The predictive value of preoperative B-type natriuretic peptide in children undergooing heart surgery.

Introduction: Low cardiac output syndrome is frequent in childrenafter heart surgery for congenital heart disease and may result in pooroutcome and increased morbidity. In the adult population, preoperativebrain natriuretic peptide (BNP) was shown to be predictive of postoperative complications. In children, the value of preoperative BNP onpostoperative outcome is not so clear. The aim of this study was todetermine the predictive value of preoperative BNP on postoperativeoutcome and low cardiac output syndrome in children after heartsurgery for congenital heart disease.Methods: We examined, retrospectively, the postoperative course of97 pediatric patients (mean age 3.7 years, range 0-14 years old) whounderwent heart surgery in a tertiary care pediatric intensive caresetting. NTproBNP was measured preoperatively in all patients(median 412 pg/ml, range 12-35'000 pg/ml). Patients were divided intothree groups according to their NTproBNP levels (group 1: 0-300 pg/ml, group 2: 300-600 pg/ml, group 3: >600 pg/ml) and then,correlations with postoperative outcomes were examined.Results: We found that patients with a high preoperative BNP requiredmore frequently prolonged (>2 days) mechanical ventilation (33%vs 40% vs 61%, p = 0.045) and stayed more frequently longer than6 days in the intensive care unit (42% vs 50% vs 71%, p = 0.03).However, high preoperative BNP was not correlated with occurrenceof low cardiac output syndrome.Conclusion: Preoperative BNP cannot be used, in children, as areliable and sole predictor of postoperative low cardiac outputsyndrome. However it may help identify, before surgery, those patientsat risk of having a difficult postoperative course.

How T cell receptors interact with peptide-MHCs: A multiple steered molecular dynamics study.

The T-cell receptor (TCR) interaction with antigenic peptides (p) presented by the major histocompatibility complex (MHC) molecule is a key determinant of immune response. In addition, TCR-pMHC interactions offer examples of features more generally pertaining to protein-protein recognition: subtle specificity and cross-reactivity. Despite their importance, molecular details determining the TCR-pMHC binding remain unsolved. However, molecular simulation provides the opportunity to investigate some of these aspects. In this study, we perform extensive equilibrium and steered molecular dynamics simulations to study the unbinding of three TCR-pMHC complexes. As a function of the dissociation reaction coordinate, we are able to obtain converged H-bond counts and energy decompositions at different levels of detail, ranging from the full proteins, to separate residues and water molecules, down to single atoms at the interface. Many observed features do not support a previously proposed two-step model for TCR recognition. Our results also provide keys to interpret experimental point-mutation results. We highlight the role of water both in terms of interface resolvation and of water molecules trapped in the bound complex. Importantly, we illustrate how two TCRs with similar reactivity and structures can have essentially different binding strategies. Proteins 2011; © 2011 Wiley-Liss, Inc.

Atrial septal pacing for the termination of atrial fibrillation: study in a biophysical model of human atria.

AIMS: While successful termination by pacing of organized atrial tachycardias has been observed in patients, single site rapid pacing has not yet led to conclusive results for the termination of atrial fibrillation (AF). The purpose of this study was to evaluate a novel atrial septal pacing algorithm for the termination of AF in a biophysical model of the human atria. METHODS AND RESULTS: Sustained AF was generated in a model based on human magnetic resonance images and membrane kinetics. Rapid pacing was applied from the septal area following a dual-stage scheme: (i) rapid pacing for 10-30 s at pacing intervals 62-70% of AF cycle length (AFCL), (ii) slow pacing for 1.5 s at 180% AFCL, initiated by a single stimulus at 130% AFCL. Atrial fibrillation termination success rates were computed. A mean success rate for AF termination of 10.2% was obtained for rapid septal pacing only. The addition of the slow pacing phase increased this rate to 20.2%. At an optimal pacing cycle length (64% AFCL) up to 29% of AF termination was observed. CONCLUSION: The proposed septal pacing algorithm could suppress AF reentries in a more robust way than classical single site rapid pacing. Experimental studies are now needed to determine whether similar termination mechanisms and rates can be observed in animals or humans, and in which types of AF this pacing strategy might be most effective.

Impact of personality on termination of short-term group psychotherapy in depressed elderly outpatients

Objective Termination in group psychotherapy is an essential phase of the psychotherapeutic process, yet its clinical determinants remain largely unknown, especially in elderly patients. The aim of this study was to assess how patients' personality traits influence their way of leaving a short-term psychotherapy group as well as a larger therapeutic community program. <p>Method Personality traits were assessed with the NEO Five-Factor Inventory in 24 elderly depressed outpatients. Patients' terminations from the group as well as from the community were ranked into four classes according to their appropriateness (completeness of experience and ability to deal with feelings of separation). Results Neuroticism was not related to the quality of termination. In contrast, agreeableness and openness to experience were strongly associated with successful termination. Conscientiousness and extraversion may have a differential impact depending on the type of group (group psychotherapy versus therapeutic community). Conclusion Personality traits may be important clinical determinants of the quality of termination process in both group psychotherapy and therapeutic community settings for elderly depressed patients.

