Hypercoagulability in Cushing Syndrome, Prevalence of Thrombotic Events: A Large, Single-Center, Retrospective Study.

BACKGROUND: The risk of Cushing syndrome (CS) patients experiencing a thrombotic event (TE) is significantly higher (odds ratio; OR 18%) than that of the general population. However, there are currently no anticoagulation guidelines. METHODS: A retrospective, single-center, longitudinal study of patients undergoing all types of treatment-surgical (pituitary, unilateral, and bilateral adrenalectomy) and medical treatment-was undertaken. TEs were recorded at any point up until last patient follow-up; myocardial infarction (MI), deep venous thrombosis (DVT), and pulmonary embolism (PE) or stroke. Patients' doses and complications of anticoagulation were recorded. RESULTS: Included were 208 patients; a total of 165 (79.3%) were women, and mean age at presentation was 44 ±â€…14.7 years. Thirty-nine (18.2%) patients had a TE; extremity DVT (38%), cerebrovascular accident (27%), MI (21%), and PE (14%). Of 56 TEs, 27 (48%) were arterial and 29 (52%) were venous. Patients who underwent bilateral adrenalectomy (BLA) had an odds ratio of 3.74 (95% CI 1.69-8.27) of developing a TE. Of patients with TEs, 40.5% experienced the event within the first 60 days after surgery. Baseline 24-hour urinary free cortisol levels did not differ in patients with or without TE after BLA. Of 197 patients who underwent surgery, 50 (25.38%) received anticoagulation after surgery, with 2% having bleeding complications. CONCLUSIONS: The risk of TEs in patients with CS was approximately 20%. Many patients had more than 1 event, with higher risk 30 to 60 days postoperatively. The optimal prophylactic anticoagulation duration is unknown, but most likely needs to continue up to 60 days postoperatively, particularly after BLA.

Evaluation of a novel PXR-knockout in HepaRG™ cells.

The nuclear pregnane X receptor (PXR) regulates the expression of genes involved in the metabolism, hepatobiliary disposition, and toxicity of drugs and endogenous compounds. PXR is a promiscuous nuclear hormone receptor (NHR) with significant ligand and DNA-binding crosstalk with the constitutive androstane receptor (CAR); hence, defining the precise role of PXR in gene regulation is challenging. Here, utilising a novel PXR-knockout (KO) HepaRG cell line, real-time PCR analysis was conducted to determine PXR involvement for a range of inducers. The selective PXR agonist rifampicin, a selective CAR activator, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO), and dual activators of CAR and PXR including phenobarbital (PB) were analyzed. HepaRG control cells (5F clone) were responsive to prototypical inducers of CYP2B6 and CYP3A4. No response was observed in the PXR-KO cells treated with rifampicin. Induction of CYP3A4 by PB, artemisinin, and phenytoin was also much reduced in PXR-KO cells, while the response to CITCO was maintained. This finding is in agreement with the abolition of functional PXR expression. The apparent EC50 values for PB were in agreement between the cell lines; however, CITCO was ~threefold (0.3 µmol/L vs. 1 µmol/L) lower in the PXR-KO cells compared with the 5F cells for CYP2B6 induction. Results presented support the application of the novel PXR-KO cells in the definitive assignment of PXR-mediated CYP2B6 and CYP3A4 induction. Utilization of such cell lines will allow advancement in composing structure activity relationships rather than relying predominantly on pharmacological manipulations and provide in-depth mechanistic evaluation.

Social media-sprint out of the gates!

Not having a Twitter account or a Facebook fan page for your employer, or for yourself, is no longer an option if you plan to participate in social media. I preface this article with the statement that many things could possibly change on Twitter and Facebook in 1 month's time from writing this article and it's publishing. We will focus on Twitter and Facebook because they can have the greatest impact with the least amount of effort, and they are the most popular. By creating an account for each and connecting them together, you ensure a "sprint" out of the social media gate versus a slow jog; so let's get started!

Association of smoking and chronic pain syndromes in Kentucky women.

