A cell therapy approach for erythropoietin delivery using encapsulated human primary fibroblasts

Summary Cell therapy has emerged as a strategy for the treatment of various human diseases. Cells can be transplanted considering their morphological and functional properties to restore a tissue damage, as represented by blood transfusion, bone marrow or pancreatic islet cells transplantation. With the advent of the gene therapy, cells also were used as biological supports for the production of therapeutic molecules that can act either locally or at distance. This strategy represents the basis of ex vivo gene therapy characterized by the removal of cells from an organism, their genetic modification and their implantation into the same or another individual in a physiologically suitable location. The tissue or biological function damage dictates the type of cells chosen for implantation and the required function of the implanted cells. The general aim of this work was to develop an ex vivo gene therapy approach for the secretion of erythropoietin (Epo) in patients suffering from Epo-responsive anemia, thus extending to humans, studies previously performed with mouse cells transplanted in mice and rats. Considering the potential clinical application, allogeneic primary human cells were chosen for practical and safety reasons. In contrast to autologous cells, the use of allogeneic cells allows to characterize a cell lineage that can be further transplanted in many individuals. Furthermore allogeneic cells avoid the potential risk of zoonosis encountered with xenogeneic cells. Accordingly, the immune reaction against this allogeneic source was prevented by cell macro- encapsulation that prevents cell-to-cell contact with the host immune system and allows to easy retrieve the implanted device. The first step consisted in testing the survival of various human primary cells that were encapsulated and implanted for one month in the subcutaneous tissue of immunocompetent and naturally or therapeutically immunodepressed mice, assuming that xenogeneic applications constitute a stringent and representative screening before human transplantation. A fibroblast lineage from the foreskin of a young donor, DARC 3.1 cells, showed the highest mean survival score. We have then performed studies to optimize the manufacturing procedures of the encapsulation device for successful engraftment. The development of calcifications on the polyvinyl alcohol (PVA) matrix serving as a scaffold for enclosed cells into the hollow fiber devices was reported after one month in vivo. Various parameters, including matrix rinsing solutions, batches of PVA and cell lineages were assessed for their respective role in the development of the phenomenon. We observed that the calcifications could be totally prevented by using ultra-pure sterile water instead of phosphate buffer saline solution in the rinsing procedure of the PVA matrix. Moreover, a higher lactate dehydrogenase activity of the cells was found to decrease calcium depositions due to more acidic microenvironment, inhibiting the calcium precipitation. After the selection of the appropriate cell lineage and the optimization of encapsulation conditions, a retroviral-based approach was applied to DARC 3.1 fibroblasts for the transduction of the human Epo cDNA. Various modifications of the retroviral vector and the infection conditions were performed to obtain clinically relevant levels of human Epo. The insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus as well as of a Kozak consensus sequence led to a 7.5-fold increase in transgene expression. Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells allowing to reach 200 IU hEpo/10E6 cells /day. These modified cells were encapsulated and implanted in vivo in the same conditions as previously described. All the mouse strains showed a sustained increase in their hematocrit and a high proportion of viable cells were observed after retrieval of the capsules. Finally, in the perspective of human application, a syngeneic model using encapsulated murine myoblasts transplanted in mice was realized to investigate the roles of both the host immune response and the cells metabolic requirements. Various loading densities and anti-inflammatory as well as immunosuppressive drugs were studied. The results showed that an immune process is responsible of cell death in capsules loaded at high cell density. A supporting matrix of PVA was shown to limit the cell density and to avoid early metabolic cell death, preventing therefore the immune reaction. This study has led to the development of encapsulated cells of human origin producing clinically relevant amounts of human EPO. This work resulted also to the optimization of cell encapsulation technical parameters allowing to begin a clinical application in end-stage renal failure patients. Résumé La thérapie cellulaire s'est imposée comme une stratégie de traitement potentiel pour diverses maladies. Si l'on considère leur morphologie et leur fonction, les cellules peuvent être transplantées dans le but de remplacer une perte tissulaire comme c'est le cas pour les transfusions sanguines ou les greffes de moelle osseuse ou de cellules pancréatiques. Avec le développement de la thérapie génique, les cellules sont également devenues des supports biologiques pour la production de molécules thérapeutiques. Cette stratégie représente le fondement de