Tumor localization in patients by radiolabeled monoclonal antibodies against colon carcinoma.

A radiolabeled monoclonal antibody (MAb) that has been shown to react specifically in vitro and ex vivo to human colorectal carcinoma and to inhibit growth of human carcinomas grafted in nude mice was administered to 52 colorectal carcinoma patients and 15 patients with other types of cancer. Of 63 colorectal carcinoma tumor sites studied, 34 showed significant accumulation of antibody by external photoscanning and tomoscintigraphy, whereas none of the 20 sites of other cancer types gave positive results. One-third of the patients received F(ab')2 fragments of the MAb, which gave a slightly higher percentage (61%) of positive results than did intact MAbs (51%). A few patients scheduled for tumor resection were given injections simultaneously of 131I-labeled MAb and 125I-labeled normal immunoglobulin G. Antibody concentration in resected tumors was 3.6 to 6.3 times higher than the average antibody concentration in adjacent normal tissues (1.5, 3.4, and 9.4 as compared with normal mucosa, serosa, and fat, respectively), and the specificity indices, calculated by differential radioactivity analysis, ranged from 2.1 to 5.1. The results show the potential value and limitations of this particular MAb for tumor detection by immunoscintigraphy.

Notch signaling is required for normal prostatic epithelial cell proliferation and differentiation.

Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. Using a transgenic cell ablation approach, we found in our previous study that cells expressing Notch1 are crucial for prostate early development and re-growth. Here, we further define the role of Notch signaling in regulating prostatic epithelial cell growth and differentiation using biochemical and genetic approaches in ex vivo or in vivo systems. Treatment of developing prostate grown in culture with inhibitors of gamma-secretase/presenilin, which is required for Notch cleavage and activation, caused a robust increase in proliferation of epithelial cells co-expressing cytokeratin 8 and 14, lack of luminal/basal layer segregation and dramatically reduced branching morphogenesis. Using conditional Notch1 gene deletion mouse models, we found that inactivation of Notch1 signaling resulted in profound prostatic alterations, including increased tufting, bridging and enhanced epithelial proliferation. Cells within these lesions co-expressed both luminal and basal cell markers, a feature of prostatic epithelial cells in predifferentiation developmental stages. Microarray analysis revealed that the gene expression in a number of genetic networks was altered following Notch1 gene deletion in prostate. Furthermore, expression of Notch1 and its effector Hey-1 gene in human prostate adenocarcinomas were found significantly down-regulated compared to normal control tissues. Taken together, these data suggest that Notch signaling is critical for normal cell proliferation and differentiation in the prostate, and deregulation of this pathway may facilitate prostatic tumorigenesis.

Oxamniquine, praziquantel and lovastatin association in the experimental Schistosomiasis mansoni

The activity of lovastatin associated with oxamniquine or praziquantel against schistosomiasis mansoni was evaluated in mice infected with Schistosoma mansoni. Forty days after infection, mice were treated with lovastatin, 400 mg/kg for five consecutive days by oral route, and on the last day of this sequence with 50 mg/kg oxamniquine or with 200 mg/kg praziquantel, both by oral route, single dose. Fifteen days later, the animals were perfused in parallel with an untreated control group. Studies were carried out in vitro, using lovastatin in culture medium containing S. mansoni worms proceeding from experimentally infected mice. In the in vivo trials, the association of lovastatin with oxamniquine or praziquantel did not show any additive action, but there were oogram changes when lovastatin was associated with oxamniquine. In vitro lovastatin was able to interrupt the maturation of S. mansoni eggs, which remained at the 1st or 2nd stages, depending on the dose used. The total number of morphologically dead eggs found in culture of worms exposed to 2 µg/ml or 4 µg/ml concentrations of lovastatin was significantly higher than the number of viable eggs. Using the probe Hoescht 33258 it was observed that 70% of the eggs considered morphologically viable in the treated groups (against 16% in the control group) were labeled, indicating that the majority of the viable eggs had membrane permeability increased due to lovastatin action.

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-²-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

An unexpected role of semaphorin3a-neuropilin-1 signaling in lymphatic vessel maturation and valve formation.

