Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

<p>Abstract</p> <p>Background</p> <p>The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression.</p> <p>Methods</p> <p>Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes.</p> <p>Results and Discussion</p> <p>We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent <it>in situ </it>hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age.</p> <p>Conclusion</p> <p>Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.</p

Requirements for F-BAR Proteins TOCA-1 and TOCA-2 in Actin Dynamics and Membrane Trafficking during Caenorhabditis elegans Oocyte Growth and Embryonic Epidermal Morphogenesis

The TOCA family of F-BAR–containing proteins bind to and remodel lipid bilayers via their conserved F-BAR domains, and regulate actin dynamics via their N-Wasp binding SH3 domains. Thus, these proteins are predicted to play a pivotal role in coordinating membrane traffic with actin dynamics during cell migration and tissue morphogenesis. By combining genetic analysis in Caenorhabditis elegans with cellular biochemical experiments in mammalian cells, we showed that: i) loss of CeTOCA proteins reduced the efficiency of Clathrin-mediated endocytosis (CME) in oocytes. Genetic interference with CeTOCAs interacting proteins WSP-1 and WVE-1, and other components of the WVE-1 complex, produced a similar effect. Oocyte endocytosis defects correlated well with reduced egg production in these mutants. ii) CeTOCA proteins localize to cell–cell junctions and are required for proper embryonic morphogenesis, to position hypodermal cells and to organize junctional actin and the junction-associated protein AJM-1. iii) Double mutant analysis indicated that the toca genes act in the same pathway as the nematode homologue of N-WASP/WASP, wsp-1. Furthermore, mammalian TOCA-1 and C. elegans CeTOCAs physically associated with N-WASP and WSP-1 directly, or WAVE2 indirectly via ABI-1. Thus, we propose that TOCA proteins control tissues morphogenesis by coordinating Clathrin-dependent membrane trafficking with WAVE and N-WASP–dependent actin-dynamics

Isolation of Fully Human Antagonistic RON Antibodies Showing Efficient Block of Downstream Signaling and Cell Migration1

RON belongs to the c-MET family of receptor tyrosine kinases. As its well-known family member MET, RON and its ligand macrophage-stimulating protein have been implicated in the progression and metastasis of tumors and have been shown to be overexpressed in cancer. We generated and tested a large number of human monoclonal antibodies (mAbs) against human RON. Our screening yielded three high-affinity antibodies that efficiently block ligand-dependent intracellular AKT and MAPK signaling. This effect correlates with the strong reduction of ligand-activated migration of T47D breast cancer cell line. By cross-competition experiments, we showed that the antagonistic antibodies fall into three distinct epitope regions of the RON extracellular Sema domain. Notably, no inhibition of tumor growth was observed in different epithelial tumor xenografts in nude mice with any of the antibodies. These results suggest that distinct properties beside ligand antagonism are required for anti-RON mAbs to exert antitumor effects in vivo

(A) Positive control: expression of Mandys106 2C6 antibody in normal human muscles

(B) Negative control: lack of expression of same antibody in the muscles of normal dog. (C, D) MT1-S shows positive staining with Mandys106 2C6 in large dystrophic fibers (white arrows). (E) Positive control: expression of C-terminal in normal human muscles. (F) C-terminal antibody in the muscles of normal dog presents weak labeling. (G) Negative control: lack of its expression in the muscles of affected dog. (H-J) MT1-S shows positive staining with C-terminal antibody in some large dystrophic fibers (arrow). K) Toluidine blue and L) HE staining shows very large fibers with multiple centrally located nuclei and splitting. A, C, E-H = Fcm + DIC, B = DIC, D,I,= EF, J,K = Light microscopy, Scale bars = 50 μm<p><b>Copyright information:</b></p><p>Taken from "Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?"</p><p>http://www.translational-medicine.com/content/6/1/35</p><p>Journal of Translational Medicine 2008;6():35-35.</p><p>Published online 3 Jul 2008</p><p>PMCID:PMC2529267.</p><p></p

GRMD puppies from a colony of dogs with X-linked muscular dystrophy were genotyped within 48 hs after birth