la thérapie génique ex vivo, caractérisée par le prélèvement de cellules d'un organisme, leur modification génétique et leur implantation dans le même individu ou dans un autre organisme. Le choix du type de cellule et la fonction qu'elle doit remplir pour un traitement spécifique dépend du tissu ou de la fonction biologique atteintes. Le but général de ce travail est de développer .une approche par thérapie génique ex vivo de sécrétion d'érythropoïétine (Epo) chez des patients souffrant d'anémie, prolongeant ainsi des travaux réalisés avec des cellules murines implantées chez des souris et des rats. Dans cette perpective, notre choix s'est porté sur des cellules humaines primaires allogéniques. En effet, contrairement aux cellules autologues, une caractérisation unique de cellules allogéniques peut déboucher sur de nombreuses applications. Par ailleurs, l'emploi de cellules allogéniques permet d'éviter les riques de zoonose que l'on peut rencontrer avec des cellules xénogéniques. Afin de protéger les cellules allogéniques soumises à une réaction immunitaire, leur confinement dans des macro-capsules cylindriques avant leur implantation permet d'éviter leur contact avec les cellules immunitaires de l'hôte, et de les retrouver sans difficulté en cas d'intolérance ou d'effet secondaire. Dans un premier temps, nous avons évalué la survie de différentes lignées cellulaires humaines primaires, une fois encapsulées et implantées dans le tissu sous-cutané de souris, soit immunocompétentes, soit immunodéprimées naturellement ou par l'intermédiaire d'un immunosuppresseur. Ce modèle in vivo correspond à des conditions xénogéniques et représente par conséquent un environnement de loin plus hostile pour les cellules qu'une transplantation allogénique. Une lignée fibroblastique issue du prépuce d'un jeune enfant, nommée DARC 3 .1, a montré une remarquable résistance avec un score de survie moyen le plus élevé parmi les lignées testées. Par la suite, nous nous sommes intéressés aux paramètres intervenant dans la réalisation du système d'implantation afin d'optimaliser les conditions pour une meilleure adaptation des cellules à ce nouvel environnement. En effet, en raison de l'apparition, après un mois in vivo, de calcifications au niveau de la matrice de polyvinyl alcohol (PVA) servant de support aux cellules encapsulées, différents paramètres ont été étudiés, tels que les procédures de fabrication, les lots de PVA ou encore les lignées cellulaires encapsulées, afin de mettre en évidence leur rôle respectif dans la survenue de ce processus. Nous avons montré que l'apparition des calcifications peut être totalement prévenue par l'utilisation d'eau pure au lieu de tampon phosphaté lors du rinçage des matrices de PVA. De plus, nous avons observe qu'un taux de lactate déshydrogénase cellulaire élevé était corrélé avec une diminution des dépôts de calcium au sein de la matrice en raison d'un micro-environnement plus acide inhibant la précipitation du calcium. Après sélection de la lignée cellulaire appropriée et de l'optimisation des conditions d'encapsulation, une modification génétique des fibroblastes DARC 3.1 a été réalisée par une approche rétrovirale, permettant l'insertion de l'ADN du gène de l'Epo dans le génome cellulaire. Diverses modifications, tant au niveau génétique qu'au niveau des conditions d'infection, ont été entreprises afin d'obtenir des taux de sécrétion d'Epo cliniquement appropriés. L'insertion dans la séquence d'ADN d'un élément de régulation post¬transcriptionnelle dérivé du virus de l'hépatite du rongeur (« woodchuck ») ainsi que d'une séquence consensus appelée « Kozak » ont abouti à une augmentation de sécrétion d'Epo 7.5 fois plus importante. De même, l'optimisation de la multiplicité d'infection et la sélection plus drastique des cellules hautement productrices ont permis finalement d'obtenir une sécrétion correspondant à 200 IU d'Epo/10E6 cells/jour. Ces cellules génétiquement modifiées ont été encapsulées et implantées in vivo dans les mêmes conditions que celles décrites plus haut. Toutes les souris transplantées ont montré une augmentation significative de leur hématocrite et une proportion importante de cellules présentait une survie conservée au moment de l'explantation des capsules. Finalement, dans la perspective d'une application humaine, un modèle syngénique a été proposé, basé sur l'implantation de myoblastes murins encapsulés dans des souris, afin d'investiguer les rôles respectifs de la réponse immunitaire du receveur et des besoins métaboliques cellulaires sur leur survie à long terme. Les cellules ont été encapsulées à différentes densités et les animaux transplantés se sont vus administrer des injections de molécules anti-inflammatoires ou immunosuppressives. Les résultats ont démontré qu'une réaction immunologique péri-capsulaire était à la base du rejet cellulaire dans le cas de capsules à haute densité cellulaire. Une matrice de PVA peut limiter cette densité et éviter une mort cellulaire précoce due à une insuffisance métabolique et par conséquent prévenir la réaction immunitaire. Ce travail a permis le développement de cellules encapsulées d'origine humaine sécrétant des taux d'Epo humaine adaptés à des traitements cliniques. De pair avec l'optimalisation des paramètres d'encapsulation, ces résultats ont abouti à l'initiation d'une application clinique destinée à des patients en insuffisance rénale terminale.