RATIONALE: Lymphatic vasculature plays important roles in tissue fluid homeostasis maintenance and in the pathology of human diseases. Yet, the molecular mechanisms that control lymphatic vessel maturation remain largely unknown. OBJECTIVE: We analyzed the gene expression profiles of ex vivo isolated lymphatic endothelial cells to identify novel lymphatic vessel expressed genes and we investigated the role of semaphorin 3A (Sema3A) and neuropilin-1 (Nrp-1) in lymphatic vessel maturation and function. METHODS AND RESULTS: Lymphatic and blood vascular endothelial cells from mouse intestine were isolated using fluorescence-activated cell sorting, and transcriptional profiling was performed. We found that the axonal guidance molecules Sema3A and Sema3D were highly expressed by lymphatic vessels. Importantly, we found that the semaphorin receptor Nrp-1 is expressed on the perivascular cells of the collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1 but not with an antibody that blocks VEGF-A binding to Nrp-1 resulted in a complex phenotype of impaired lymphatic vessel function, enhanced perivascular cell coverage, and abnormal lymphatic vessel and valve morphology. CONCLUSIONS: Together, these results reveal an unanticipated role of Sema3A-Nrp-1 signaling in the maturation of the lymphatic vascular network likely via regulating the perivascular cell coverage of the vessels thus affecting lymphatic vessel function and lymphatic valve development.

PD-1 is a regulator of NY-ESO-1-specific CD8+ T cell expansion in melanoma patients.

The programmed death 1 (PD-1) receptor is a negative regulator of activated T cells and is up-regulated on exhausted virus-specific CD8(+) T cells in chronically infected mice and humans. Programmed death ligand 1 (PD-L1) is expressed by multiple tumors, and its interaction with PD-1 resulted in tumor escape in experimental models. To investigate the role of PD-1 in impairing spontaneous tumor Ag-specific CD8(+) T cells in melanoma patients, we have examined the effect of PD-1 expression on ex vivo detectable CD8(+) T cells specific to the tumor Ag NY-ESO-1. In contrast to EBV, influenza, or Melan-A/MART-1-specific CD8(+) T cells, NY-ESO-1-specific CD8(+) T cells up-regulated PD-1 expression. PD-1 up-regulation on spontaneous NY-ESO-1-specific CD8(+) T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines. Importantly, blockade of the PD-1/PD-L1 pathway in combination with prolonged Ag stimulation with PD-L1(+) APCs or melanoma cells augmented the number of cytokine-producing, proliferating, and total NY-ESO-1-specific CD8(+) T cells. Collectively, our findings support the role of PD-1 as a regulator of NY-ESO-1-specific CD8(+) T cell expansion in the context of chronic Ag stimulation. They further support the use of PD-1/PD-L1 pathway blockade in cancer patients to partially restore NY-ESO-1-specific CD8(+) T cell numbers and functions, increasing the likelihood of tumor regression.

Plasmodium vivax antigen discovery based on alpha-helical coiled coil protein motif.

Protein α-helical coiled coil structures that elicit antibody responses, which block critical functions of medically important microorganisms, represent a means for vaccine development. By using bioinformatics algorithms, a total of 50 antigens with α-helical coiled coil motifs orthologous to Plasmodium falciparum were identified in the P. vivax genome. The peptides identified in silico were chemically synthesized; circular dichroism studies indicated partial or high α-helical content. Antigenicity was evaluated using human sera samples from malaria-endemic areas of Colombia and Papua New Guinea. Eight of these fragments were selected and used to assess immunogenicity in BALB/c mice. ELISA assays indicated strong reactivity of serum samples from individuals residing in malaria-endemic regions and sera of immunized mice, with the α-helical coiled coil structures. In addition, ex vivo production of IFN-γ by murine mononuclear cells confirmed the immunogenicity of these structures and the presence of T-cell epitopes in the peptide sequences. Moreover, sera of mice immunized with four of the eight antigens recognized native proteins on blood-stage P. vivax parasites, and antigenic cross-reactivity with three of the peptides was observed when reacted with both the P. falciparum orthologous fragments and whole parasites. Results here point to the α-helical coiled coil peptides as possible P. vivax malaria vaccine candidates as were observed for P. falciparum. Fragments selected here warrant further study in humans and non-human primate models to assess their protective efficacy as single components or assembled as hybrid linear epitopes.