UNLABELLED: The objective of this project was to determine the relationship between cigarette smoking and the reporting of chronic pain syndromes among participants in the Kentucky Women's Health Registry. Data was analyzed on 6,092 women over 18 years of age who responded to survey questions on pain and smoking. The chronic pain syndromes included in the analysis were fibromyalgia, sciatica, chronic neck pain, chronic back pain, joint pain, chronic head pain, nerve problems, and pain all over the body. Analyses controlled for age, body mass index, and Appalachian versus non-Appalachian county of residence. Results showed that women who were daily smokers reported more chronic pain (defined as the presence of any reported chronic pain syndromes) than women who were never smokers (adjusted odds ratio [aOR] = 2.04 and 95% confidence interval [CI] 1.67, 2.49). An increased risk was also seen for "some-day" smokers (aOR 1.68, 95% CI 1.24, 2.27), and former smokers (aOR 1.20, 95% CI 1.06, 1.37), though with less of an association in the latter group. This study provides evidence of an association between chronic pain and cigarette smoking that is reduced in former smokers. PERSPECTIVE: This paper presents the association between smoking and musculoskeletal pain syndromes among Kentucky women. This finding may provide additional opportunities for intervention in patients with chronic pain.

Interaction forces between waterborne bacteria and activated carbon particles.

Activated carbons remove waterborne bacteria from potable water systems through attractive Lifshitz-van der Waals forces despite electrostatic repulsion between negatively charged cells and carbon surfaces. In this paper we quantify the interaction forces between bacteria with negatively and positively charged, mesoporous wood-based carbons, as well as with a microporous coconut carbon. To this end, we glued carbon particles to the cantilever of an atomic force microscope and measured the interaction forces upon approach and retraction of thus made tips. Waterborne Raoultella terrigena and Escherichia coli adhered weakly (1-2 nN) to different activated carbon particles, and the main difference between the activated carbons was the percentage of curves with attractive sites revealed upon traversing of a carbon particle through the bacterial EPS layer. The percentage of curves showing adhesion forces upon retraction varied between 21% and 69%, and was highest for R. terrigena with positively charged carbon (66%) and a coconut carbon (69%). Macroscopic bacterial removal by the mesoporous carbon particles increased with increasing percentages of attractive sites revealed upon traversing a carbon particle through the outer bacterial surface layer.

Peptide-based in vitro assay for the detection of reactive metabolites.

We describe a novel peptide-based in vitro method for the detection of reactive metabolites that is amenable for use with microsomal or purified enzyme systems. Covalently bound adducts are detected by mass spectrometry using a surface-enhanced laser desorption ionizationtime of flight detector. The trapping molecule is an 11 amino acid peptide (ECGHDRKAHYK) that contains cysteine and other nucleophilic amino acid residues, as well as charged residues to enhance binding to a weak cation exchange chip surface used with the detection system. The assay concept was initially tested using rat or human liver microsomes with a series of benzodioxolanes. The assay was refined using human recombinant cytochrome P450 3A4 as the bioactivation system and validated with a series of positive and negative reference compounds. Alternative individual human recombinant P450 enzymes (e.g., 1A1, 2C9, or 2D6) may be used in place of 3A4 as the bioactivation system, or several P450 enzymes can be combined together into a single bioactivation system. We found that a mixture of P450s 3A4, 2C9, and 2D6 was suitable as a rapid general screen for the detection of reactive metabolites that covalently bind to proteins. Combining results from assays of individual P450 enzymes with microsomal systems allows the rapid profiling of metabolic pathways involved in reactive metabolite generation and provides valuable information that can be used to guide structural modifications to minimize the potential for metabolic bioactivation. In addition, non-P450 enzymes may be used as activation systems, such as peroxidases or alcohol dehydrogenase. In summary, this peptide-based assay system is able to detect reactive metabolites generated from a structurally diverse set of drugs and xenobiotics using a variety of microsomal or purified enzyme activation systems.

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow.

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination.

Adhesion and viability of waterborne pathogens on p-DADMAC coatings.

