Expression of neuroectodermal antigens common to melanomas, gliomas, and neuroblastomas. I. Identification by monoclonal anti-melanoma and anti-glioma antibodies.

The reactivity spectrum of five different monoclonal anti-melanoma antibodies cross-reacting with gliomas and neuroblastomas and one monoclonal anti-glioma antibody cross-reacting with melanomas and neuroblastomas was investigated. Comparison of the binding activity of these monoclonal antibodies for 11 melanoma, seven glioma, and three neuroblastoma cell lines showed that each of these clones had a different pattern of cross-reactivity. The results indicated that the antigenic determinants detected by these antibodies were not associated with the same antigen and thus suggested the existence of at least six different antigens common to melanomas, gliomas, and neuroblastomas. Since all these tumors are known to derive from cells originating embryologically from the neural crest, it can be assumed that the antigens recognized by our monoclonal antibodies are neuroectodermal differentiation antigens. However, absorption with fetal brain homogenates abolished only the binding of monoclonal anti-glioma antibody, but did not modify the binding of monoclonal anti-melanoma antibodies.

A comparative analysis by means of quantum molecular similarity measures of density distributions derived from conventional ab initio and density functional methods

A procedure based on quantum molecular similarity measures (QMSM) has been used to compare electron densities obtained from conventional ab initio and density functional methodologies at their respective optimized geometries. This method has been applied to a series of small molecules which have experimentally known properties and molecular bonds of diverse degrees of ionicity and covalency. Results show that in most cases the electron densities obtained from density functional methodologies are of a similar quality than post-Hartree-Fock generalized densities. For molecules where Hartree-Fock methodology yields erroneous results, the density functional methodology is shown to yield usually more accurate densities than those provided by the second order Møller-Plesset perturbation theory

Ab initio benchmark study for the oxidative addition of CH4 to Pd: importance of basis-set flexibility and polarization

To obtain a state-of-the-art benchmark potential energy surface (PES) for the archetypal oxidative addition of the methane C-H bond to the palladium atom, we have explored this PES using a hierarchical series of ab initio methods (Hartree-Fock, second-order Møller-Plesset perturbation theory, fourth-order Møller-Plesset perturbation theory with single, double and quadruple excitations, coupled cluster theory with single and double excitations (CCSD), and with triple excitations treated perturbatively [CCSD(T)]) and hybrid density functional theory using the B3LYP functional, in combination with a hierarchical series of ten Gaussian-type basis sets, up to g polarization. Relativistic effects are taken into account either through a relativistic effective core potential for palladium or through a full four-component all-electron approach. Counterpoise corrected relative energies of stationary points are converged to within 0.1-0.2 kcal/mol as a function of the basis-set size. Our best estimate of kinetic and thermodynamic parameters is -8.1 (-8.3) kcal/mol for the formation of the reactant complex, 5.8 (3.1) kcal/mol for the activation energy relative to the separate reactants, and 0.8 (-1.2) kcal/mol for the reaction energy (zero-point vibrational energy-corrected values in parentheses). This agrees well with available experimental data. Our work highlights the importance of sufficient higher angular momentum polarization functions, f and g, for correctly describing metal-d-electron correlation and, thus, for obtaining reliable relative energies. We show that standard basis sets, such as LANL2DZ+ 1f for palladium, are not sufficiently polarized for this purpose and lead to erroneous CCSD(T) results. B3LYP is associated with smaller basis set superposition errors and shows faster convergence with basis-set size but yields relative energies (in particular, a reaction barrier) that are ca. 3.5 kcal/mol higher than the corresponding CCSD(T) values

A representação do portador do vírus da imunodeficiência humana sobre o tratamento com os anti-retrovirais

