Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales

Synthesis of peptidoglycan precursors ending in d-lactate (d-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by l,d-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in d-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide {"type":"entrez-nucleotide","attrs":{"text":"A40296","term_id":"2296392","term_text":"A40296"}}A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of d-Lac into cytoplasmic precursors. This was due to a d,d-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of d-Lac for d-Ala and Gly. The contribution of l,d-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-d,d-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal d-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by d,d-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of l,d-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-d,d-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that l,d-transpeptidases merely act as a tolerance mechanism in this bacterium.     

Automated LC-MS method for the fast stereoselective determination of methadone in plasma.

Methadone (MTD) is a chiral drug widely used for the treatment of opioid dependence for which a rapid analytical method for the determination of each enantiomer would be advantageous. In order to improve method sensitivity and to automate the entire analytical process, a column-switching configuration has been developed. An online extraction system coupled to a cellulose-based chiral stationary phase (CSP), namely Chiralcel OJ-R, was used and detection was performed by mass spectrometry. Fifty microl of plasma were injected into the liquid chromatography-mass spectrometry (LC-MS) system after addition of acetonitrile (ACN) containing methadone deuterated D9 (MTD-D9) (internal standard) and centrifugation. For the rapid extraction step, a large particle size support was selected. A baseline separation of MTD enantiomers was obtained in less than 12 min. Trueness and precision were evaluated with control samples at 500 ng/ml of (R,S)-methadone. Trueness values were 106.6% and 103.0% for (R)-MTD and (S)-MTD, respectively, with a coefficient of variation inferior to 2.5% for both compounds. Finally, a good concordance was found using this method for analysis of plasma samples from patients in maintenance treatment as compared to a previously described HPLC-UV method after liquid-liquid extraction.     

Molecular aspects of human FcgammaR interactions with IgG: functional and therapeutic consequences

The binding of IgG antibodies to receptors for the Fc region of IgG (FcgammaR) is a critical step for the initiation and/or the control of effector immune functions once immune complexes have been formed. Site-directed and random mutagenesis as well as domain-swapping, NMR and X-ray cristallography have made it possible to get detailed insights in the molecular mechanisms that govern IgG/FcgammaR interactions and to define some of the structural determinants that impact IgG binding to the various FcgammaR. It has demonstrated the role of particular stretches and individual residues located in the lower hinge region of the CH2 domain and in the CH2 and CH3 domains of the Fc region. The importance of the sugar components linked to asparagine 297 in the binding properties of IgG1, the human IgG isotype the most widely used in antibody-based therapies, has been also highlighted. These data have led to the engineering of a new generation of monoclonal antibodies for therapeutic use with optimized effector functions.     

Diisocyanate asthma and gene-environment interactions with IL4RA, CD-14, and IL-13 genes.

BACKGROUND: Diisocyanate asthma (DA) affects 2% to 10% of exposed workers, yet the pathogenetic mechanisms underlying this disorder remain ill defined. OBJECTIVE: To determine if specific single nucleotide polymorphisms (SNPs) of interleukin 4 receptor alpha (IL4RA), IL-13, and CD14 promoter genes are associated with DA. METHODS: Sixty-two workers with DA confirmed by specific inhalation challenge (SIC) and 75 diisocyanate-exposed, SIC-negative workers were analyzed for SNPs associated with IL4RA, IL-13, and CD14 promoter genes. RESULTS: No associations were found with individual SNPs and DA. When stratified according to specific diisocyanate exposure, a significant association was found between IL4RA (I50V) II and DA among individuals exposed to hexamethylene diisocyanate (HDI) (odds ratio [OR], 3.29; 95% confidence interval [CI], 1.33-8.14; P = .01) only. Similarly, the IL4RA (I50V) II and IL-13 (R110Q) RR combination was significantly associated with DA in HDI-exposed workers (OR, 4.13; 95% CI, 1.35-12.68; P = .01), as was the IL4RA (I50V) II and CD14 (C159T) CT genotype combination (OR, 5.2; 95% CI, 1.82-14.88; P = .002) and the triple genotype combination IL4RA (I50V) II, IL-13 (R110Q) RR, and CD14 (C159T) CT (OR, 6.4; 95% CI, 1.57-26.12; P = .01). CONCLUSIONS: Gene-environmental interactions may contribute to the pathogenesis of DA, and gene-gene interactions may modulate this relationship.     

Intima-Media Thickness in Severe Obesity: Links with BMI and metabolic status but not with systemic or adipose tissue inflammation

OBJECTIVE

Obesity is associated with cardiovascular risk and a low-grade inflammatory state in both blood and adipose tissue (AT). Whether inflammation contributes to vascular alteration remains an open question. To test this hypothesis, we measured arterial intima-media thickness (IMT), which reflects subclinical atherosclerosis, in severely obese subjects and explored associations with systemic inflammation and AT inflammation.

RESEARCH DESIGN AND METHODS

IMT of the carotid artery (C-IMT) and IMT of the femoral artery (F-IMT) were measured in 132 nonobese (control) subjects (BMI 22.3 kg/m²; mean age 44.8 years) and 232 subjects who were severely obese without diabetes (OB/ND; n = 146; BMI 48.3 kg/m²; age 38.2 years) or severely obese with type 2 diabetes (OB/D; n = 86; BMI 47.0; age 49.4 years). In 57 OB/ND subjects, circulating soluble E-selectin, matrix metalloproteinase 9, myeloperoxidase, soluble intracellular adhesion molecule 1, soluble vascular cell adhesion molecule 1, tissue plasminogen activator inhibitor 1, cystatin C, cathepsin S, and soluble CD14 were measured in serum. AT macrophages were quantified by CD68 immunochemistry.

