and large polypoid masses with a mixture of juvenile and adenomatous features. Several typical small hyperplastic polyps were also found in the stomach. This case contrasts with the previous three cases in that the gastrointestinal tract is more widely involved and in that there is an unusual marked hyperplasia of argentaffin-and argyrophil-positive cells. The case reported here strengthens the relation between adenomatous polyposis and juvenile polyposis.     

Lack of acute effect of amylin (islet associated polypeptide) on insulin sensitivity during hyperinsulinaemic euglycaemic clamp in humans

Summary It is suggested that amylin (islet associated polypeptide), co-secreted with insulin from the pancreatic beta cells acts as a circulating hormone which opposes the action of insulin on muscle and increases hepatic glucose production. We have tested the effect of amylin in human subjects on postabsorptive glucose homeostasis and on insulin sensitivity using the euglycaemic hyperinsulinaemic clamp. The amylin used opposed insulin-mediated glucose disposal in rat soleus muscle at concentrations of 10 nmol/l. Seven subjects were studied on two occasions and infused with either amylin or placebo for 6 h, initially when postabsorptive and then during a euglycaemic hyperinsulinaemic clamp. Mean plasma amylin concentrations during the first 3 h were 2006±327 pmol/l during amylin infusion and 20±9 pmol/l during the control infusion. Amylin infusion had no effect on postabsorptive plasma concentrations of insulin (control: 32±16 vs amylin: 25±8 pmol/l) or glucose (5.1±0.1 vs 5.3±0.1 mmol/l). During the clamp, amylin concentrations were 1636 ±422 pmol/l when it was infused and 24±6 during control infusions. Plasma glucose and insulin concentrations were well matched during the control and amylin infusions (glucose: 4.7±0.1 vs 4.8±0.1 mmol/l; insulin: 198±37 vs 195±22 pmol/l). Exogenous glucose infusion rates were a mean of 13 % lower than control values during the amylin infusion but were not statistically different (p =0.17). Therefore, an approximately 100-fold elevation of plasma amylin concentration failed to consistently alter glucose metabolism. Our data suggest that amylin does not act as a circulating hormone to influence glucose metabolism in humans. [Diabetologia (1994) 37: 166–169] Received: 1 June 1993 and in revised form: 16 August 1993     

Impact of benign breast biopsy upon breast self-examination

Current American Cancer Society guidelines recommend monthly performance of breast self-examination (BSE) for women over 20 years of age. While the experience of a benign breast biopsy can result in elevated levels of distress, the impact of benign biopsy upon breast cancer (BC) screening behavior is not well known. The present study examined frequency of BSE practice in 102 women after benign breast biopsy (biopsy group). Telephone interviews were completed a mean of 21 days (initial interview) and 8 months after biopsy (follow-up interview). A healthy comparison (HC) group of women (n = 76) without a history of breast biopsy completed an initial interview only. Information regarding distress, dispositional characteristics, BC screening-related attitudes and behaviors, and subjective and objective risk for BC was collected. Results indicated that the biopsy and HC groups did not differ in typical (i.e., prebiopsy) practice of BSE. However, practice of BSE changed after biopsy with a general trend toward a decrease in BSE frequency. Only 8% of women in the biopsy group reported appropriate (once per month) practice of BSE at the 8-month Follow-up while 28% reported appropriate practice at the initial interview. Decreases in BSE performance after biopsy were characteristic of younger women, women who lacked confidence in the ability to perform BSE correctly, and women whose biopsy was preceded by discovery of a breast lump or abnormality during BSE. Results suggest the potential value of a psychoeducational intervention after biopsy to enhance appropriate performance of BSE.     

Differential effect of aspirin on thromboxane and prostaglandin biosynthesis in man.

1. Effects of a single intravenous dose of aspirin (600 mg) on bradykinin-stimulated prostaglandin (PG) and on thromboxane (TX) biosynthesis were determined in nine healthy male volunteers. Plasma concentrations of 6-oxo-PGF1 alpha and 13,14-dihydro-15-oxo-PGF2 alpha were measured in samples obtained during repeated 10 min intravenous infusions of bradykinin before and up to 6 h after the dose of aspirin. TXB2 was measured in serum from blood allowed to clot at 37 degrees C. 2. Aspirin inhibited bradykinin stimulated PG and platelet TX biosynthesis 0.5 h after the dose. Serum TXB2 remained low, whereas PG synthesis recovered within 6 h. 3. Effects of intravenous sodium salicylate (600 mg) were studied identically in eight subjects. Prostanoid biosynthesis was not inhibited. 4. Biosynthesis of prostacyclin and TXA2 under basal conditions was studied in eight subjects by measuring 2,3-dinor-6-oxo-PGF1 alpha and 2,3-dinor-TXB2 in hourly urine samples obtained during and after intravenous infusion of aspirin and, on a separate occasion, of vehicle. 5. Aspirin infusion reduced urinary excretion of both metabolites greater than 90%, but excretion of 2,3-dinor-6-oxo-PGF1 alpha recovered more rapidly than did that of 2,3-dinor-TXB2. 6. We conclude that cyclo-oxygenase is rapidly synthesised in bradykinin-responsive tissues in vivo and that this reflects similarly rapid enzyme biosynthesis in tissues that produce PGI2 under basal conditions.