Intrachannel versus interchannel application of angiogenic factors in transmyocardial laser revascularization.

BACKGROUND AND OBJECTIVE: Theoretically myocardial angiogenesis of laser injury can be further enhanced by the addition of angiogenic growth factors. The influence of the way of administration of these factors on vascular growth around the channels is still unclear. MATERIALS AND METHODS: 18 pigs (mean weight 72 +/- 5.2 kg) were randomized to either triads of transmyocardial laser revascularization (TMLR) channels (group 1, n = 6) or isolated channels (group 2, n = 6), or a control group (n = 6). The animals had injections of bovine bone derived growth factor mixture either in the center of the triads in group 1 or within the channels themselves in group 2. Animals were sacrificed one month later for histological analysis. RESULTS: The vascular densities of myocardial areas within the triads of group 1 and around the channels in group 2 were significantly larger than in the control group: 15.2 +/- 3.7/mm2 and 14.2 +/- 3.5/mm2 respectively vs 5.3 +/- 1.6/mm2 (p < 0.001 for both differences). Differences of densities between group 1 and 2 were not statistically significant (p = 0.6). CONCLUSIONS: In this porcine model, the addition of a bovine bone derived growth factor mixture to TMLR significantly stimulates angiogenesis in the areas adjacent to the channels. The place of injection does not influence the angiogenesis intensity.

Combinatorial peptide library-based identification of peptide ligands for tumor-reactive cytolytic T lymphocytes of unknown specificity.

A novel approach for the identification of tumor antigen-derived sequences recognized by CD8(+) cytolytic T lymphocytes (CTL) consists in using synthetic combinatorial peptide libraries. Here we have screened a library composed of 3.1 x 10(11) nonapeptides arranged in a positional scanning format, in a cytotoxicity assay, to search the antigen recognized by melanoma-reactive CTL of unknown specificity. The results of this analysis enabled the identification of several optimal peptide ligands, as most of the individual nonapeptides deduced from the primary screening were efficiently recognized by the CTL. The results of the library screening were also analyzed with a mathematical approach based on a model of independent and additive contribution of individual amino acids to antigen recognition. This biometrical data analysis enabled the retrieval, in public databases, of the native antigenic peptide SSX-2(41-49), whose sequence is highly homologous to the ones deduced from the library screening, among the ones with the highest stimulatory score. These results underline the high predictive value of positional scanning synthetic combinatorial peptide library analysis and encourage its use for the identification of CTL ligands.