High frequency of functionally active Melan-a-specific T cells in a patient with progressive immunoproteasome-deficient melanoma.

Tumor-reactive T cells play an important role in cancer immunosurveillance. Applying the multimer technology, we report here an unexpected high frequency of Melan-A-specific CTLs in a melanoma patient with progressive lymph node metastases, consisting of 18 and 12.8% of total peripheral blood and tumor-infiltrating CD8+ T cells, respectively. Melan-A-specific CTLs revealed a high cytolytic activity against allogeneic Melan-A-expressing target cells but failed to kill the autologous tumor cells. Loading of the tumor cells with Melan-A peptide reversed the resistance to killing, suggesting impaired function of the MHC class I antigen processing and presentation pathway. Mutations of the coding region of the HLA-A2 binding Melan-A26-35 peptide or down-regulation of the MHC class I heavy chain, the antigenic peptide TAP, and tapasin could be excluded. However, PCR and immunohistochemical analysis revealed a deficiency of the immunoproteasomes low molecular weight protein 2 and low molecular weight protein 7 in the primary tumor cells, which affects the quantity and quality of generated T-cell epitopes and might explain the resistance to killing. This is supported by our data, demonstrating that the resistance to killing can be partially reversed by pre-exposure of the tumor cells to IFN-gamma, which is known to induce the immunoproteasomes. Overall, this is the first report of an extremely high frequency of tumor-specific CTLs that exhibit competent T-cell-effector functions but fail to lyse the autologous tumor cells. Immunotherapeutic approaches should not only focus on the induction of a robust antitumor immune response, but should also have to target tumor immune escape mechanisms.

Characterization of the enzyme involved in the processing of big endothelin-1 in human lung epithelial cells.

Biosynthesis of active endothelin-1 (ET-1) implies an enzymatic processing of the inactive precursor Big ET-1 (1-39) into the mature, 21 amino acid peptide. The aim of this study was to characterize in airway and alveolar epithelial cells the enzymes responsible for this activation. BEAS-2B and A549 cells, which both produce ET-1, were studied in vitro as models for bronchiolar and alveolar cells, respectively. Both cell lines were able to convert exogenously added Big ET-1 (0.1 microM) into ET-1, suggesting a cell surface or an extracellular processing. The conversion was inhibited by phosphoramidon in both cell lines with an IC50 approximately 1 microM, but not by thiorphan, a specific inhibitor of neutral endopeptidase 24.11 (NEP). The endogenous production of serum-stimulated BEAS-2B and A549 cells was not inhibited by thiorphan, and phosphoramidon showed inhibition only at high concentration (>100 microM). Western blotting following electrophoresis in reducing conditions demonstrated a protein of MR 110 corresponding to the ECE-1 monomer in both BEAS-2B and A549 cells, as well as in whole lung extracts. By RT-PCR we revealed the mRNA encoding for the ECE-1b and/or -1c subtype, but not ECE-1a, in both cell lines. We conclude that BEAS-2B and A549 cells are able to process either endogenous or exogenous Big ET-1 by ECE-1 and that isoforms 1b and 1c could be involved in this processing with no significant role of NEP.

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness.

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ∼1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ∼1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.

Connexins, diabetes and the metabolic syndrome.

Diabetes and the related metabolic syndrome are multi system disorders that result from improper interactions between various cell types. Even though the underlying mechanism remains to be fully understood, it is most likely that both the long and the short distance range cell interactions, which normally ensure the physiologic functioning of the pancreas, and its relationships with the insulin-targeted organs, are altered. This review focuses on the short-range type of interactions that depend on the contact between adjacent cells and, specifically, on the interactions that are dependent on connexins. The widespread distribution of these membrane proteins, their multiple modes of action, and their interactions with conditions/molecules associated to both the pathogenesis and the treatment of the 2 main forms of diabetes and the metabolic syndrome, make connexins an essential part of the chain of events that leads to metabolic diseases. Here, we review the present state of knowledge about the molecular and cell biology of the connexin genes and proteins, their general mechanisms of action, the roles specific connexin species play in the endocrine pancreas and the major insulin-targeted organs, under physiological and patho-physiological conditions.

Mutational analysis of class A and class B penicillin-binding proteins in Streptococcus gordonii.

High-molecular-weight (HMW) penicillin-binding proteins (PBPs) are divided into class A and class B PBPs, which are bifunctional transpeptidases/transglycosylases and monofunctional transpeptidases, respectively. We determined the sequences for the HMW PBP genes of Streptococcus gordonii, a gingivo-dental commensal related to Streptococcus pneumoniae. Five HMW PBPs were identified, including three class A (PBPs 1A, 1B, and 2A) and two class B (PBPs 2B and 2X) PBPs, by homology with those of S. pneumoniae and by radiolabeling with [3H]penicillin. Single and double deletions of each of them were achieved by allelic replacement. All could be deleted, except for PBP 2X, which was essential. Morphological alterations occurred after deletion of PBP 1A (lozenge shape), PBP 2A (separation defect and chaining), and PBP 2B (aberrant septation and premature lysis) but not PBP 1B. The muropeptide cross-link patterns remained similar in all strains, indicating that cross-linkage for one missing PBP could be replaced by others. However, PBP 1A mutants presented shorter glycan chains (by 30%) and a relative decrease (25%) in one monomer stem peptide. Growth rate and viability under aeration, hyperosmolarity, and penicillin exposure were affected primarily in PBP 2B-deleted mutants. In contrast, chain-forming PBP 2A-deleted mutants withstood better aeration, probably because they formed clusters that impaired oxygen diffusion. Double deletion could be generated with any PBP combination and resulted in more-altered mutants. Thus, single deletion of four of the five HMW genes had a detectable effect on the bacterial morphology and/or physiology, and only PBP 1B seemed redundant a priori.