The genomic PCR product digested with Sau96I produces the wild type band (310 pb) and the mutant band (150 pb) labelled with arrows. ■ = Affected male. ● = Affected female. ◉ = Carrier female.<p><b>Copyright information:</b></p><p>Taken from "Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?"</p><p>http://www.translational-medicine.com/content/6/1/35</p><p>Journal of Translational Medicine 2008;6():35-35.</p><p>Published online 3 Jul 2008</p><p>PMCID:PMC2529267.</p><p></p

Ticagrelor in patients with diabetes and stable coronary artery disease with a history of previous percutaneous coronary intervention (THEMIS-PCI) : a phase 3, placebo-controlled, randomised trial

Background: Patients with stable coronary artery disease and diabetes with previous percutaneous coronary intervention (PCI), particularly those with previous stenting, are at high risk of ischaemic events. These patients are generally treated with aspirin. In this trial, we aimed to investigate if these patients would benefit from treatment with aspirin plus ticagrelor. Methods: The Effect of Ticagrelor on Health Outcomes in diabEtes Mellitus patients Intervention Study (THEMIS) was a phase 3 randomised, double-blinded, placebo-controlled trial, done in 1315 sites in 42 countries. Patients were eligible if 50 years or older, with type 2 diabetes, receiving anti-hyperglycaemic drugs for at least 6 months, with stable coronary artery disease, and one of three other mutually non-exclusive criteria: a history of previous PCI or of coronary artery bypass grafting, or documentation of angiographic stenosis of 50% or more in at least one coronary artery. Eligible patients were randomly assigned (1:1) to either ticagrelor or placebo, by use of an interactive voice-response or web-response system. The THEMIS-PCI trial comprised a prespecified subgroup of patients with previous PCI. The primary efficacy outcome was a composite of cardiovascular death, myocardial infarction, or stroke (measured in the intention-to-treat population). Findings: Between Feb 17, 2014, and May 24, 2016, 11 154 patients (58% of the overall THEMIS trial) with a history of previous PCI were enrolled in the THEMIS-PCI trial. Median follow-up was 3·3 years (IQR 2·8–3·8). In the previous PCI group, fewer patients receiving ticagrelor had a primary efficacy outcome event than in the placebo group (404 [7·3%] of 5558 vs 480 [8·6%] of 5596; HR 0·85 [95% CI 0·74–0·97], p=0·013). The same effect was not observed in patients without PCI (p=0·76, p interaction=0·16). The proportion of patients with cardiovascular death was similar in both treatment groups (174 [3·1%] with ticagrelor vs 183 (3·3%) with placebo; HR 0·96 [95% CI 0·78–1·18], p=0·68), as well as all-cause death (282 [5·1%] vs 323 [5·8%]; 0·88 [0·75–1·03], p=0·11). TIMI major bleeding occurred in 111 (2·0%) of 5536 patients receiving ticagrelor and 62 (1·1%) of 5564 patients receiving placebo (HR 2·03 [95% CI 1·48–2·76], p<0·0001), and fatal bleeding in 6 (0·1%) of 5536 patients with ticagrelor and 6 (0·1%) of 5564 with placebo (1·13 [0·36–3·50], p=0·83). Intracranial haemorrhage occurred in 33 (0·6%) and 31 (0·6%) patients (1·21 [0·74–1·97], p=0·45). Ticagrelor improved net clinical benefit: 519/5558 (9·3%) versus 617/5596 (11·0%), HR=0·85, 95% CI 0·75–0·95, p=0·005, in contrast to patients without PCI where it did not, p interaction=0·012. Benefit was present irrespective of time from most recent PCI. Interpretation: In patients with diabetes, stable coronary artery disease, and previous PCI, ticagrelor added to aspirin reduced cardiovascular death, myocardial infarction, and stroke, although with increased major bleeding. In that large, easily identified population, ticagrelor provided a favourable net clinical benefit (more than in patients without history of PCI). This effect shows that long-term therapy with ticagrelor in addition to aspirin should be considered in patients with diabetes and a history of PCI who have tolerated antiplatelet therapy, have high ischaemic risk, and low bleeding risk