Expression of MYC, IgM, As Well As Non-Germinal Centre B-Cell Like Immunophenotype and Positive Immunofish Index Predict a Worse Progression Free Survival and Overall Survival in a Series of 670 De Novo Diffuse Large B-Cell Lymphomas Included in Clinical Trials: A GELA Study of the 2003 Program

Introduction: Diffuse large B-cell lymphomas (DLBCL) represent a heterogeneous disease with variable clinical outcome. Identifying phenotypic biomarkers of tumor cells on paraffin sections that predict different clinical outcome remain an important goal that may also help to better understand the biology of this lymphoma. Differentiating non-germinal centre B-cell-like (non-GCB) from Germinal Centre B-cell-like (GCB) DLBCL according to Hans algorithm has been considered as an important immunohistochemical biomarker with prognostic value among patients treated with R-CHOP although not reproducibly found by all groups. Gene expression studies have also shown that IgM expression might be used as a surrogate for the GCB and ABC subtypes with a strong preferential expression of IgM in ABC DLBCL subtype. ImmunoFISH index based on the differential expression of MUM-1, FOXP1 by immunohistochemistry and on the BCL6 rearrangement by FISH has been previously reported (C Copie-Bergman, J Clin Oncol. 2009;27:5573-9) as prognostic in an homogeneous series of DLBCL treated with R-CHOP. In addition, oncogenic MYC protein overexpression by immunohistochemistry may represent an easy tool to identify the consequences of MYC deregulation in DLBCL. Our aim was to analyse by immunohistochemistry the prognostic relevance of MYC, IgM, GCB/nonGCB subtype and ImmunoFISH index in a large series of de novo DLBCL treated with Rituximab (R)-chemotherapy (anthracyclin based) included in the 2003 program of the Groupe d'Etude des Lymphomes de l'Adulte (GELA) trials. Methods: The 2003 program included patients with de novo CD20+ DLBCL enrolled in 6 different LNH-03 GELA trials (LNH-03-1B, -B, -3B, 39B, -6B, 7B) stratifying patients according to age and age-adjusted IPI. Tumor samples were analyzed by immunohistochemistry using CD10, BCL6, MUM1, FOXP1 (according to Barrans threshold), MYC, IgM antibodies on tissue microarrays and by FISH using BCL6 split signal DNA probes. Considering evaluable Hans score, 670 patients were included in the study with 237 (35.4%) receiving intensive R-ACVBP regimen and 433 (64.6%) R-CHOP/R-mini-CHOP. Results: 304 (45.4%) DLBCL were classified as GCB and 366 (54.6%) as non-GCB according to Hans algorithm. 337/567 cases (59.4%) were positive for the ImmunoFISH index (i.e. two out of the three markers positive: MUM1 protein positive, FOXP1 protein Variable or Strong, BCL6 rearrangement). Immunofish index was preferentially positive in the non-GCB subtype (81.3%) compared to the GCB subtype (31.2%), (p<0.001). IgM was recorded as positive in tumor cells in 351/637 (52.4%) DLBCL cases with a preferential expression in non-GCB 195 (53.3%) vs GCB subtype 100(32.9%), p<0.001). MYC was positive in 170/577 (29.5%) cases with a 40% cut-off and in 44/577 (14.2%) cases with a cut-off of 70%. There was no preferential expression of MYC among GCB or non-GCB subtype (p>0.4) for both cut-offs. Progression-free Survival (PFS) was significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p<0.0001), MYC positive tumor with a 40% threshold (p<0.001), ImmunoFISH positive index (p<0.002), non-GCB DLBCL subtype (p<0.0001). Overall Survival (OS) was also significantly worse among patients with high IPI score (p<0.0001), IgM positive tumor (p=0.02), MYC positive tumor with a 40% threshold (p<0.01), ImmunoFISH positive index (p=0.02), non-GCB DLBCL subtype (p<0.0001). All significant parameters were included in a multivariate analysis using Cox Model and in addition to IPI, only the GCB/non-GCB subtype according to Hans algorithm predicted significantly a worse PFS among non-GCB subgroup (HR 1.9 [1.3-2.8] p=0.002) as well as a worse OS (HR 2.0 [1.3-3.2], p=0.003). This strong prognostic value of non-GCB subtyping was confirmed considering only patients treated with R- CHOP for PFS (HR 2.1 [1.4-3.3], p=0.001) and for OS (HR 2.3 [1.3-3.8], p=0.002). Conclusion: Our study on a large series of patients included in trials confirmed the relevance of immunohistochemistry as a useful tool to identify significant prognostic biomarkers for clinical use. We show here that IgM and MYC might be useful prognostic biomarkers. In addition, we confirmed in this series the prognostic value of the ImmunoFISH index. Above all, we fully validated the strong and independent prognostic value of the Hans algorithm, daily used by the pathologists to subtype DLBCL.

