High-performance liquid chromatographic assay of hydroxyl free radical using salicylic acid hydroxylation during in vitro experiments involving thiols.

A HPLC method was developed to monitor the production of hydroxyl free radical (*OH) produced during in vitro experiments: (i) a chemical reaction involving EDTA chelated ferric ion and various exogenous and endogenous thiols [glutathione (GSH) and its metabolites], and (ii) an enzymatic reaction corresponding to the breakdown of GSH catalyzed by gamma-glutamyltransferase (GGT). The method relies upon the use of a selective trapping reagent of *OH: salicylic acid (SA). The three resulting dihydroxylated products, i.e., 2,3-dihydroxybenzoic acid (DHB), 2,5-DHB and catechol, were measured in an ion-pairing reversed-phase HPLC system coupled with amperometric detection; the sum of the three concentrations was used to quantify the production of *OH during in vitro experiments. Resulting data demonstrate that *OH is produced during Fenton-like reactions involving thiols and GSH catabolism via GGT.     

Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to extracellular matrix proteins

Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, beta-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.     

Postorthodontic stability of the new attachment achieved by guided tissue regeneration following orthodontic movement: report of 2 cases.

Orthodontic treatment was provided after guided tissue regeneration of intrabony defects in 2 patients with periodontal disease. The new attachment obtained by guided tissue regeneration remained stable when subjected to orthodontic forces.     

Markers of Inflammation and Coagulation after Long-Term Exposure to Coarse Particulate Matter: A Cross-Sectional Analysis from the Multi-Ethnic Study of Atherosclerosis

Background

Toxicological research suggests that coarse particles (PM_10–2.5) are inflammatory, but responses are complex and may be best summarized by multiple inflammatory markers. Few human studies have investigated associations with PM_10–2.5 and, of those, none have explored long-term exposures. Here we examine long-term associations with inflammation and coagulation in the Multi-Ethnic Study of Atherosclerosis.

Methods

Participants included 3,295 adults (45–84 years of age) from three metropolitan areas. Site-specific spatial models were used to estimate 5-year concentrations of PM_10–2.5 mass and copper, zinc, phosphorus, silicon, and endotoxin found in PM_10–2.5. Outcomes included interleukin-6, C-reactive protein, fibrinogen, total homocysteine, D-dimer, factor VIII, plasmin–antiplasmin complex, and inflammation and coagulation scores. We used multivariable regression with multiply imputed data to estimate associations while controlling for potential confounders, including co-pollutants such as fine particulate matter.

Results

Some limited evidence was found of relationships between inflammation and coagulation and PM_10–2.5. Endotoxin was the PM_10–2.5 component most strongly associated with inflammation, with an interquartile range (IQR) increase (0.08 EU/m³) associated with 0.15 (95% CI: 0.01, 0.28; p = 0.03) and 0.08 (95% CI: –0.07, 0.23; p = 0.28) higher inflammation scores before and after control for city, respectively. Copper was the component with the strongest association with coagulation, with a 4-ng/m³ increase associated with 0.19 (95% CI: 0.08, 0.30; p = 0.0008) and 0.12 (95% CI: –0.05, 0.30; p = 0.16) unit higher coagulation scores before and after city adjustment, respectively.

Conclusions

Our cross-sectional analysis provided some evidence that long-term PM_10–2.5 exposure was associated with inflammation and coagulation, but associations were modest and depended on particle composition.

Citation

Adar SD, D’Souza J, Mendelsohn-Victor K, Jacobs DR Jr, Cushman M, Sheppard L, Thorne PS, Burke GL, Daviglus ML, Szpiro AA, Diez Roux AV, Kaufman JD, Larson TV. 2015. Markers of inflammation and coagulation after long-term exposure to coarse particulate matter: a cross-sectional analysis from the Multi-Ethnic Study of Atherosclerosis. Environ Health Perspect 123:541–548; http://dx.doi.org/10.1289/ehp.1308069     

Serum insulin-like growth factor type 1, insulin-like growth factor-binding protein-1, and insulin-like growth factor-binding protein-3 concentrations in patients with thyroid dysfunction.

