Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments.

Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats.

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.

PPAR gamma: ally and foe in bone metabolism.

Bone homeostasis is a well-balanced process that is largely dependent on the contribution of both bone-forming osteoblasts and bone-resorbing osteoclasts. A new study (Wan et al., 2007) suggests a previously unsuspected role for the transcription factor PPARgamma in promoting bone progenitors to the osteoclastic lineage.

Revision anterior cruciate ligament reconstruction with quadriceps tendon-patellar bone autograft.

PURPOSE: To evaluate the cause of recurrent pathologic instability after anterior cruciate ligament (ACL) surgery and the effectiveness of revision reconstruction using a quadriceps tendon autograft using a 2-incision technique. TYPE OF STUDY: Retrospective follow-up study. METHODS: Between 1999 and 2001, 31 patients underwent ACL revision reconstruction because of recurrent pathologic instability during sports or daily activities. Twenty-eight patients were reviewed after a mean follow-up of 4.2 years (range, 3.3 to 5.6 years). The mean age at revision surgery was 27 years (range, 18 to 41 years). The average time from primary procedure to revision surgery was 26 months (range, 9 to 45 months). A clinical, functional, and radiographic evaluation was performed. Also magnetic resonance imaging (MRI) or computed tomography (CT) scanning was performed. The International Knee Documentation Committee (IKDC), Lysholm, and Tegner scales were used. A KT-1000 arthrometer measurement (MEDmetric, San Diego, CA) by an experienced physician was made. RESULTS: Of the failures, 79% had radiographic evidence of malposition of their tunnels. In only 6 cases (21%) was the radiologic anatomy of tunnel placement judged to be correct on both the femoral and tibial side. The MRI or CT showed, in 6 cases, a too-centrally placed femoral tunnel. After revision surgery, the position of tunnels was corrected. A significant improvement of Lachman and pivot-shift phenomenon was observed. In particular, 17 patients had a negative Lachman test, and 11 patients had a grade I Lachman with a firm end point. Preoperatively, the pivot-shift test was positive in all cases, and at last follow-up in 7 patients (25%) a grade 1+ was found. Postoperatively, KT-1000 testing showed a mean manual maximum translation of 8.6 mm (SD, 2.34) for the affected knee; 97% of patients had a maximum manual side-to-side translation <5 mm. At the final postoperative evaluation, 26 patients (93%) graded their knees as normal or nearly normal according to the IKDC score. The mean Lysholm score was 93.6 (SD, 8.77) and the mean Tegner activity score was 6.1 (SD, 1.37). No patient required further revision. Five patients (18%) complained of hypersensitive scars from the reconstructive surgery that made kneeling difficult. CONCLUSIONS: There were satisfactory results after ACL revision surgery using quadriceps tendon and a 2-incision technique at a minimum 3 years' follow-up; 93% of patients returned to sports activities. LEVEL OF EVIDENCE: Level IV, case series, no control group.

Rapid death and regeneration of NKT cells in anti-CD3epsilon- or IL-12-treated mice: a major role for bone marrow in NKT cell homeostasis.

Natural killer T (NKT) cells express a T cell receptor (TCR) and markers common to NK cells, including NK1.1. In vivo, NKT cells are triggered by anti-CD3epsilon MAb to rapidly produce large amounts of IL-4 and by IL-12 to reject tumors. We show here that anti-CD3epsilon MAb treatment rapidly depletes the liver (and partially the spleen) of NKT cells and that homeostasis is achieved 1 to 2 days later via NKT cell proliferation that occurs mainly in bone marrow. Similar results were obtained in mice treated with IL-12. Collectively, our data demonstrate that peripheral NKT cells are highly sensitive to activation-induced cell death and that bone marrow plays a major role in restoring NKT cell homeostasis.

Evaluation of the potential use of trabecular bone score to complement bone mineral density in the diagnosis of osteoporosis: a preliminary spine BMD-matched, case-control study.

