MM-GBSA binding free energy decomposition and T cell receptor engineering.

Recognition by the T-cell receptor (TCR) of immunogenic peptides (p) presented by class I major histocompatibility complexes (MHC) is the key event in the immune response against virus infected cells or tumor cells. The major determinant of T cell activation is the affinity of the TCR for the peptide-MHC complex, though kinetic parameters are also important. A study of the 2C TCR/SIYR/H-2Kb system using a binding free energy decomposition (BFED) based on the MM-GBSA approach had been performed to assess the performance of the approach on this system. The results showed that the TCR-p-MHC BFED including entropic terms provides a detailed and reliable description of the energetics of the interaction (Zoete and Michielin, 2007). Based on these results, we have developed a new approach to design sequence modifications for a TCR recognizing the human leukocyte antigen (HLA)-A2 restricted tumor epitope NY-ESO-1. NY-ESO-1 is a cancer testis antigen expressed not only in melanoma, but also on several other types of cancers. It has been observed at high frequencies in melanoma patients with unusually positive clinical outcome and, therefore, represents an interesting target for adoptive transfer with modified TCR. Sequence modifications of TCR potentially increasing the affinity for this epitope have been proposed and tested in vitro. T cells expressing some of the proposed TCR mutants showed better T cell functionality, with improved killing of peptide-loaded T2 cells and better proliferative capacity compared to the wild type TCR expressing cells. These results open the door of rational TCR design for adoptive transfer cancer therapy.

Validation de la mesure de l'oedème postopératoire par bioimpédance chez les patients opérés d'une prothèse totale du genou : une expérience de collaboration entre physiothérapeutes et ingénieurs

Fondements : La recherche sur l'oedème postopératoire consécutif à la chirurgie prothétique du genou est peu développée, notamment en raison de l'absence d'une méthode de mesure adaptée. Une collaboration entre physiothérapeutes et ingénieurs a permis de développer et valider une méthode de mesure innovante et facilement applicable. Les physiothérapeutes ont identifié un besoin clinique, les ingénieurs ont apporté leur savoir technologique, et l'équipe a conjointement élaboré le protocole de mesure et effectué l'étude de validation. Introduction : La bioimpédance est fréquemment utilisée pour évaluer l'oedème par l'analyse d'un signal électrique passant au travers du corps, en extrapolant la résistance théorique à une fréquence égale à zéro (R0). La mesure s'avère fiable et rapide, mais n'a jamais été appliquée et validée pour l'évaluation de l'oedème en chirurgie orthopédique. Objectif : L'objectif de l'étude est de valider la mesure de l'oedème du membre inférieur par bioimpédance, chez des patients ayant bénéficié d'une prothèse totale de genou (PTG). Questionnement : Après nous être assurés de l'absence d'influence de l'implant métallique de la PTG sur la mesure, nous nous questionnions sur la validité et la fiabilité des mesures de bioimpédance dans ce contexte. Méthodes : Deux évaluateurs ont mesuré à tour de rôle et à deux reprises successives l'oedème chez 24 patients opérés d'une PTG, à trois temps différents (préopératoire, J+2, J+8). L'oedème a été évalué par bioimpédance (R0) et par conversion en volume de mesures centimétriques du membre inférieur (MI). Nous avons calculé le ratio moyen des MI pour chaque méthode. Nous avons évalué la reproductibilité intra- et inter-observateurs de la bioimpédance (coefficient de corrélation intraclasse, CCI) et la corrélation entre méthodes (Spearman). Résultats : Le ratio moyen opéré/sain du volume des MI est de 1.04 (SD ± 0.06) en préopératoire, 1.18 (SD ± 0.09) à J+2 et 1.17 (SD ± 0.10) à J+8. Le ratio sain/opéré des MI de R0 est de 1.04 (SD ± 0.07) en préopératoire, 1.51 (SD ± 0.22) à J+2 et 1.65 (SD ± 0.21) à J+8. En préopératoire, à J+2 et J+8, les CCI tous supérieurs à 0.95 pour la reproductibilité intra- et inter-observateurs de la bioimpédance. La corrélation entre méthodes est de 0.71 en préopératoire, 0.61 à J2 et 0.33 à J8. Analyse et conclusion : La variation du ratio des MI entre les temps préopératoire, J+2 et J+8 est plus marquée pour R0. La mesure de bioimpédance bénéficie d'une excellente reproductibilité intra- et inter-observateurs. L'évolution dans le temps de la corrélation entre méthodes peut être expliquée par l'influence potentielle de facteurs confondants sur R0 (modification de la composition liquidienne) et par l'influence de l'atrophie musculaire postopératoire sur la mesure de volume. La collaboration physiothérapeutes-ingénieurs a permis le développement et l'évaluation d'une nouvelle méthode de mesure.

CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion.

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Current features of infective endocarditis in elderly patients: results of the International Collaboration on Endocarditis Prospective Cohort Study.

BACKGROUND: Elderly patients are emerging as a population at high risk for infective endocarditis (IE). However, adequately sized prospective studies on the features of IE in elderly patients are lacking. METHODS: In this multinational, prospective, observational cohort study within the International Collaboration on Endocarditis, 2759 consecutive patients were enrolled from June 15, 2000, to December 1, 2005; 1056 patients with IE 65 years or older were compared with 1703 patients younger than 65 years. Risk factors, predisposing conditions, origin, clinical features, course, and outcome of IE were comprehensively analyzed. RESULTS: Elderly patients reported more frequently a hospitalization or an invasive procedure before IE onset. Diabetes mellitus and genitourinary and gastrointestinal cancer were the major predisposing conditions. Blood culture yield was higher among elderly patients with IE. The leading causative organism was Staphylococcus aureus, with a higher rate of methicillin resistance. Streptococcus bovis and enterococci were also significantly more prevalent. The clinical presentation of elderly patients with IE was remarkable for lower rates of embolism, immune-mediated phenomena, or septic complications. At both echocardiography and surgery, fewer vegetations and more abscesses were found, and the gain in the diagnostic yield of transesophageal echocardiography was significantly larger. Significantly fewer elderly patients underwent cardiac surgery (38.9% vs 53.5%; P < .001). Elderly patients with IE showed a higher rate of in-hospital death (24.9% vs 12.8%; P < .001), and age older than 65 years was an independent predictor of mortality. CONCLUSIONS: In this large prospective study, increasing age emerges as a major determinant of the clinical characteristics of IE. Lower rates of surgical treatment and high mortality are the most prominent features of elderly patients with IE. Efforts should be made to prevent health care-associated acquisition and improve outcomes in this major subgroup of patients with IE.

Use of nucleoside reverse transcriptase inhibitors and risk of myocardial infarction in HIV-infected patients enrolled in the D:A:D study: a multi-cohort collaboration.

BACKGROUND: Whether nucleoside reverse transcriptase inhibitors increase the risk of myocardial infarction in HIV-infected individuals is unclear. Our aim was to explore whether exposure to such drugs was associated with an excess risk of myocardial infarction in a large, prospective observational cohort of HIV-infected patients. METHODS: We used Poisson regression models to quantify the relation between cumulative, recent (currently or within the preceding 6 months), and past use of zidovudine, didanosine, stavudine, lamivudine, and abacavir and development of myocardial infarction in 33 347 patients enrolled in the D:A:D study. We adjusted for cardiovascular risk factors that are unlikely to be affected by antiretroviral therapy, cohort, calendar year, and use of other antiretrovirals. FINDINGS: Over 157,912 person-years, 517 patients had a myocardial infarction. We found no associations between the rate of myocardial infarction and cumulative or recent use of zidovudine, stavudine, or lamivudine. By contrast, recent-but not cumulative-use of abacavir or didanosine was associated with an increased rate of myocardial infarction (compared with those with no recent use of the drugs, relative rate 1.90, 95% CI 1.47-2.45 [p=0.0001] with abacavir and 1.49, 1.14-1.95 [p=0.003] with didanosine); rates were not significantly increased in those who stopped these drugs more than 6 months previously compared with those who had never received these drugs. After adjustment for predicted 10-year risk of coronary heart disease, recent use of both didanosine and abacavir remained associated with increased rates of myocardial infarction (1.49, 1.14-1.95 [p=0.004] with didanosine; 1.89, 1.47-2.45 [p=0.0001] with abacavir). INTERPRETATION: There exists an increased risk of myocardial infarction in patients exposed to abacavir and didanosine within the preceding 6 months. The excess risk does not seem to be explained by underlying established cardiovascular risk factors and was not present beyond 6 months after drug cessation.

How can continuing professional development better promote shared decision-making? Perspectives from an international collaboration.

ABSTRACT: BACKGROUND: Shared decision-making is not widely implemented in healthcare. We aimed to set a research agenda about promoting shared decision-making through continuing professional development. METHODS: Thirty-six participants met for two days. RESULTS: Participants suggested ways to improve an environmental scan that had inventoried 53 shared decision-making training programs from 14 countries. Their proposed research agenda included reaching an international consensus on shared decision-making competencies and creating a framework for accrediting continuing professional development initiatives in shared decision-making. CONCLUSIONS: Variability in shared decision-making training programs showcases the need for quality assurance frameworks.