Thyroid hormones play a role in the regulation of insulin-like growth factor type 1 (IGF-1) and insulin-like growth factor-binding protein-3 (IGFBP-3) expression, and both IGF-1 and IGFBPs have been shown to be related to the function and growth of the thyroid. Our aim was to evaluate serum concentrations of IGF-1, IGFBP-1, and IGFBP-3 in patients with thyroid dysfunction before and after normalization of thyroid function. The study was performed in 86 patients with thyroid dysfunction (43 hyperthyroid and 43 hypothyroid patients) and 17 euthyroid subjects. Serum growth hormone (GH), insulin, IGF-1, IGFBP-1, and IGFBP-3 were measured in all patients before and after normalizing serum thyroid hormone concentrations. Hyperthyroid patients showed IGF-1 (198.8 +/- 17.0 microg/L) and IGFBP-3 levels (4.2 +/- 0.2 mg/L) similar to those found in the control group (217.9 +/- 20.3 microg/L and 4.2 +/- 0.3 mg/L, respectively). After therapy these levels significantly decreased to 156.6 + 11.1 microg/L (p < 0.01) and 3.3 +/- 0.1 mg/L (p < 0.001), respectively. IGFBP-1 concentrations were clearly higher than those found in controls (22.7+/- 2.6 vs. 5.7 +/- 1.5 microg/L, p < 0.001) and exhibited a significant reduction after therapy for thyroid hyperfunction (11.0 +/- 1.7 microg/L, p < 0.001). Patients with hypothyroidism showed serum concentrations of IGF-1 (161.5 +/- 13.1 microg/L, p < 0.05) and IGFBP-3 (3.2 +/- 0.3 microg/L, p < 0.05) significantly lower than those found in healthy volunteers. However, replacement therapy with levothyroxine did not induce any significant modification of these concentrations (152.6 +/- 10.6 microg/L and 3.2 +/- 0.2 mg/L, respectively). Similarly, patients with thyroid hypofunction exhibited raised levels of IGFBP-1 (15.5 +/- 0.9 microg/L, p < 0.05 vs. control group) that were significantly decreased after therapy (8.8 +/- 1.4 microg/L, p < 0.01). The results of the present study show that thyroid status affects GH/IGF axis. Hypothyroidism is associated with significant reductions of IGF-1 and IGFBP-3, and IGFBP-1 is elevated in both hypothyroidism and hyperthyroidism.     

Three peroxisome proliferator-activated receptor isotypes from each of two species of marine fish

The cloning and characterization of cDNAs and genes encoding three peroxisome proliferator-activated receptor (PPAR) isotypes from two species of marine fish, the plaice (Pleuronectes platessa) and the gilthead sea bream (Sparus aurata), are reported for the first time. Although differences in the genomic organization of the fish PPAR genes compared with their mammalian counterparts are evident, sequence alignments and phylogenetic comparisons show the fish genes to be homologs of mammalian PPARalpha, PPARbeta/delta, and PPARgamma. Like their mammalian homologs, fish PPARs bind to a variety of natural PPAR response elements (PPREs) present in the promoters of mammalian or piscine genes. In contrast, the mRNA expression pattern of PPARs in the two fish species differs from that observed in other vertebrates. Thus, PPARgamma is expressed more widely in fish tissues than in mammals, whereas PPARalpha and beta are expressed similarly in profile to mammals. Furthermore, nutritional status strongly influences the expression of all three PPAR isotypes in liver, whereas it has no effect on PPAR expression in intestinal and adipose tissues. Fish PPARalpha and beta exhibit an activation profile similar to that of the mammalian PPAR in response to a variety of activators/ligands, whereas PPARgamma is not activated by mammalian PPARgamma-specific ligands. Amino acid residues shown to be critical for ligand binding in mammalian PPARs are not conserved in fish PPARgamma and therefore, together with the distinct tissue expression profile of this receptor, suggest potential differences in the function of PPARgamma in fish compared with mammals.     

Pharmacoinvasive management of acute coronary syndrome: incorporating the 2007 ACC/AHA guidelines: the CATH (cardiac catheterization and antithrombotic therapy in the hospital) Clinical Consensus Panel Report--III

