Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells.

Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the TAT-RasGAP(317-326) peptide on apoptosis of human malignant mesothelioma cells and fibroblasts exposed to meso-tetra-hydroxyphenyl-chlorin and light.

BACKGROUND: 5,10,15,20-Tetrakis(m-hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy (PDT) has shown insufficient tumor selectivity for the treatment of pleural mesothelioma. Tumor selectivity of mTHPC-PDT may be enhanced in the presence of the TAT-RasGAP(317-326) peptide which has the potential to specifically sensitize tumor cells to cytostatic agents. MATERIALS AND METHODS: H-meso-1 and human fibroblast cell cultures, respectively, were exposed to two different mTHPC doses followed by light delivery with and without TAT-RasGAP(317-326) administration. mTHPC was added to the cultures at a concentration of 0.04microg/ml and 0.10microg/ml, respectively, 24h before laser light illumination at 652nm (3J/cm(2), 40mW/cm(2)). TAT-RasGAP(317-326) was added to the cultures immediately after light delivery at a concentration of 20microM. The apoptosis rate was determined by scoring the cells displaying pycnotic nuclei. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Light delivery associated with 0.04microg/ml mTHPC resulted in a significantly higher apoptosis rate in the presence of TAT-RasGAP(317-326) than without in H-meso-1 cells (p<0.05) but not in fibroblasts. In contrast, 1.0microg/ml mTHPC and light resulted in a significantly higher apoptosis rate in both H-meso-1 cells and fibroblasts as compared to controls (p<0.05) but the addition of TAT-RasGAP(317-326) did not lead to a further significant increase of the apoptosis rate of both H-meso-1 cells and fibroblasts as compared to mTHPC and light delivery alone. CONCLUSION: TAT-RasGAP(317-326) selectively enhanced the effect of mTHPC and light delivery on H-meso-1 cells but not on fibroblasts. However, this effect was mTHPC dose-dependent and occurred only at a low sensitizer dose.

Frequent cytolytic T-cell responses to peptide MAGE-A10(254-262) in melanoma.

MAGE genes encode tumor-specific shared antigens that are among the most interesting candidates for cancer vaccines. Despite extensive studies, however, CD8+ T-cell responses to MAGE-derived epitopes have been detected only occasionally in cancer patients, even after vaccination. In contrast with these findings, we report here that HLA-A2 melanoma patients respond frequently to the recently identified peptide MAGE-A10(254-262). Indeed, as assessed by staining with fluorescent HLA-A2/peptide MAGE-A10(254-262) tetramers, CD8+ T cells directed against this peptide were readily detectable in a large proportion of HLA-A2+ melanoma patients. These results provide new insight into the immunogenicity of MAGE antigens and underline the potential usefulness of MAGE-A10 peptide-based cancer vaccines.

Modulation du métabolisme du glucose astrocytaire par les cytokines pro-inflammatoires et le peptide beta-amyloïde. A la recherche d'une composante gliale aux processus neurodégénératifs