Tendo como objeto de investigação a percepção dos portadores do HIV/AIDS sobre a adesão à terapêutica com medicamentos anti-retrovirais, este estudo de abordagem qualitativa teve como objetivos: conhecer as representações sociais dos portadores do HIV/AIDS sobre a terapêutica medicamentosa e analisar a relação entre a percepção do portador do HIV/AIDS sobre a terapêutica medicamentosa e a motivação para aderir ao tratamento. Os sujeitos do estudo foram nove usuários, internados na unidade de doenças infecciosas de um hospital de ensino na cidade de Juiz de Fora. Os dados foram colhidos através de entrevista semi-estruturada. A categoria de análise permitiu discutir as diversas facetas da adesão aos anti-retrovirais, partindo das representações elaboradas pelos sujeitos que fazem o tratamento, os quais, tomando como mediação o diálogo, falam dos aspectos dificultadores e as motivações para a gestão do tratamento.

In vivo localisation of radiolabelled monoclonal antibody in human gliomas.

F(ab')2-fragments of the anti-melanoma monoclonal antibody MeI-14 were labelled with 123I for external scanning and with 125I for tissue measurement of radioactivity and injected intravenously into patients scheduled for surgical resection of a glioma. The paired-label study was performed by injecting simultaneously 131I-labelled control (F(ab')2-fragments. The patients were scanned by computerised tomoscintigraphy. After surgery, the activities of 125I and 131I were counted in tumour and normal tissues. The results indicate that there was a low but definite uptake of the antibody in the tumour due to its specificity. The external detection was difficult because of accumulation of antibody fragments in the skull.

T-cell activation by treatment of cancer patients with EMD 521873 (Selectikine), an IL-2/anti-DNA fusion protein.

ABSTRACT: BACKGROUND: EMD 521873 (Selectikine or NHS-IL2LT) is a fusion protein consisting of modified human IL-2 which binds specifically to the high-affinity IL-2 receptor, and an antibody specific for both single- and double-stranded DNA, designed to facilitate the enrichment of IL-2 in tumor tissue. METHODS: An extensive analysis of pharmacodynamic (PD) markers associated with target modulation was assessed during a first-in-human phase I dose-escalation trial of Selectikine. RESULTS: Thirty-nine patients with metastatic or locally advanced tumors refractory to standard treatments were treated with increasing doses of Selectikine, and nine further patients received additional cyclophosphamide. PD analysis, assessed during the first two treatment cycles, revealed strong activation of both CD4+ and CD8+ T-cells and only weak NK cell activation. No dose response was observed. As expected, Treg cells responded actively to Selectikine but remained at lower frequency than effector CD4+ T-cells. Interestingly, patient survival correlated positively with both high lymphocyte counts and low levels of activated CD8+ T-cells at baseline, the latter of which was associated with enhanced T-cell responses to the treatment. CONCLUSIONS: The results confirm the selectivity of Selectikine with predominant T-cell and low NK cell activation, supporting follow-up studies assessing the clinical efficacy of Selectikine for cancer patients.

Cytokine targeting in tumors using a bispecific antibody directed against carcinoembryonic antigen and tumor necrosis factor alpha.