This paper provides a comprehensive up-to-date review of the medical and invasive management of patients with non-ST-segment elevation acute coronary syndromes (NSTE-ACS), and ST-elevation myocardial infarction (STEMI), as supported by recent updates to the ACC/AHA Guidelines. The authors have summarized findings from key clinical trials published in recent years that contribute to clinician's understanding of how best to optimize therapy. The goals for the management of NSTE-ACS and STEMI are rapid and accurate risk stratification, appropriate and institution-specific triage to interventional versus medical strategies and optimal pharmacologic therapy - all of which provide for a smooth and seamless transition of care between the emergency department and the cardiology service. High-risk features or absolute treatment trigger criteria that support more aggressive medical therapy (i.e., addition of a small molecule gylcoprotein [GP] IIb/IIIa inhibitor to a core regimen of aspirin, enoxaparin or other anticoagulants, and in most cases, clopidogrel) and/or that would direct clinicians toward percutaneous interventional strategies as the preferred modality include, but are not limited to the presence of one or more of the following: 1) elevatedcardiac markers (troponin and/or CK-MB); 2) age older than 65 years; 3) presence of ST-T-wave changes; 4) TIMI Risk Score >/= 5; 5) clinical instability in the setting of suspected NSTE-ACS. Although additional refinements and changes in ACS management are still to come, evidence-based strategies suggest that prompt mechanical revascularization is associated with the best possible clinical outcomes, particularly for patients with high-risk features and in whom benefits of adjunctive, pharmacoinvasive antithrombotic therapies can be consolidated. Transfer for cardiac catheterization/percutaneous coronary intervention (PCI) is strongly recommended in patients who manifest high-risk features and/or aggressive treatment trigger criteria, so that this high-risk subgroup may receive definitive, interventional and/or cardiology-directed specialty care at appropriate sites of care. When available, interventional management is preferred in these patients. The importance of safe and effective anticoagulation in the spectrum of management strategies has been confirmed, and the evidence in support of enoxaparin and other antithrombotic agents has been reviewed. Dosing recommendations for enoxaparin use in the setting of PCI have been issued by the CATH Panel and have been summarized in this consensus report. Similar recommendations have been presented for the use of oral antiplatelet agents and GP IIb/IIIa antagonists. The addition of statins, angiotensin-converting enzyme (ACE) inhibitors and beta-blockers is also stressed as part of a comprehensive secondary cardioprotective strategy for patients with coronary heart disease.     

Why Clinicians Should Care About the Cardiac Interstitium

Interstitial heart disease, whether primarily from myocardial fibrosis or cardiac amyloidosis, indicates excess protein accumulation in the interstitium and constitutes a major source of heart failure with excess cardiac morbidity and mortality. Myocardial fibrosis (defined as excess myocardial collagen concentration that distorts myocardial architecture) is prevalent and causes cardiac symptoms and ultimately adverse cardiac events, such as heart failure, arrhythmia, and death. Conversely, cardiac amyloidosis is far less prevalent than myocardial fibrosis but represents a more extreme form of interstitial heart disease with marked interstitial expansion, profound architectural distortion, and then rapid clinical decline. Myocardial extracellular volume measures fundamentally advance the understanding of myocardium and specifically highlights the role of the interstitium. Rather than conceptualizing myocardium as a homogenous tissue, dichotomizing the myocardium into its interstitial (including the microvasculature) and cardiomyocyte phenotypes promotes additional understanding of heart failure pathophysiology that may spur the development of more effective therapies.     

Correlates of hepatitis C virus (HCV) RNA negativity among HCV-seropositive blood donors

BACKGROUND: Approximately 20 percent of persons infected with hepatitis C virus (HCV) clear viremia. Factors associated with resolution of viremia are not well defined. Implementation of routine nucleic acid testing (NAT) of blood donors has yielded a large data set for analysis of demographic correlates of resolved viremia. STUDY DESIGN AND METHODS: HCV antibody and NAT data, liver enzyme (alanine aminotransferase [ALT]) results, and donor demographic characteristics were compiled for 2,579,290 allogeneic donations given at five large blood centers after NAT implementation in 1999 through December 2001. Donation HCV RNA status was compared between first-time donors categorized by ALT levels, sex, age, race and/or ethnicity, country of birth, level of education, blood center location, and blood group, with chi-square tests and multivariable logistic regression methods. RESULTS: Of 35 confirmed-seropositive repeat donors, 19 (54.3%) tested negative for the presence of HCV RNA; there was no association between RNA status and preseroconversion intervals (p = 0.74). Of 2105 RIBA-positive, first-time donors, 402 (19.1%) tested negative for the presence of HCV RNA by NAT (presumptive resolved infections). There were significant differences in the frequency of RNA negativity among first-time donors categorized by ALT levels and by race and/or ethnicity. ALT levels were more likely to be elevated in RNA-positive, first-time donors (p < 0.0001). Viremia was less likely to resolve in Asian (8.2%) and black non-Hispanic (14.4%) donors than in white non-Hispanic (20.7%), Hispanic (22.1%), and other race and/or ethnicity (22.1%) donors (p = 0.02). No significant associations were found for age, sex, country of origin, level of education, blood type, and donor center location. CONCLUSION: These results confirm that the frequency of HCV RNA negativity among seropositive persons differs by race and/or ethnicity. Follow-up studies of donors with resolved viremia are warranted to further elucidate viral, immunologic, and genetic factors underlying spontaneous viral clearance.