Résumé large public: Une altération localisée du métabolisme du glucose, le substrat énergétique préférentiellement utilisé dans le cerveau, est un trait caractéristique précoce de la maladie d'Alzheimer (MA). Il est maintenant largement admis que le beta-amyloïde, la neuroinflammation et le stress oxydatif participent au développement de la MA. Cependant les mécanismes cellulaires de la pathogenèse restent à identifier. Le métabolisme cérébral a ceci de remarquable qu'il repose sur la coopération entre deux types cellulaires, ainsi les astrocytes et les neurones constituent une unité métabolique. Les astrocytes sont notamment responsables de fournir aux neurones des substrats énergétiques, ainsi que des précurseurs du glutathion pour la défense contre le stress oxydatif. Ces fonctions astrocytaires sont essentielles au bon fonctionnement et à la survie neuronale; par conséquent, une altération de ces fonctions astrocytaires pourrait participer au développement de certaines maladies cérébrales. Le but de ce travail est, dans un premier temps, d'explorer les effets de médiateurs de la neuroinflammation (les cytokines pro-inflammatoires) et du peptide beta-amyloïde sur le métabolisme des astrocytes corticaux, en se focalisant sur les éléments en lien avec le métabolisme énergétique et le stress oxydatif. Puis, dans un second temps, de caractériser les conséquences pour les neurones des modifications du métabolisme astrocytaire induites par ces substances. Les résultats obtenus ici montrent que les cytokines pro-inflammatoires et le beta-amyloïde induisent une profonde altération du métabolisme astrocytaire, selon deux profils distincts. Les cytokines pro-inflammatoires, particulièrement en combinaison, agissent comme « découpleurs » du métabolisme énergétique du glucose, en diminuant l'apport potentiel de substrats énergétiques aux neurones. En plus de son effet propre, le peptide beta-amyloïde potentialise les effets des cytokines pro-inflammatoires. Or, dans le cerveau de patients atteints de la MA, les astrocytes sont exposés simultanément à ces deux types de substances. Les deux types de substances ont un effet ambivalent en termes de stress oxydatif. Ils induisent à la fois une augmentation de la libération de glutathion (potentiellement protecteur pour les neurones voisins) et la production d'espèces réactives de l'oxygène (potentiellement toxiques). Etant donné l'importance de la coopération entre astrocytes et neurones, ces modulations du métabolisme astrocytaire pourraient donc avoir un retentissement majeur sur les cellules environnantes, et en particulier sur la fonction et la survie neuronale. Résumé Les astrocytes et les neurones constituent une unité métabolique. Les astrocytes sont notamment responsables de fournir aux neurones des substrats énergétiques, tels que le lactate, ainsi que des précurseurs du glutathion pour la défense contre le stress oxydatif. Une altération localisée du métabolisme du glucose, le substrat énergétique préférentiellement utilisé dans le cerveau, est un trait caractéristique, précoce, de la maladie d'Alzheimer (MA). Il est maintenant largement admis que le beta-amyloïde, la neuroinflammation et le stress oxydatif participent au développement de la MA. Cependant, les mécanismes cellulaires de la pathogenèse restent à identifier. Le but de ce travail est d'explorer les effets des cytokines pro-inflammatoires (Il-1 ß et TNFα) et du beta-amyloïde (Aß) sur le métabolisme du glucose des astrocytes corticaux en culture primaire ainsi que de caractériser les conséquences, pour la viabilité des neurones voisins, des modifications du métabolisme astrocytaire induites par ces substances. Les résultats obtenus montrent que les cytokines pro-inflammatoires et le beta-amyloïde induisent une profonde altération du métabolisme astrocytaire, selon deux profils distincts. Les cytokines pro-inflammatoires, particulièrement en combinaison, agissent comme « découpleurs » du métabolisme glycolytique astrocytaire. Après 48 heures, le traitement avec TNFα et Il-lß cause une augmentation de la capture de glucose et de son métabolisme dans la voie des pentoses phosphates et dans le cycle de Krebs. A l'inverse, il cause une diminution de la libération de lactate et des stocks cellulaires de glycogène. En combinaison avec les cytokines tel qu'in vivo dans les cerveaux de patients atteints de MA, le peptide betaamyloïde potentialise les effets décrits ci-dessus. Isolément, le Aß cause une augmentation coordonnée de la capture de glucose et de toutes les voies de son métabolisme (libération de lactate, glycogenèse, voie des pentoses phosphate et cycle de Krebs). Les traitements altèrent peu les taux de glutathion intracellulaires, par contre ils augmentent massivement la libération de glutathion dans le milieu extracellulaire. A l'inverse, les deux types de traitements augmentent la production intracellulaire d'espèces réactives de l'oxygène (ROS). De plus, les cytokines pro-inflammatoires en combinaison augmentent massivement la production des ROS dans l'espace extracellulaire. Afin de caractériser l'impact de ces altérations métaboliques sur la viabilité des neurones environnants, un modèle de co-culture et des milieux conditionnés astrocytaires ont été utilisés. Les résultats montrent qu'en l'absence d'une source exogène d'antioxydants, la présence d'astrocytes favorise la viabilité neuronale ainsi que leur défense contre le stress oxydatif. Cette propriété n'est cependant pas modulée par les différents traitements. D'autre part, la présence d'astrocytes, et non de milieu conditionné, protège les neurones contre l'excitotoxicité due au glutamate. Les astrocytes prétraités (aussi bien avec le beta-amyloïde qu'avec les cytokines pro-inflammatoires) perdent cette propriété. Cet élément suggère que la perturbation du métabolisme astrocytaire causé par les cytokines pro-inflammatoires ou le beta-amyloïde pourrait participer à l'atteinte de la viabilité neuronale associée à certaines pathologies neurodégénératives.