Structure-function characterization and optimization of a plant-derived antibacterial peptide.

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.

Subconjunctival Injection of XG-102, a c-Jun N-Terminal Kinase Inhibitor Peptide, in the Treatment of Endotoxin-Induced Uveitis in Rats.

Abstract Purpose: XG-102, a TAT-coupled dextrogyre peptide inhibiting the c-Jun N-terminal kinase, was shown efficient in the treatment of experimental uveitis. Preclinical studies are now performed to determine optimal XG-102 dose and route of administration in endotoxin-induced uveitis (EIU) in rats with the purpose of clinical study design. METHODS: EIU was induced in Lewis rats by lipopolysaccharides (LPS) injection. XG-102 was administered at the time of LPS challenge by intravenous (IV; 3.2, 35 or 355 μg/injection), intravitreal (IVT; 0.08, 0.2 or 2.2 μg/eye), or subconjunctival (SCJ; 0.2, 1.8 or 22 μg/eye) routes. Controls received either the vehicle (saline) or dexamethasone phosphate injections. Efficacy was assessed by clinical scoring, infiltrating cells count, and expression of inflammatory mediators [inducible nitric oxide synthase (iNOS), cytokine-induced neutrophil chemoattractant-1 (CINC-1)]. The effect of XG-102 on phosphorylation of c-Jun was evaluated by Western blot. RESULTS: XG-102 demonstrated a dose-dependent anti-inflammatory effect in EIU after IV and SCJ administrations. Respective doses of 35 and 1.8 μg were efficient as compared with the vehicle-injected controls, but only the highest doses, respectively 355 and 22 μg, were as efficient as dexamethasone phosphate. After IVT injections, the anti-inflammatory effect of XG-102 was clinically evaluated similar to the corticoid's effect with all the tested doses. Regardless of the administration route, the lowest efficient doses of XG-102 significantly decreased the ration of phospho c-Jun/total c-Jun, reduced cells infiltration in the treated eyes, and significantly downregulated iNOS and CINC-1 expression in the retina. CONCLUSION: These results confirm that XG-102 peptide has potential for treating intraocular inflammation. SCJ injection appears as a good compromise to provide a therapeutic effect while limiting side effects.

Adaptive frequency tracking of the baseline ECG identifies the site of atrial fibrillation termination by catheter ablation

Multiple organization indices have been used to predict the outcome of stepwise catheter ablation in long-standing persistent atrial fibrillation (AF), however with limited success. Our study aims at developinginnovative organization indices from baseline ECG (i.e. during the procedure, before ablation) in orderto identify the site of AF termination by catheter ablation. Seventeen consecutive male patients (age60 ± 5 years, AF duration 7 ± 5 years) underwent a stepwise catheter ablation. Chest lead V6 was placedin the back (V6b). QRST cancelation was performed from chest leads V1 to V6b. Using an innovativeadaptive harmonic frequency tracking, two measures of AF organization were computed to quantify theharmonics components of ECG activity: (1) the adaptive phase difference variance (APD) between theAF harmonic components as a measure of AF regularity, and (2) and adaptive organization index (AOI)evaluating the cyclicity of the AF oscillations. Both adaptive indices were compared to indices computedusing a time-invariant approach: (1) ECG AF cycle length (AFCL), (2) the spectrum based organizationindex (OI), and (3) the time-invariant phase difference TIPD. Long-standing persistent AF was terminatedinto sinus rhythm or atrial tachycardia in 13/17 patients during stepwise ablation, 11 during left atriumablation (left terminated patients - LT), 2 during the right atrium ablation (right terminated patients -RT), and 4 were non terminated (NT) and required electrical cardioversion. Our findings showed that LTpatients were best separated from RT/NT before ablation by the duration of sustained AF and by AOI onchest lead V1 and APD from the dorsal lead V6b as compared to ECG AFCL, OI and TIPD, respectively. Ourresults suggest that adaptive measures of AF organization computed before ablation perform better thantime-invariant based indices for identifying patients whose AF will terminate during ablation within theleft atrium. These findings are indicative of a higher baseline organization in these patients that could beused to select candidates for the termination of AF by stepwise catheter ablation.© 2013 Elsevier Ltd. All rights reserved.

Induction of potent antitumor CTL responses by recombinant vaccinia encoding a melan-A peptide analogue.

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.

The final N-terminal trimming of a subaminoterminal proline-containing HLA class I-restricted antigenic peptide in the cytosol is mediated by two peptidases.

The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.

Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.