Association of major and minor ECG abnormalities with coronary heart disease events.

CONTEXT: In populations of older adults, prediction of coronary heart disease (CHD) events through traditional risk factors is less accurate than in middle-aged adults. Electrocardiographic (ECG) abnormalities are common in older adults and might be of value for CHD prediction. OBJECTIVE: To determine whether baseline ECG abnormalities or development of new and persistent ECG abnormalities are associated with increased CHD events. DESIGN, SETTING, AND PARTICIPANTS: A population-based study of 2192 white and black older adults aged 70 to 79 years from the Health, Aging, and Body Composition Study (Health ABC Study) without known cardiovascular disease. Adjudicated CHD events were collected over 8 years between 1997-1998 and 2006-2007. Baseline and 4-year ECG abnormalities were classified according to the Minnesota Code as major and minor. Using Cox proportional hazards regression models, the addition of ECG abnormalities to traditional risk factors were examined to predict CHD events. MAIN OUTCOME MEASURE: Adjudicated CHD events (acute myocardial infarction [MI], CHD death, and hospitalization for angina or coronary revascularization). RESULTS: At baseline, 276 participants (13%) had minor and 506 (23%) had major ECG abnormalities. During follow-up, 351 participants had CHD events (96 CHD deaths, 101 acute MIs, and 154 hospitalizations for angina or coronary revascularizations). Both baseline minor and major ECG abnormalities were associated with an increased risk of CHD after adjustment for traditional risk factors (17.2 per 1000 person-years among those with no abnormalities; 29.3 per 1000 person-years; hazard ratio [HR], 1.35; 95% CI, 1.02-1.81; for minor abnormalities; and 31.6 per 1000 person-years; HR, 1.51; 95% CI, 1.20-1.90; for major abnormalities). When ECG abnormalities were added to a model containing traditional risk factors alone, 13.6% of intermediate-risk participants with both major and minor ECG abnormalities were correctly reclassified (overall net reclassification improvement [NRI], 7.4%; 95% CI, 3.1%-19.0%; integrated discrimination improvement, 0.99%; 95% CI, 0.32%-2.15%). After 4 years, 208 participants had new and 416 had persistent abnormalities. Both new and persistent ECG abnormalities were associated with an increased risk of subsequent CHD events (HR, 2.01; 95% CI, 1.33-3.02; and HR, 1.66; 95% CI, 1.18-2.34; respectively). When added to the Framingham Risk Score, the NRI was not significant (5.7%; 95% CI, -0.4% to 11.8%). CONCLUSIONS: Major and minor ECG abnormalities among older adults were associated with an increased risk of CHD events. Depending on the model, adding ECG abnormalities was associated with improved risk prediction beyond traditional risk factors.

Predicting present and future intra-specific genetic structure through niche hindcasting across 24 millennia.

Paleoclimatic reconstructions coupled with species distribution models and identification of extant spatial genetic structure have the potential to provide insights into the demographic events that shape the distribution of intra-specific genetic variation across time. Using the globeflower Trollius europaeus as a case-study, we combined (1) Amplified Fragment Length Polymorphisms, (2) suites of 1000-years stepwise hindcasted species distributions and (3) a model of diffusion through time over the last 24,000 years, to trace the spatial dynamics that most likely fits the species' current genetic structure. We show that the globeflower comprises four gene pools in Europe which, from the dry period preceding the Last Glacial Maximum, dispersed while tracking the conditions fitting its climatic niche. Among these four gene pools, two are predicted to experience drastic range retraction in the near future. Our interdisciplinary approach, applicable to virtually any taxon, is an advance in inferring how climate change impacts species' genetic structures.

Synthesis of a non-peptidic PET tracer designed for α5β1 integrin receptor.