The trabecular bone score (TBS) is a new parameter that is determined from gray-level analysis of dual-energy X-ray absorptiometry (DXA) images. It relies on the mean thickness and volume fraction of trabecular bone microarchitecture. This was a preliminary case-control study to evaluate the potential diagnostic value of TBS as a complement to bone mineral density (BMD), by comparing postmenopausal women with and without fractures. The sample consisted of 45 women with osteoporotic fractures (5 hip fractures, 20 vertebral fractures, and 20 other types of fracture) and 155 women without a fracture. Stratification was performed, taking into account each type of fracture (except hip), and women with and without fractures were matched for age and spine BMD. BMD and TBS were measured at the total spine. TBS measured at the total spine revealed a significant difference between the fracture and age- and spine BMD-matched nonfracture group, when considering all types of fractures and vertebral fractures. In these cases, the diagnostic value of the combination of BMD and TBS likely will be higher compared with that of BMD alone. TBS, as evaluated from standard DXA scans directly, potentially complements BMD in the detection of osteoporotic fractures. Prospective studies are necessary to fully evaluate the potential role of TBS as a complementary risk factor for fracture.

Correlations between trabecular bone score, measured using anteroposterior dual-energy x-ray absorptiometry acquisition, and 3-dimensional parameters of bone microarchitecture: an experimental study on human cadaver vertebrae.

Developing a novel technique for the efficient, noninvasive clinical evaluation of bone microarchitecture remains both crucial and challenging. The trabecular bone score (TBS) is a new gray-level texture measurement that is applicable to dual-energy X-ray absorptiometry (DXA) images. Significant correlations between TBS and standard 3-dimensional (3D) parameters of bone microarchitecture have been obtained using a numerical simulation approach. The main objective of this study was to empirically evaluate such correlations in anteroposterior spine DXA images. Thirty dried human cadaver vertebrae were evaluated. Micro-computed tomography acquisitions of the bone pieces were obtained at an isotropic resolution of 93μm. Standard parameters of bone microarchitecture were evaluated in a defined region within the vertebral body, excluding cortical bone. The bone pieces were measured on a Prodigy DXA system (GE Medical-Lunar, Madison, WI), using a custom-made positioning device and experimental setup. Significant correlations were detected between TBS and 3D parameters of bone microarchitecture, mostly independent of any correlation between TBS and bone mineral density (BMD). The greatest correlation was between TBS and connectivity density, with TBS explaining roughly 67.2% of the variance. Based on multivariate linear regression modeling, we have established a model to allow for the interpretation of the relationship between TBS and 3D bone microarchitecture parameters. This model indicates that TBS adds greater value and power of differentiation between samples with similar BMDs but different bone microarchitectures. It has been shown that it is possible to estimate bone microarchitecture status derived from DXA imaging using TBS.

CD34/QBEND10 immunostaining in bone marrow biopsies: an additional parameter for the diagnosis and classification of myelodysplastic syndromes.

CD34/QBEND10 immunostaining has been assessed in 150 bone marrow biopsies (BMB) including 91 myelodysplastic syndromes (MDS), 16 MDS-related AML, 25 reactive BMB, and 18 cases where RA could neither be established nor ruled out. All cases were reviewed and classified according to the clinical and morphological FAB criteria. The percentage of CD34-positive (CD34 +) hematopoietic cells and the number of clusters of CD34+ cells in 10 HPF were determined. In most cases the CD34+ cell count was similar to the blast percentage determined morphologically. In RA, however, not only typical blasts but also less immature hemopoietic cells lying morphologically between blasts and promyelocytes were stained with CD34. The CD34+ cell count and cluster values were significantly higher in RA than in BMB with reactive changes (p&lt;0.0001 for both), in RAEB than in RA (p=0.0006 and p=0.0189, respectively), in RAEBt than in RAEB (p=0.0001 and p=0.0038), and in MDS-AML than in RAEBt (p&lt;0.0001 and p=0.0007). Presence of CD34+ cell clusters in RA correlated with increased risk of progression of the disease. We conclude that CD34 immunostaining in BMB is a useful tool for distinguishing RA from other anemias, assessing blast percentage in MDS cases, classifying them according to FAB, and following their evolution.