Novel murine dendritic cell lines: a powerful auxiliary tool for dendritic cell research.

Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.

Functional analysis of different haematopoietic stem cell subsets

RÉSUMÉ : Elucider les bases moléculaires et cellulaires du fonctionnement des cellules souches s'avère crucial dans la compréhension de l'organisation cellulaire au sein des tissus et des organes ainsi que pour le développement de nouvelles stratégies thérapeutiques en médecine régénérative et en oncologie. Les cellules souches adultes les mieux connues sont celles responsables de l'hématopoïèse, les cellules souches hématopoïétiques (CSH). Durant ces dernières années, la recherche a porté une attention particulière à l'isolation prospective de CSH dérivées de la moelle osseuse de souris en utilisant des marqueurs de surface cellulaire ainsi que des propriétés fonctionnelles alléguées. Par la suite, la capacité fonctionnelle des CSH a été vérifiée classiquement par leur transplantation intraveineuse dans des souris réceptrices conditionnées et par l'analyse de leur aptitude à reconstituer le système hématopoïétique à long terme. Des études récentes suggèrent que la transplantation des cellules directement dans la moelle osseuse pourrait non seulement aboutir à une prise de greffe plus rapide et plus efficace, mais pourrait même aider à l'identification de cellules qui ont certes des propriétés intrinsèques de CSH, mais qui n'ont pas la capacité de trouver leur niche au sein de la moelle osseuse et ont donc échoué dans les analyses classiques de reconstitution. Dans cette étude, nous comparons à deux niveaux la fonction de différents sous-groupes de cellules souches de la moelle osseuse, définis par leur phénotype de surface cellulaire. Premièrement, nous étudions leur capacité à reconstituer des souris létalement irradiées après injection soit intraveineuse soit intrafémorale. Deuxièmement, par analyse cytométrique de flux à 8 couleurs, nous comparons leur activité relative de « side population » (SP) par exclusion du colorant fluorescent Hoechst 33342. Nos résultats préliminaires renforcent en effet l'idée que la transplantation intrafémorale aboutit à une greffe plus rapide et plus efficace. Par contre, en utilisant cette approche, nous n'arrivons pas à identifier des cellules capables de prendre greffe spécifiquement quand elles sont injectées en intrafémorale. Finalement, bien qu'une confirmation in vivo soit encore nécessaire, nous suggérons sur la base de nos analyses cytométriques de flux, que les cellules SP Sca1t~és éie~~ CD48t~és bas sont très enrichies en CSH. Ceci permettrait l'isolation ex vivo de CSH de la moelle osseuse de souris par une stratégie à la fois nouvelle et simple. SUMMARY : Elucidating the molecular and cellular bases of stem cell function is crucial for the understanding of cellular organisation within tissues and organs as well as for the development of new therapeutic strategies in regenerative medicine and oncology. The best-known adult stem cells are those responsible for haematopoiesis, the haematopoietic stem cells (HSCs). In recent years, much effort has been put into the prospective isolation of mouse bone marrow (BM)-derived HSCs using cell-surface markers and alleged functional properties. Upon isolation, the functional capacity of putative HSCs has been classically assessed by intravenous transplantation into conditioned recipient mice and analysis of their ability to reconstitute the haematopoietic system at long-term. It has recently been suggested that transplanting the cells directly into the BM might not only result in more rapid and more effective engraftment, but even help to identify cells that have intrinsic HSC properties but lack the ability to home to their BM niche and have thus failed to succeed in classical reconstitution assays. In this study, we compare the function of different BM cell subsets, as defined by their cell surface phenotype, on two levels. Firstly, we assess their ability to reconstitute lethally irradiated mice, when injected either intravenously or intrafemorally. Secondly, using 8-colour flow cytometric analysis, we compare their relative side population (SP) activity by exclusion of the fluorescent dye Hoechst 33342. Our preliminary results indeed reinforce the idea that intrafemoral transplantation results in faster and more effective engraftment, however, using this approach, we are unable to identify cells that are capable of engrafting specifically when injected intrafemorally. Finally, although in vivo confirmation is still required, we propose, based on the results of our flow cytometric analyses, that SP Scat Very h'9h CD48Very'°W cells should be highly enriched for HSCs. This would allow for a simple new strategy for the isolation of mouse BM HSCs ex vivo.