Arginine-glycine-aspartic acid (RGD)-containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α5 β1 integrin receptor have been developed with promising results. Sixty-one antagonists were screened, and tert-butyl (S)-3-(2-((3R,5S)-1-(3-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)propanoyl)-5-((pyridin-2-ylamino)methyl)pyrrolidin-3-yloxy)acetamido)-2-(2,4,6-trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α5 β1 integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide-alkyne copper(II)-catalyzed Huisgen's cycloaddition by using 1-azido-2-[(18)F]fluoroethane ([(18)F]12). Different reaction conditions between PMt and [(18)F]12 were investigated, but all of them afforded [(18)F]FPMt in 15 min with similar radiochemical yields (80-83%, decay corrected). Overall, the final radiopharmaceutical ([(18)F]FPMt) was obtained after a synthesis time of 60-70 min in 42-44% decay-corrected radiochemical yield.

Predictive Value of Readiness, Importance, and Confidence in Ability to Change Drinking and Smoking

Background: Visual analog scales (VAS) are used to assess readiness to changeconstructs, which are often considered critical for change.Objective: We studied whether 3 constructs -readiness to change, importance of changing and confidence inability to change- predict risk status 6 months later in 20 year-old men with either orboth of two behaviors: risky drinking and smoking. Methods: 577 participants in abrief intervention randomized trial were assessed at baseline and 6 months later onalcohol and tobacco consumption and with three 1-10 VAS (readiness, importance,confidence) for each behavior. For each behavior, we used one regression model foreach constructs. Models controlled for receipt of a brief intervention and used thelowest level (1-4) in each construct as the reference group (vs medium (5-7) and high(8-10) levels).Results: Among the 475 risky drinkers, mean (SD) readiness, importance and confidence to change drinking were 4.0 (3.1), 2.8 (2.2) and 7.2 (3.0).Readiness was not associated with being alcohol-risk free 6 months later (OR 1.3[0.7; 2.2] and 1.4 [0.8; 2.6] for medium and high readiness). High importance andhigh confidence were associated with being risk free (OR 0.9 [0.5; 1.8] and 2.9 [1.2;7.5] for medium and high importance; 2.1 [1.0;4.8] and 2.8 [1.5;5.6] for medium andhigh confidence). Among the 320 smokers, mean readiness, importance andconfidence to change smoking were 4.6 (2.6), 5.3 (2.6) and 5.9 (2.6). Neitherreadiness nor importance were associated with being smoking free (OR 2.1 [0.9; 4.7]and 2.1 [0.8; 5.8] for medium and high readiness; 1.4 [0.6; 3.4] and 2.1 [0.8; 5.4] formedium and high importance). High confidence was associated with being smokingfree (OR 2.2 [0.8;6.6] and 3.4 [1.2;9.8] for medium and high confidence).Conclusions: For drinking and smoking, high confidence in ability to change wasassociated -with similar magnitude- with a favorable outcome. This points to thevalue of confidence as an important predictor of successful change.

Population specific and up to date cardiovascular risk charts can be efficiently obtained with record linkage of routine and observational data.

BACKGROUND: Only few countries have cohorts enabling specific and up-to-date cardiovascular disease (CVD) risk estimation. Individual risk assessment based on study samples that differ too much from the target population could jeopardize the benefit of risk charts in general practice. Our aim was to provide up-to-date and valid CVD risk estimation for a Swiss population using a novel record linkage approach. METHODS: Anonymous record linkage was used to follow-up (for mortality, until 2008) 9,853 men and women aged 25-74 years who participated in the Swiss MONICA (MONItoring of trends and determinants in CVD) study of 1983-92. The linkage success was 97.8%, loss to follow-up 1990-2000 was 4.7%. Based on the ESC SCORE methodology (Weibull regression), we used age, sex, blood pressure, smoking, and cholesterol to generate three models. We compared the 1) original SCORE model with a 2) recalibrated and a 3) new model using the Brier score (BS) and cross-validation. RESULTS: Based on the cross-validated BS, the new model (BS = 14107×10(-6)) was somewhat more appropriate for risk estimation than the original (BS = 14190×10(-6)) and the recalibrated (BS = 14172×10(-6)) model. Particularly at younger age, derived absolute risks were consistently lower than those from the original and the recalibrated model which was mainly due to a smaller impact of total cholesterol. CONCLUSION: Using record linkage of observational and routine data is an efficient procedure to obtain valid and up-to-date CVD risk estimates for a specific population.