Vergleich von oraler und parenteraler Verabreichung von Glucagon-like Peptide-1 : Pilotprojekt

Résumé : Le Glucagon-Like Peptide 1 (GLP-1) est synthétisé par les cellules L du tractus gastro-intestinal et est sécrété dans le sang lors du repas. Grâce à ses fonctions d'hormone de satiété et d'incrétine, il joue un rôle important dans le système complexe de l'homéostase énergétique. Dans ce contexte, cette molécule est intéressante dans la thérapie du diabète sucré de type 2 et de l'obésité. Comme tous les peptides, le GLP-1 est rapidement dégradé par l'acidité gastrique et les enzymes digestifs lors de son administration orale ;c'est pourquoi il est administré uniquement par voie intra-veineuse ou sous-cutanée. Le but de cette étude était d'analyser l'absorption intestinale du GLP-1 administré simultanément avec une molécule de type «promoteur de l'absorption». C'était une étude phase 1, ouverte, placébo-contrôlée, avec un «cross-ovér» à 5 bras à des doses croissantes. Le promoteur de l'absorption était une molécule appelée SNAC (sodium N-(8-(2-hydroxybenzoyl)amino) caprylate) qui protège les peptides de la dégradation intestinale et améliore leur absorption. Les valeurs de GLP-1, d'insuline et de glucose mesurées dans les prises de sang ont montré clairement que le peptide a pu être absorbé grâce à la molécule SNAC et a de ce fait stimulé la sécrétion d'insuline. Lors du deuxième dosage, cette sécrétion d'insuline avait déjà atteint un niveau maximal qui n'augmentait plus, même avec des dosages plus élevés. Les concentrations de GLP-1 ont atteint des valeurs pharmacologiques et ont augmenté en fonction de la dose (pour la AUC et pour Cmax). En comparaison avec l'administration veineuse de GLP-1, l'administration orale a démontré une biodisponibilité calculée de 4% en moyenne. L'étude montre que le GLP-1 peut être administré par voie orale grâce à des promoteurs de l'absorption. Dès lors, des études thérapeutiques avec le GLP-1 administré oralement peuvent être entreprises.

Degeneracy of antigen recognition as the molecular basis for the high frequency of naive A2/Melan-a peptide multimer(+) CD8(+) T cells in humans.

In contrast with the low frequency of most single epitope reactive T cells in the preimmune repertoire, up to 1 of 1,000 naive CD8(+) T cells from A2(+) individuals specifically bind fluorescent A2/peptide multimers incorporating the A27L analogue of the immunodominant 26-35 peptide from the melanocyte differentiation and melanoma associated antigen Melan-A. This represents the only naive antigen-specific T cell repertoire accessible to direct analysis in humans up to date. To get insight into the molecular basis for the selection and maintenance of such an abundant repertoire, we analyzed the functional diversity of T cells composing this repertoire ex vivo at the clonal level. Surprisingly, we found a significant proportion of multimer(+) clonotypes that failed to recognize both Melan-A analogue and parental peptides in a functional assay but efficiently recognized peptides from proteins of self- or pathogen origin selected for their potential functional cross-reactivity with Melan-A. Consistent with these data, multimers incorporating some of the most frequently recognized peptides specifically stained a proportion of naive CD8(+) T cells similar to that observed with Melan-A multimers. Altogether these results indicate that the high frequency of Melan-A multimer(+) T cells can be explained by the existence of largely cross-reactive subsets of naive CD8(+) T cells displaying multiple specificities.

Crystal structures of two closely related but antigenically distinct HLA-A2/melanocyte-melanoma tumor-antigen peptide complexes.

We have determined high-resolution crystal structures of the complexes of HLA-A2 molecules with two modified immunodominant peptides from the melanoma tumor-associated protein Melan-A/Melanoma Ag recognized by T cells-1. The two peptides, a decamer and nonamer with overlapping sequences (ELAGIGILTV and ALGIGILTV), are modified in the second residue to increase their affinity for HLA-A2. The modified decamer is more immunogenic than the natural peptide and a candidate for peptide-based melanoma immunotherapy. The crystal structures at 1.8 and 2.15 A resolution define the differences in binding modes of the modified peptides, including different clusters of water molecules that appear to stabilize the peptide-HLA interaction. The structures suggest both how the wild-type peptides would bind and how three categories of cytotoxic T lymphocytes with differing fine specificity might recognize the two peptides.

The CD8 beta polypeptide is required for the recognition of an altered peptide ligand as an agonist.

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 alpha and beta chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8beta in the CD8 co-receptor dimers expressed at the T cell surface.

Activation of electrical triggers of atrial fibrillation in hyperthyroidism.