The attachment of waterborne pathogens onto surfaces can be increased by coating the surfaces with positive charge-enhancing polymers. In this paper, the increased efficacy of polydiallyldimethylammonium chloride (p-DADMAC) coatings on glass was evaluated in a parallel plate flow chamber with the use of waterborne pathogens (Raoultella terrigena, Escherichia coli, and Brevundimonas diminuta). p-DADMAC coatings strongly compensated the highly negative charges on the glass surface and even yielded a positively charged surface when applied from a 500 ppm solution. Whereas none of the strains adhered from water to glass due to electrostatic repulsion, R. terrigena and E. coli readily adhered in high numbers to p-DADMAC coated glass slides applied from 1, 100, or 500 ppm aqueous solutions. B. diminuta only adhered to a positively charged p-DADMAC coating applied from a 500 ppm solution. In addition, all p-DADMAC coatings indicated strong contact killing with the bacterial species used in this study by live/dead staining techniques. In summary, this paper demonstrates the potential of p-DADMAC coatings to strongly enhance bacterial adhesion. Moreover, once adhered, bacterial viability can be reduced by the positively charged ammonium groups in the coating.

Evaluation of two novel peptide safety markers for exocrine pancreatic toxicity.

Current markers of exocrine pancreatic toxicity have historically been poor indicators for both early diagnosis of disease and prediction of disease severity. Recently we identified two peptide markers (RA1609 and RT2864) of pancreatic toxicity that are target organ specific. In order to evaluate sensitivity of these markers versus current standard tests for pancreatic damage (i.e., lipase), we measured amylase and lipase, as well as RA1609 and RT2864 marker levels, in serum from rats treated with four doses (50-200 mg/kg) of the model pancreatic toxicant cyanohydroxybutene (CHB). In addition, to determine whether these peptide markers could detect pancreatic injury induced by different toxicants and in different species, we measured RA1609 and RT2864 marker levels in rats treated with the pancreatic toxicant caerulein, and in mice treated with CHB. RA1609 and RT2864 peptide markers proved to be more sensitive than amylase or lipase in detecting pancreatic damage, especially at an early time point (8 h) following CHB administration. The peptide markers also accurately predicted pancreatic injury induced by caerulein in rats. These markers were sensitive in detecting very mild pancreatic damage following CHB administration in mice, which are less susceptible to CHB-induced pancreatic toxicity. In addition, a species comparison of the RA1609 albumin fragment sequence indicated that cleavage of albumin from pancreatic proteases produces a similar fragment marker in several species, including humans. To determine whether the comparable human albumin fragment could be detected in sera from pancreatitis patients, we analyzed sera from normal individuals and from patients with diabetes, vasculitis, pancreatic cancer, and pancreatitis. It was found that markers corresponding to the fragments found in rat serum (RA1609 and RT2864) were present in human serum, and changes in these were indicative of and specific to pancreatitis. In conclusion, the RA1609 and RT2864 peptides are sensitive indicators of exocrine pancreatic damage that may be useful as safety markers for general pancreatic toxicity in multiple species.

Identification of novel peptide safety markers for exocrine pancreatic toxicity induced by cyanohydroxybutene.

Historically, serum amylase and lipase levels have been used to indicate pancreas injury; however, these enzyme levels are often not predictive of pathology. In an effort to discover novel biomarkers of pancreatic acinar cell injury, we analyzed serum and pancreas tissue from cyanohydroxybutene (CHB)-treated male IGS rats using proteomics methods. CHB produces an "edematous pancreatitis," characterized by depletion of zymogen granules, acinar cell apoptosis, and mild to moderate inflammation. Secondary necrosis occurs at higher doses. Rats were treated with 150 mg/kg of CHB and samples were collected at 4, 8, and 24 h. Analyses of serum tryptic digests by surface-enhanced laser desorption-ionization mass spectrometry revealed two novel peptide biomarkers (RA1609 and RT2864) that were predictive of pancreatic damage. Levels of RA1609 decreased, while levels of RT2864 increased by 8 h following CHB treatment. The changes in RA1609 and RT2864 were detected in media from CHB-treated primary rat acini, demonstrating that these peptides are either of pancreatic cell origin or are produced by proteases released from acinar cells. Sequencing revealed that RA1609 is a fragment of rat albumin (accession number P02770, residues 348-360) and RT2864 is a portion of either rat trypsin III (accession number P08426, residues 39-65) or bovine trypsin (accession number P00760, residues 35-61). These two peptides, and possibly other fragments of serum proteins that are digested by pancreatic proteases, may be useful as safety markers for exocrine pancreatic toxicity during drug development or as biomarkers for the diagnosis and/or grading of severity of pancreatic disease.