The use of tumor necrosis factor alpha (TNFalpha) in cancer therapy is limited by its short circulatory half-life and its severe systemic side effects. To overcome these limitations, we evaluated the capability of a bispecific antibody (BAb) directed against carcinoembryonic antigen (CEA) and human TNFalpha to target this cytokine in tumors. A BAb was constructed by coupling the Fab' fragments from an anti-CEA monoclonal antibody (MAb) to the Fab' fragments from an anti-TNFalpha MAb via a stable thioether linkage. The double specificity of the BAb for CEA and TNFalpha was demonstrated using a BIAcoreTM two-step analysis. The affinity constants of the BAb for CEA immobilized on a sensor chip and for soluble TNFalpha added to the CEA-BAb complex were as high as those of the parental MAbs (1.7 x 10(9) M-1 and 6.6 x 10(8) M-1, respectively). The radiolabeled 125I-labeled BAb retained high immunoreactivity with both CEA and TNFalpha immobilized on a solid phase. In nude mice xenografted with the human colorectal carcinoma T380, the 125I-labeled BAb showed a tumor localization and biodistribution comparable to that of 131I-labeled anti-CEA parental F(ab')2 with 25-30% of the injected dose (ID)/g tumor at 24 h and 20% ID/g tumor at 48 h. To target TNFalpha to the tumor, a two-step i.v. injection protocol was used first, in which a variable dose of 125I-labeled BAb was injected, followed 24 or 48 h later by a constant dose of 131I-labeled TNFalpha (1 microg). Mice pretreated with 3 microg of BAb and sacrificed 2, 4, 6, or 8 h after the injection of TNFalpha showed a 1.5- to 2-fold increased concentration of 131I-labeled TNFalpha in the tumor as compared to control mice, which received TNFalpha alone. With a higher dose of BAb (25 microg), mice showed a better targeting of TNFalpha with a 3.2-fold increased concentration of 131I-labeled TNFalpha in the tumor: 9.3% versus 2.9% ID/g in control mice 6 h after TNFa injection. In a one-step injection protocol using a premixed BAb-TNFalpha preparation, similar results were obtained 6 h postinjection (3.5-fold increased TNFalpha tumor concentration). A longer retention time of TNFalpha was observed leading to an 8.1-fold increased concentration of TNFalpha in the tumor 14 h postinjection (4.4 versus 0.5% ID/g tumor for BAb-treated and control mice, respectively). These results show that our BAb is able, first, to localize in a human colon carcinoma and, there, to immunoabsorb the i.v.-injected TNFalpha, leading to its increased concentration at the tumor site.

Anti-tumour effect of monoclonal antibodies against selected antigens expressed by B-cells in Chronic Lymphocytic Leukaemia : strategies to enhance the efficacy of immunotherapy