CONTEXT: A shortening of the atrial refractory period has been considered as the main mechanism for the increased risk of atrial fibrillation in hyperthyroidism. However, other important factors may be involved. OBJECTIVE: Our objective was to determine the activity of abnormal supraventricular electrical depolarizations in response to elevated thyroid hormones in patients without structural heart disease. PATIENTS AND DESIGN: Twenty-eight patients (25 females, three males, mean age 43+/-11 yr) with newly diagnosed and untreated hyperthyroidism were enrolled in a prospective trial after exclusion of heart disease. Patients were followed up for 16 +/- 6 months and studied at baseline and 6 months after normalization of serum TSH levels. MAIN OUTCOME MEASURES: The incidence of abnormal premature supraventricular depolarizations (SVPD) and the number of episodes of supraventricular tachycardia was defined as primary outcome measurements before the start of the study. In addition, heart rate oscillations (turbulence) after premature depolarizations and heart rate variability were compared at baseline and follow-up. RESULTS: SVPDs decreased from 59 +/- 29 to 21 +/- 8 per 24 h (P = 0.003), very early SVPDs (so called P on T) decreased from 36 +/- 24 to 3 +/- 1 per 24 h (P < 0.0001), respectively, and nonsustained supraventricular tachycardias decreased from 22 +/- 11 to 0.5 +/- 0.2 per 24 h (P = 0.01) after normalization of serum thyrotropin levels. The hyperthyroid phase was characterized by an increased heart rate (93 +/- 14 vs. 79 +/- 8 beats/min, P < 0.0001) and a decreased turbulence slope (3.6 vs. 9.2, P = 0.003), consistent with decreased vagal tone. This was confirmed by a significant decrease of heart rate variability. CONCLUSION: Hyperthyroidism is associated with an increased supraventricular ectopic activity in patients with normal hearts. The activation of these arrhythmogenic foci by elevated thyroid hormones may be an important causal link between hyperthyroidism and atrial fibrillation.

A biophysical model of atrial fibrillation ablation: what can a surgeon learn from a computer model?

AIMS: Surgical ablation procedures for treating atrial fibrillation have been shown to be highly successful. However, the ideal ablation pattern still remains to be determined. This article reports on a systematic study of the effectiveness of the performance of different ablation line patterns. METHODS AND RESULTS: This study of ablation line patterns was performed in a biophysical model of human atria by combining basic lines: (i) in the right atrium: isthmus line, line between vena cavae and appendage line and (ii) in the left atrium: several versions of pulmonary vein isolation, connection of pulmonary veins, isthmus line, and appendage line. Success rates and the presence of residual atrial flutter were documented. Basic patterns yielded conversion rates of only 10-25 and 10-55% in the right and the left atria, respectively. The best result for pulmonary vein isolation was obtained when a single closed line encompassed all veins (55%). Combination of lines in the right/left atrium only led to a success rate of 65/80%. Higher rates, up to 90-100%, could be obtained if right and left lines were combined. The inclusion of a left isthmus line was found to be essential for avoiding uncommon left atrial flutter. CONCLUSION: Some patterns studied achieved a high conversion rate, although using a smaller number of lines than those of the Maze III procedure. The biophysical atrial model is shown to be effective in the search for promising alternative ablation strategies.

Atrial arrhythmias in adult patients with right- versus left-sided congenital heart disease anomalies.

Atrial arrhythmias (AAs) are a common complication in adult patients with congenital heart disease. We sought to compare the lifetime prevalence of AAs in patients with right- versus left-sided congenital cardiac lesions and their effect on the prognosis. A congenital heart disease diagnosis was assigned using the International Disease Classification, Ninth Revision, diagnostic codes in the administrative databases of Quebec, from 1983 to 2005. Patients with AAs were those diagnosed with an International Disease Classification, Ninth Revision, code for atrial fibrillation or intra-atrial reentry tachycardia. To ensure that the diagnosis of AA was new, a washout period of 5 years after entry into the database was used, a period during which the patient could not have received an International Disease Classification, Ninth Revision, code for AA. The cumulative lifetime risk of AA was estimated using the Practical Incidence Estimators method. The hazard ratios (HRs) for mortality, morbidity, and cardiac interventions were compared between those with right- and left-sided lesions after adjustment for age, gender, disease severity, and cardiac risk factors. In a population of 71,467 patients, 7,756 adults developed AAs (isolated right-sided, 2,229; isolated left-sided, 1,725). The lifetime risk of developing AAs was significantly greater in patients with right- sided than in patients with left-sided lesions (61.0% vs 55.4%, p <0.001). The HR for mortality and the development of stroke or heart failure was similar in both groups (HR 0.96, 95% confidence interval [CI] 0.86 to 1.09; HR 0.94, 95% CI 0.80 to 1.09; and HR 1.10, 95% CI 0.98 to 1.23, respectively). However, the rates of cardiac catheterization (HR 0.63, 95% CI 0.55 to 0.72), cardiac surgery (HR 0.40, 95% CI 0.36 to 0.45), and arrhythmia surgery (HR 0.77, 95% CI 0.6 to 0.98) were significantly less for patients with right-sided lesions. In conclusion, patients with right-sided lesions had a greater lifetime burden of AAs. However, their morbidity and mortality were no less than those with left-sided lesions, although the rate of intervention was substantially different.