Removal of two waterborne pathogenic bacterial strains by activated carbon particles prior to and after charge modification.

Waterborne diseases constitute a threat to public health despite costly treatment measures aimed at removing pathogenic microorganisms from potable water supplies. This paper compared the removal of Raoultella terrigena ATCC 33257 and Escherichia coli ATCC 25922 by negatively and positively charged types of activated carbon particles. Both strains display bimodal negative zeta-potential distributions in stabilized water. Carbon particles were suspended to an equivalent external geometric surface area of 700 cm2 in 250 mL of a bacterial suspension, with shaking. Samples were taken after different durations for plate counting. Initial removal rates were less elevated for the positively charged carbon particle than expected, yielding the conclusion that bacterial adhesion under shaking is mass-transport limited. After 360 min, however, the log-reduction of the more negatively charged R. terrigena in suspension was largest for the positively charged carbon particles as compared with the negatively charged ones, although conditioning in ultrapure or tap water of positively charged carbon particles for 21 days eliminated the favorable effect of the positive charge due to counterion adsorption from the water. Removal of the less negatively charged E. coli was less affected by aging of the (positively charged) carbon particles, confirming the role of electrostatic interactions in bacterial removal by activated carbon particles. The microporous, negatively charged coconut carbon performed less than the mesoporous, positively charged carbon particle prior to conditioning but did not suffer from loss of effect after conditioning in ultrapure or tap water.

Proteomics: recent applications and new technologies.

Interest in proteomics as a tool for drug development and a myriad of other applications continues to expand at a rapid rate. Proteomic analyses have recently been conducted on tissues, biofluids, subcellular components and enzymatic pathways as well as various disease and toxicological states, in both animal models and man. In addition, several recent studies have attempted to integrate proteomics data with genomics and/or metabonomics data in a systems biology approach. The translation of proteomic technology and bioinformatics tools to clinical samples, such as in the areas of disease and toxicity biomarkers, represents one of the major opportunities and challenges facing this field. An ongoing challenge in proteomics continues to be the analysis of the serum proteome due to the vast number and complexity of proteins estimated to be present in this biofluid. Aside from the removal of the most abundant proteins, a number of interesting approaches have recently been suggested that may help reduce the overall complexity of serum analysis. In keeping with the increasing interest in applications of proteomics, the tools available for proteomic analyses continue to improve and expand. For example, enhanced tools (such as software and labeling procedures) continue to be developed for the analysis of 2D gels and protein quantification. In addition, activity-based probes are now being used to tag, enrich and isolate distinct sets of proteins based on enzymatic activity. One of the most active areas of development involves microarrays. Antibody-based microarrays have recently been released as commercial products while numerous additional capture agents (e.g. aptamers) and many additional types of microarrays are being explored.

Role of metabolism in drug-induced idiosyncratic hepatotoxicity.

Rare adverse reactions to drugs that are of unknown etiology, or idiosyncratic reactions, can produce severe medical complications or even death in patients. Current hypotheses suggest that metabolic activation of a drug to a reactive intermediate is a necessary, yet insufficient, step in the generation of an idiosyncratic reaction. We review evidence for this hypothesis with drugs that are associated with hepatotoxicity, one of the most common types of idiosyncratic reactions in humans. We identified 21 drugs that have either been withdrawn from the U.S. market due to hepatotoxicity or have a black box warning for hepatotoxicity. Evidence for the formation of reactive metabolites was found for 5 out of 6 drugs that were withdrawn, and 8 out of 15 drugs that have black box warnings. For the other drugs, either evidence was not available or suitable studies have not been carried out. We also review evidence for reactive intermediate formation from a number of additional drugs that have been associated with idiosyncratic hepatotoxicity but do not have black box warnings. Finally, we consider the potential role that high dosages may play in these adverse reactions.