Résumé : Les anticorps monoclonaux ont une place de plus en plus prépondérante dans le traitement des lymphomes et leucémies. Dans cette étude, trois anticorps monoclonaux murins, dirigés contre les antigènes CDS, CD71 et HLA-DR exprimés à la surface des cellules de leucémies lymphoïdes chroniques (LLC), ont été évalués. In vitro, les anticorps radiomarqués ont montrés des bonnes liaisons spécifiques sur les différentes cellules cibles. L'anti-CD71 inhibait la prolifération de la plupart des lignées cellulaires testées avec une accumulation des cellules en phase S précoce du cycle cellulaire. L'anti-HLA-DR inhibait aussi la prolifération des lignées leucémique JOK1-5.3 et lymphoïde Daudi. Cette inhibition était associée à une agrégation des cellules. Aucune induction d'apoptose n'a pu être clairement observée avec ces anticorps. L'anti-CD5 n'a montré aucun effet d'inhibition de croissance in vitro. In vivo, l'injection des anticorps individuellement augmentait significativement la survie médiane de souris SCID greffées avec des cellules JOK1-5.3 en i.p. De plus, l'anticorps antiCD5 combiné à l'anti-HLA-DR ou l'anti-CD71, sous certaines conditions, inhibait complètement le développement tumoral dans la quasi totalité des souris traitées avec une augmentation significative de l'efficacité comparée aux anticorps seuls. L'augmentation de l'efficacité thérapeutique des anticorps monoclonaux par les cytokines, dont l'IL-2, a déjà été montrée dans la littérature. Au regard du meilleur comportement de l'IL-2 sous la forme complexée à un anticorps anti-IL-2, nous avons évalué l'efficacité de l'IL-2/anti-IL-2 seul ou combinés au rituximab chez différents modèles tumoraux s.c. (BL60.2, Daudi, Ramos) ou i.p. (JOK15.3) de souris SCID. Le complexe IL-2/anti-IL-2 a montré un effet anti-tumoral dans les souris greffées avec BL60.2 et Daudi. Le traitement IL-2/anti-IL-2 combiné au rituximab a montré une efficacité accrue chez des souris avec BL60.2 par rapport au rituximab seul. En revanche, nous n'avons pas observé de différence avec IL-2/anti-IL-2 seul.Aussi, nous avons évalué l'utilisation de l'agent couplant tri-fonctionnel TMEA pour produire des anticorps bispecifiques. Les expériences préliminaires avec les anticorps rituximab et herceptine, ont mis en évidence sur gel SDS-Page la formation de dimers (~100kDa) et de trimers (~150kDa). Les anticorps bispecifiques sont composés d'un fragment Fab' d'une spécificité et de un ou deux fragments Fab' de l'autre spécificité permettant de moduler la capacité de liaison. Nous avons enfin montré qu'une construction anti-CD5/anti-CD20 était capable de se lier indépendamment ou simultanément à ses antigènes cibles. En conclusion, ce travail a montré l'efficacité thérapeutique des trois anticorps monoclonaux étudiés dans un model de LLC in vivo, et plus particulièrement l'intérêt de certaines combinaisons. D'autre part, nous avons montré l'efficacité anti-tumorale du complexe IL-2/anti-IL-2 in vivo. Des études futures devront permettre de définir un régime favorable pour augmenter l'efficacité de la thérapie avec les anticorps monoclonaux. Enfin, nous avons montré la faisabilité d'utiliser l'agent couplant TMEA pour produire des anticorps bispécifiques fonctionnels.Abstract : Monoclonal antibody (mAb) therapy has become an integral part in different treatments of lymphomas and leukaemias. In this study, we describe three murine mAbs directed against the CD5, CD71 and HLA-DR antigens expressed on chronic lymphocytic leukaemia cells (CLL). In vitro, radiolabeled purified mAbs showed good specific binding on live target cells. Anti-CD71 mAb inhibited proliferation of most cell lines with an accumulation of responding cells in early S-phase of the cell cycle, but without induction of apoptosis. Anti-HLA-DR mAb showed proliferation inhibition of leukaemia JOK1-5.3 and lymphoid Daudi cells, associated with cell aggregation, but again no specific sign of apoptosis was observed. Anti-CD5 mAb did not show any growth inhibitory effect in vitro. In vivo, in a model of SCID mice grafted i.p. with JOK1-5.3 cells, injection of individual mAbs induced significant prolongation of median survival, up to complete inhibition of tumour growth in some mice. Antibody combination of anti-CD5 with anti-HLA-DR or anti-CD71, evaluated in an early treatment, completely inhibited tumour growth in most mice, with a significant efficacy enhancement as compared to mAb used as single agents. Previous reports described the improved efficacy of mAb therapy when combined with cytokines such as IL-2. Relying further on the improved efficacy of IL-2 when administered as an immune complex with anti-IL-2 mAb, we evaluated the anti-tumour effect of the IL-2/anti-IL-2 complex alone or combined with rituximab in subcutaneous (BL60.2, Daudi, Ramos) or i.p. (JOK1-5.3) tumour models in SCID mice. The IL-2/anti-IL-2 complex demonstrated an anti-tumour effect in BL60.2 and Daudi grafted SCID mice. Combination of IL-2/anti-IL-2 treatment with rituximab showed increased efficacy as compared to rituximab alone in BL60.2 grafted mice. However, no difference was observed with IL-2/anti-IL-2 complex alone in these experiments. Finally, we evaluated the feasibility of producing bispecific antibodies (bsAbs) using a trifunctional coupling agent, called TMEA. In preliminary experiments coupling rituximab with herceptine Fab' fragments we obtained the formation of dimers (~100kDa) and trimers (~150kDa) as observed on SDS-Page gel. This method allowed us to produce bsAb with one Fab' fragments of one specificity and one or two Fab' fragments of the second specificity. An anti-CD5/anti-CD20 bsAb was shown to bind targeted antigen either independently or simultaneously. In conclusion, these data show that the three mAbs were all able to induce significant growth inhibition of the JOK1-5.3 cell line in vivo, and efficacy was enhanced when used in combination. IL2/anti-IL-2 complex displayed anti-tumour efficacy in vivo. Further evaluation is necessary to define the most favourable combination to improve mAb therapy. BsAb were produced using the tri-functional agent allowing antibody fragments with relatively good binding. The poor yield obtained with such chemical couplings limited the use of these constructs in preclinical experiments.