Effects of epitope modification on T cell receptor-ligand binding and antigen recognition by seven H-2Kd-restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative.

We tested for antigen recognition and T cell receptor (TCR)-ligand binding 12 peptide derivative variants on seven H-2Kd-restricted cytotoxic T lymphocytes (CTL) clones specific for a bifunctional photoreactive derivative of the Plasmodium berghei circumsporozoite peptide 252-260 (SYIPSAEKI). The derivative contained iodo-4-azidosalicylic acid in place of PbCS S-252 and 4-azidobenzoic acid on PbCS K-259. Selective photoactivation of the N-terminal photoreactive group allowed crosslinking to Kd molecules and photoactivation of the orthogonal group to TCR. TCR photoaffinity labeling with covalent Kd-peptide derivative complexes allowed direct assessment of TCR-ligand binding on living CTL. In most cases (over 80%) cytotoxicity (chromium release) and TCR-ligand binding differed by less than fivefold. The exceptions included (a) partial TCR agonists (8 cases), for which antigen recognition was five-tenfold less efficient than TCR-ligand binding, (b) TCR antagonists (2 cases), which were not recognized and capable of inhibiting recognition of the wild-type conjugate, (c) heteroclitic agonists (2 cases), for which antigen recognition was more efficient than TCR-ligand binding, and (d) one partial TCR agonist, which activated only Fas (C1)95), but not perforin/granzyme-mediated cytotoxicity. There was no correlation between these divergences and the avidity of TCR-ligand binding, indicating that other factors than binding avidity determine the nature of the CTL response. An unexpected and novel finding was that CD8-dependent clones clearly incline more to TCR antagonism than CD8-independent ones. As there was no correlation between CD8 dependence and the avidity of TCR-ligand binding, the possibility is suggested that CD8 plays a critical role in aberrant CTL function.

Intermittent atrial tachycardia facilitates atrial fibrillation by a shortening of activation recovery interval

Introduction: We recently observed in a chronic ovine model that a shortening of action potential duration (APD) as assessed by the activation recovery interval (ARI) may be a mechanism whereby pacing-induced atrial tachycardia (PIAT) facilitates atrial fibrillation (AF), mediated by a return to 1:1 atrial capture after the effective refractory period has been reached. The aim of the present study is to evaluate the effect of long term intermittent burst pacing on ARI before induction of AF.Methods: We specifically developed a chronic ovine model of PIAT using two pacemakers (PM) each with a right atrial (RA) lead separated by ∼2cm. The 1st PM (Vitatron T70) was used to record a broadband unipolar RA EGM (800 Hz, 0.4 Hz high pass filter). The 2nd was used to deliver PIAT during electrophysiological protocols at decremental pacing CL (400 beats, from 400 to 110ms) and long term intermittent RA burst pacing to promote electrical remodeling (5s of burst followed by 2s of sinus rhythm) until onset of sustained AF. ARI was defined as the time difference between the peak of the atrial repolarization wave and the first atrial depolarization. The mean ARIs of paired sequences (before and after remodeling), each consisting of 20 beats were compared.Results: As shown in the figure, ARIs (n=4 sheep, 46 recordings) decreased post remodeling compared to baseline (86±19 vs 103±12 ms, p<0.05). There was no difference in atrial structure as assessed by light microscopy between control and remodeled sheep.Conclusions: Using standard pacemaker technology, atrial ARIs as a surrogate of APDs were successfully measured in vivo during the electrical remodeling process leading to AF. The facilitation of AF by PIAT mimicking salvos from pulmonary veins is heralded by a significant shortening of ARI.