Activity of bone cement loaded with daptomycin alone or in combination with gentamicin or PEG600 against Staphylococcus epidermidis biofilms.

Daptomycin is a promising candidate for local treatment of bone infection due to its activity against multi-resistant staphylococci. We investigated the activity of antibiotic-loaded PMMA against Staphylococcus epidermidis biofilms using an ultra-sensitive method bacterial heat detection method (microcalorimetry). PMMA cylinders loaded with daptomycin alone or in combination with gentamicin or PEG600, vancomycin and gentamicin were incubated with S. epidermidis-RP62A in tryptic soy broth (TSB) for 72h. Cylinders were thereafter washed and transferred in microcalorimetry ampoules pre-filled with TSB. Bacterial heat production, proportional to the quantity of biofilm on the PMMA, was measured by isothermal microcalorimetry at 37°C. Heat detection time was considered time to reach 20μW. Experiments were performed in duplicate. The heat detection time was 5.7-7.0h for PMMA without antibiotics. When loaded with 5% of daptomycin, vancomycin or gentamicin, detection times were 5.6-16.4h, 16.8-35.7h and 4.7-6.2h, respectively. No heat was detected when 5% gentamicin or 0.5% PEG600 was added to the daptomycin-loaded PMMA. The study showed that vancomycin was superior to daptomycin and gentamicin in inhbiting staphylococcal adherence in vitro. However, PMMA loaded with daptomycin combined with gentamicin or PEG600 completely inhibited S. epidermidis-biofilm formation. PMMA loaded with these combinations may represent effective strategies for local treatment in the presence of multi-resistant staphylococci.

Success of a general school-based physical activity intervention on bone mineral content depends on pubertal stage but not on gender

Aims: We performed a randomised controlled trial in children of both gender and different pubertal stages to determine whether a school-based physical activity (PA) program during a full schoolyear influences bone mineral content (BMC) and whether there are differences in response for boys and girls before and during puberty. Methods: Twenty-eight 1st and 5th grade classes were cluster randomised to an intervention (INT, 16 classes, n=297) and control (CON; 12 classes, n=205) group. The intervention consisted of a multi-component PA intervention including daily physical education during a full school year. Each lesson was predetermined, included about ten minutes of jumping or strength training exercises of various intensity and was the same for all children. Measurements included anthropometry (height and weight), tanner stages (by self-assessment), PA (by accelerometry) and BMC for total body, femoral neck, total hip and lumbar spine using dualenergy X-ray absorptiometry (DXA). Bone parameters were normalized for gender and tanner stage (pre- vs. puberty). Analyses were performed by a regression model adjusted for gender, baseline height, baseline weight, baseline PA, post-intervention tanner stage, baseline BMC, and cluster. Researchers were blinded to group allocation. Children in the control group did not know about the intervention arm. Results: 217 (57%) of 380 children who initially agreed to have DXA measurements had also post-intervention DXA and PA data. Mean age of prepubertal and pubertal children at baseline was 9.0±2.1 and 11.2±0.6 years, respectively. 47/114 girls and 68/103 boys were prepubertal at the end of the intervention. Compared to CON, children in INT showed statistically significant increases in BMC of total body (adjusted z-score differences: 0.123; 95%>CI 0.035 to 0.212), femoral neck (0.155; 95%>CI 0.007 to 0.302), and lumbar spine (0.127; 95%>CI 0.026 to 0.228). Importantly, there was no gender*group, but a tanner*group interaction consistently favoring prepubertal children. Conclusions: Our findings show that a general, but stringent school-based PA intervention can improve BMC in elementary school children. Pubertal stage, but not gender seems to determine bone sensitivity to physical activity loading.

Reggies/flotillins regulate E-cadherin-mediated cell contact formation by affecting EGFR trafficking.

The reggie/flotillin proteins are implicated in membrane trafficking and, together with the cellular prion protein (PrP), in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies, as well as PrP down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.