Human synthetic lethal inference as potential anti-cancer target gene detection

Background: Two genes are called synthetic lethal (SL) if mutation of either alone is not lethal, but mutation of both leads to death or a significant decrease in organism's fitness. The detection of SL gene pairs constitutes a promising alternative for anti-cancer therapy. As cancer cells exhibit a large number of mutations, the identification of these mutated genes' SL partners may provide specific anti-cancer drug candidates, with minor perturbations to the healthy cells. Since existent SL data is mainly restricted to yeast screenings, the road towards human SL candidates is limited to inference methods. Results: In the present work, we use phylogenetic analysis and database manipulation (BioGRID for interactions, Ensembl and NCBI for homology, Gene Ontology for GO attributes) in order to reconstruct the phylogenetically-inferred SL gene network for human. In addition, available data on cancer mutated genes (COSMIC and Cancer Gene Census databases) as well as on existent approved drugs (DrugBank database) supports our selection of cancer-therapy candidates.Conclusions: Our work provides a complementary alternative to the current methods for drug discovering and gene target identification in anti-cancer research. Novel SL screening analysis and the use of highly curated databases would contribute to improve the results of this methodology.

Respecte-t-on les bonnes pratiques de prescriptions des anti-inflammatoires non stéroïdiens en Suisse?

Introduction :¦Les anti-inflammatoires non stéroïdiens (AINS) sont une classe de médicaments fréquemment utilisée. Bien qu'ils soient utiles pour leurs propriétés analgésiques, antipyrétiques et anti-inflammatoires, ils sont à l'origine d'effets indésirables nombreux et potentiellement graves. Neuf recommandations de bonne pratique ont été établies en France en 1994 dans le but de limiter les prescriptions inutiles ou dangereuses. Nous avons examiné ces recommandations, pour savoir si elles sont encore pertinentes, et avons évalué leur suivi en Suisse.¦Méthode :¦La population étudiée consiste en 53 891 patients de plus de 16 ans, suivis en 2005 et 2006, qui ont eu au moins une fois la délivrance d'un AINS (voie non locale).¦Résultats :¦60% des prescriptions d'AINS excèdent 14 jours de traitement standard et 25% des renouvellements de traitement surviennent avant le nombre de jours correspondant à une prescription journalière standard. 2,7% des prescriptions contiennent deux ou plus d'AINS. Un tiers des prescriptions d'AINS sont associées à un protecteur gastrique dans les 3 mois. Cette proportion augmente avec les facteurs de risque des complications gastro-intestinales : l'âge, la prescription d'autres médicaments gastro-toxiques et la dose d'AINS prescrite. Toutefois, moins d'un patient sur deux de plus de 70 ans bénéficie d'un protecteur gastrique. Une prescription d'AINS sur mille concerne une femme au 3ème trimestre de grossesse. La prescription concomitante d'un médicament susceptible d'augmenter la fréquence des événements indésirables des AINS est fréquente : 25% d'anticoagulant ou anti-thrombotique, 30,5% d'inhibiteur de l'enzyme de conversion, diurétique ou antagoniste des récepteurs à l'angiotensine II, et 5,3% de corticothérapie. Ces proportions augmentent avec l'âge. La plupart des taux définis ci-dessus varient en fonction du canton de résidence, après ajustement sur le sexe et l'âge.¦Conclusion:¦Nos résultats suggèrent qu'en Suisse, les AINS sont fréquemment prescrits à doses trop élevées ou pendant une trop longue durée et souvent associés à d'autres médicaments susceptibles d'augmenter le risque d'effets secondaires, et ce particulièrement chez les sujets les plus âgés. La fréquence de prescriptions chez les femmes au 3ème trimestre de grossesse est inférieure à celles publiées ailleurs. Il est probable que les protecteurs gastriques soient sous-utilisés chez les personnes âgées. Les variations inter-cantonales suggèrent que certaines pratiques pourraient être améliorées