Long-term, multilineage hematopoiesis occurs in the combined absence of beta-catenin and gamma-catenin

The canonical Wnt signaling pathway plays key roles in stem-cell maintenance, progenitor cell expansion, and lineage decisions. Transcriptional responses induced by Wnt depend on the association of either beta-catenin or gamma-catenin with lymphoid enhancer factor/T cell factor transcription factors. Here we show that hematopoiesis, including thymopoiesis, is normal in the combined absence of beta- and gamma-catenin. Double-deficient hematopoietic stem cells maintain long-term repopulation capacity and multilineage differentiation potential. Unexpectedly, 2 independent ex vivo reporter gene assays show that Wnt signal transmission is maintained in double-deficient hematopoietic stem cells, thymocytes, or peripheral T cells. In contrast, Wnt signaling is strongly reduced in thymocytes lacking TCF-1 or in nonhematopoietic cells devoid of beta-catenin. These data provide the first evidence that hematopoietic cells can transduce canonical Wnt signals in the combined absence of beta- and gamma-catenin

Modulation of the airway allergic response : characterization of the cellular and molecular mechanisms

ABSTRACT Allergic asthma is a major complication of atopy. Its severity correlates with the presence of activated T lymphocytes and eosinophils in the bronchoalveolar lavage fluid (BALF). Mechanisms that protect against asthma are poorly understood. Based on oral models of mucosal tolerance induction, models using the nasal route showed that uptake of important amounts of antigen can induce tolerance and reverse the allergic phenotype. 1L-10 producing regulatory T cells were proposed as key players in tolerance induction, but other players, e.g. dendritic cells (DC), B cells and epithelial cells may have to be taken into consideration. The objective of the present study is to characterize the effects of a therapeutic intranasal treatment (INT) in a murine model of asthma and to determine, in this model, the cellular and molecular mechanisms leading to protection against asthma. First, we established an asthma model by sensitizing the BALB/c mouse to ovalbumin (OVA) by two intraperitoneal injections of alum-adsorbed OVA and three inhalations of aerosolized OVA. Then OVA was applied to the nasal mucosa of OVA- sensitized mice. Mice were later re-exposed to OVA aerosols to assess the protection induced by OVA INT. OVA sensitization induced strong eosinophil recruitment, OVA-specific T cell proliferation and IgE production. Three intranasal treatments at 24-hour intervals with 1.5 mg OVA drastically reduced inflammatory cell recruitment into the BALF and inhibited OVA-specific IgE production upon allergen re-exposure. T cell proliferation in ex vivo bronchial lymph node (BLN) cells was inhibited, as well as TH2 cytokine production. Protection against OVA-induced bronchial inflammation was effective for an extended period of time and treated mice resisted a second re-exposure. Transfer of CD4+ cells from BLN and lungs of OVA-treated mice protected asthmatic recipient mice from subsequent aerosol challenge indicating an involvement of CD4+ T regulatory cells in this protection. RESUME L'asthme allergique est une manifestation clinique majeure de l'atopie. La sévérité de l'asthme est liée à la présence de lymphocytes T activés ainsi que d'éosinophiles dans le lavage broncho-alvéolaire (LBA). Les mécanismes permettant de se prémunir contre l'asthme sont mal connus. Basés sur des modèles muqueux d'induction de tolérance par la voie orale, des modèles utilisant la voie nasale ont montré que d'importantes quantités d'antigène peuvent induire une tolérance et ainsi reverser le phénotype allergique. Des cellules régulatrices produisant de l'IL-10 pourraient jouer un rôle clé dans l'induction de la tolérance mais d'autres acteurs tels que les cellules dendritiques, les cellules B et les cellules épithéliales doivent