RESULTS

Both C-IMT and F-IMT increased in OB/ND and OB/D patients. In OB/ND patients, age was the sole independent determinant of IMT. No significant association was found with circulating inflammation-related molecules, number of CD68⁺ cells, or the presence of crown-like structures in visceral or subcutaneous AT of OB/ND patients.

CONCLUSIONS

IMT increased with severe obesity but was not influenced by the degree of systemic inflammation or AT macrophage accumulation.Obesity is well-recognized as a major risk factor for the development of metabolic disorders and cardiovascular disease (CVD), such as heart failure, myocardial infarction, and stroke (1–4). Two recent studies have identified an increased risk of cardiovascular events in subjects with extreme BMI (4,5), and the number of these subjects is increasing rapidly (6,7). As shown in the Prospective Studies Collaboration (4), cardiovascular risk factors (CV-RFs) increase with BMI. In a French study conducted using the general population (6), the proportion of subjects treated for three CV-RFs (dyslipidemia, diabetes, and hypertension) was 14-fold higher for obese subjects compared with subjects of normal weight. In addition to BMI, altered body-fat distribution and ectopic fat deposition are strongly associated with mortality and morbidity attributable to CVD (8,9). It is well-established that visceral fat accumulation is associated with CV-RFs, such as hypertension, hypertriglyceridemia, or low HDL cholesterol (HDL-c), and with insulin resistance, type 2 diabetes, prothrombotic and proinflammatory states, sleep apnea, and cardiac hypertrophy, which can have potentially deleterious effects on the cardiovascular system.Although the association between obesity and increased CV-RFs is recognized, the pathophysiological pathways that link expansion of fat mass (FM) to atherosclerosis are less clearly established. Growing evidence attributes a major role to the altered biology of adipose tissue (AT) as a cause of comorbidities in obesity. Part of the systemic inflammation that characterizes obesity originates from AT, where inflammatory cells, mainly macrophages, accumulate and create local inflammation (10,11). Adipose-derived inflammatory factors produced by enlarged adipocytes or AT macrophages (or both) are often increased in the serum of obese subjects and thought to contribute to the metabolic complications of obesity, including insulin resistance and liver disease (12). In this context, it is tempting to hypothesize that products released by AT impinge on vascular cells to promote the development of atherosclerotic lesions in obesity. In line with this hypothesis, recent studies of humans report a positive association between AT inflammation, as assessed by the presence of macrophages in crown-like structures (CLS), and endothelial dysfunction, as evaluated by brachial artery flow-mediated dilatation in obese subjects (13,14).The measure of intima-media thickness (IMT) is a noninvasive marker for subclinical atherosclerosis and provides a reliable and predictive value for later cardiovascular events (15–17). Previous studies of humans have shown correlations between BMI and increased IMT (18–22). However, only one previous study (23) has investigated IMT in morbidly obese subjects and only in a limited sample.The objectives of our study were to describe the relationships between obesity-related phenotypes and IMT values at two arterial sites in a large group of massively obese subjects and to assess the potential links between systemic inflammation and AT inflammation.     

Prevalence of, associations with, and prognostic value of tricuspid annular plane systolic excursion (TAPSE) among out-patients referred for the evaluation of heart failure

BackgroundPrevalence, predictors, and prognostic value of right ventricular (RV) function measured by the tricuspid annular plane systolic excursion (TAPSE) in patients with chronic heart failure (CHF) symptoms with a broad range of left ventricular ejection fraction (LVEF) are unknown.Methods and ResultsOf 1,547 patients, mean (±SD) age was 71 ± 11 years, 48% were women, median (interquartile range [IQR]) TAPSE was 18.5 (14.0–22.7) mm, mean LVEF was 47 ± 16%, 47% had LVEF ≤45% and 67% were diagnosed with CHF, defined as systolic (S-HF) if LVEF was ≤45% and as heart failure with preserved ejection fraction (HFPEF) if LVEF was >45% and treated with a loop diuretic. During a median (IQR) follow-up of 63 (41–75) months, mortality was 34%. In multivariable analysis, increasing age, N-terminal pro–B-type natriuretic peptide (NT-proBNP), New York Heart Association functional class, right atrial volume index, and transtricuspid pressure gradient; lower TAPSE, diastolic blood pressure, and hemoglobin; and atrial fibrillation (AF) or COPD were associated with an adverse prognosis. Receiver operating characteristic curve analysis identified a TAPSE of 15.9 mm as the best prognostic threshold (P = .0001); 47% of S-HF and 20% of HFPEF had a TAPSE of <15.9 mm. The main associations with a TAPSE <15.9 mm were higher NT-proBNP, presence of atrial fibrillation and presence of LV systolic dysfunction.ConclusionsIn patients with CHF, low values for TAPSE are common, especially in those with reduced LVEF. TAPSE, unlike LVEF, was an independent predictor of outcome.