aussi être prises en compte. L'objectif de la présente étude est de caractériser les effets d'un traitement intranasal thérapeutique dans un modèle murin d'asthme et de déterminer dans ce modèle les mécanismes cellulaires et moléculaires conférant une protection contre l'asthme. En premier lieu, un modèle d'asthme allergique a été établi en sensibilisant des souris BALB/c à l'ovalbumine (OVA) par deux injections intraperitonéales d'OVA adsorbé sur de l'alum et trois séances d'OVA en aérosol. Dans un second temps, de l'OVA a été administrée sur la muqueuse nasale des souris sensibilisées à l'OVA. Les souris furent ensuite challengées par des aérosols d'OVA afin d'évaluer la protection conférée par le traitement intranasal à l'OVA. La sensibilisation à l'OVA a induit un fort recrutement d'éosinophiles, une réponse proliférative des cellules T à l'OVA ainsi qu'une production d'lgE spécifiques. Trois traitements intranasaux à 24 heures d'intervalle avec 1.5 mg d'OVA ont permis de réduire drastiquement le recrutement des cellules inflammatoires dans le LBA ainsi que d'inhiber la production d'lgE spécifiques à l'OVA produits lors d'une ré-exposition à l'OVA. La prolifération en réponse à l'OVA de cellules extraites ex vivo de ganglions bronchiques a, elle aussi, été inhibée de même que la production de cytokines TH2. La protection contre l'inflammation provoquée par l'aérosol est efficace pour une longue période et les souris traitées résistent à une seconde ré- exposition. Le transfert de cellules CD4+ issues de ganglions bronchiques et de poumons de souris traitées à l'OVA protège les souris asthmatiques receveuses contre les effets inflammatoires d'un aérosol, indiquant que des cellules T CD4+ régulatrices pourraient être impliquées dans cette protection. RESUME DESTINE A UN LARGE PUBLIC L'asthme est une affection des voies respiratoires qui se caractérise par une contraction de la musculature des voies aériennes, une production de mucus et d'anticorps de l'allergie (IgE). On parle d'asthme allergique lorsque les facteurs déclenchant l'asthme sont des allergènes inhalés tels que acariens, pollens ou poils d'animaux. Le système immunitaire des patients asthmatiques a un défaut de programmation qui le rend réactif à des substances qui sont normalement inoffensives. Le traitement actuel de l'asthme repose sur le soulagement des symptômes grâce à des produits à base de stéroïdes. Les techniques permettant de reprogrammer le système immunitaire (immunothérapie) ne sont pas efficaces pour tous les antigènes et prennent beaucoup de temps. En conséquence, il est nécessaire de mieux comprendre les mécanismes sous-tendant une telle reprogrammation afin d'en améliorer le rendement et l'efficacité. Dans ce but, des modèles d'immunothérapie ont été mis au point chez la souris. Ils permettent une plus grande liberté d'investigation. Dans cette étude, un modèle d'asthme allergique dans la souris a été établi par une sensibilisation à un antigène particulier : l'ovalbumine (OVA). Ce modèle présente les caractéristiques principales de l'asthme humain : recrutement de cellules inflammatoires dans les poumons, augmentation de la production d'anticorps et de la résistance des bronches aux flux respiratoires. Cette souris asthmatique a ensuite été traitée par application nasale d'OVA. Comparées aux souris non traitées, les souris traitées à l'OVA ont moins de cellules inflammatoires dans leurs poumons et produisent moins d'anticorps IgE. D'autres marqueurs inflammatoires sont aussi fortement diminués. Des cellules de poumons ou de ganglions bronchiques prélevées sur des souris traitées injectées dans des souris asthmatiques améliorent les symptômes de l'asthme. Ces cellules pourraient donc avoir un rôle régulateur dans l'asthme. Les caractériser et les étudier afin d'être capable de les générer est crucial pour les futures thérapies de l'asthme.

Mechanisms of the anti-obesity effects of oxytocin in diet-induced obese rats.

Apart from its role during labor and lactation, oxytocin is involved in several other functions. Interestingly, oxytocin- and oxytocin receptor-deficient mice develop late-onset obesity with normal food intake, suggesting that the hormone might exert a series of beneficial metabolic effects. This was recently confirmed by data showing that central oxytocin infusion causes weight loss in diet-induced obese mice. The aim of the present study was to unravel the mechanisms underlying such beneficial effects of oxytocin. Chronic central oxytocin infusion was carried out in high fat diet-induced obese rats. Its impact on body weight, lipid metabolism and insulin sensitivity was determined. We observed a dose-dependent decrease in body weight gain, increased adipose tissue lipolysis and fatty acid β-oxidation, as well as reduced glucose intolerance and insulin resistance. The additional observation that plasma oxytocin levels increased upon central infusion suggested that the hormone might affect adipose tissue metabolism by direct action. This was demonstrated using in vitro, ex vivo, as well as in vivo experiments. With regard to its mechanism of action in adipose tissue, oxytocin increased the expression of stearoyl-coenzyme A desaturase 1, as well as the tissue content of the phospholipid precursor, N-oleoyl-phosphatidylethanolamine, the biosynthetic precursor of the oleic acid-derived PPAR-alpha activator, oleoylethanolamide. Because PPAR-alpha regulates fatty acid β-oxidation, we hypothesized that this transcription factor might mediate the oxytocin effects. This was substantiated by the observation that, in contrast to its effects in wild-type mice, oxytocin infusion failed to induce weight loss and fat oxidation in PPAR-alpha-deficient animals. Altogether, these results suggest that oxytocin administration could represent a promising therapeutic approach for the treatment of human obesity and type 2 diabetes.

Radiolabeled chimeric anti-CEA monoclonal antibody compared with the original mouse monoclonal antibody for surgically treated colorectal carcinoma.

Biodistribution and tumor uptake of a chimeric human-mouse monoclonal antibody (MAb) and the original mouse MAb have been comparatively studied. METHODS: Eighteen patients with suspected colorectal cancer scheduled for surgery underwent immunoscintigraphy with 123I-labeled chimeric anti-CEA MAb. Iodine-125 and 131I trace-labeled chimeric and original mouse MAb were simultaneously injected for biodistribution studies. RESULTS: Similar serum kinetics and a low immunogenicity were observed for both antibodies. Mean binding capacity to CEA measured in PBS after radiolabeling was identical for both MAbs and it was slightly decreased when measured in serum 1-4 hr after injection. Radiochromatograms of patients sera showed immune complex formation related to the amount of circulating CEA. Postoperative ex vivo radioactivity counting in tissue samples revealed similar antibody distributions with notably similar antibody uptakes in tumors. High tumor uptakes (between 0.02 to 0.06% injected dose per g) were observed in 3 of 13 patients operated for primary or metastatic colorectal cancer. CONCLUSION: In this dual-label technique, the radioiodinated anti-CEA IgG4 chimeric MAb and the original mouse IgG1 MAb were shown to have very similar behavior in colorectal cancer patients.

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.

Two specific populations of GABAergic neurons originating from the medial and the caudal ganglionic eminences aid in proper navigation of callosal axons.

The corpus callosum (CC) plays a crucial role in interhemispheric communication. It has been shown that CC formation relies on the guidepost cells located in the midline region that include glutamatergic and GABAergic neurons as well as glial cells. However, the origin of these guidepost GABAergic neurons and their precise function in callosal axon pathfinding remain to be investigated. Here, we show that two distinct GABAergic neuronal subpopulations converge toward the midline prior to the arrival of callosal axons. Using in vivo and ex vivo fate mapping we show that CC GABAergic neurons originate in the caudal and medial ganglionic eminences (CGE and MGE) but not in the lateral ganglionic eminence (LGE). Time lapse imaging on organotypic slices and in vivo analyses further revealed that CC GABAergic neurons contribute to the normal navigation of callosal axons. The use of Nkx2.1 knockout (KO) mice confirmed a role of these neurons in the maintenance of proper behavior of callosal axons while growing through the CC. Indeed, using in vitro transplantation assays, we demonstrated that both MGE- and CGE-derived GABAergic neurons exert an attractive activity on callosal axons. Furthermore, by combining a sensitive RT-PCR technique with in situ hybridization, we demonstrate that CC neurons express multiple short and long range guidance cues. This study strongly suggests that MGE- and CGE-derived interneurons may guide CC axons by multiple guidance mechanisms and signaling pathways. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 647-672, 2013.