This study indicates that CD34(+) cells obtained from dental pulp can be used for engineering bone, without the need for prior culture expanding procedures. Using autologous stem cells, this schedule could be used to provide the basis for bone regenerative surgery, with limited sacrifice of tissue, low morbidity at the collection site, and significant reduction in time needed for clinical recovery.

Mucopolysaccharidosis type I is a lysosomal disease due to mutations in the IDUA gene, resulting in deficiency of alpha-L-iduronidase and accumulation of glycosaminoglycans (GAGs). Bone marrow transplantation and enzyme replacement are two therapies considered only moderately successful for affected patients, making the development of novel treatments necessary. We have previously shown the efficacy of lentivirus-mediated gene transfer to correct patient fibroblasts in vitro. Here we tested lentiviral-IDUA vector gene therapy in vivo on a murine MPS I model. Eight- to 10 week-old mice were injected with increasing lentiviral doses via the tail vein and analyzed 1 month after treatment. A single injection of lentiviral-IDUA vector resulted in transgene expression in several murine tissues, with the highest level reached in liver and spleen. Expression of 1% normal activity was sufficient in treated animals to normalize the GAG level in urine, liver, and spleen and was able to reduce the GAG level in kidney, heart, and lung. Polymerase chain reaction assays showed integration of the viral genome only in liver and spleen of treated animals, suggesting that the correction of the pathology in other tissues was due to secretion into the plasma by liver and spleen and uptake of corrective enzyme by distant tissues. Long-term (6 months) analysis showed the presence of enzyme-specific antibodies and the loss of enzyme activity and vector sequence in the target tissue, suggesting that the transgene-specific immune response interfered with long-term therapeutic correction and led to clearance of transduced cells. In conclusion, our results show the promising potential and the limitations of lentiviral-IDUA vector-mediated gene therapy in an in vivo model.

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Mucopolysaccharidosis (MPS) IIIB is a lysosomal disease due to the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The disease is characterized by mild somatic features and severe neurological disorders. Very little is known on the cardiac dysfunctions in MPS IIIB. In this study, we used the murine model of MPS IIIB (NAGLU knockout mice, NAGLU-/-) in order to investigate the cardiac involvement in the disease. Echocardiographic analysis showed a marked increase in left ventricular (LV) mass, reduced cardiac function and valvular defects in NAGLU-/- mice as compared to wild-type (WT) littermates. The NAGLU-/- mice exhibited a significant increase in aortic and mitral annulus dimension with a progressive elongation and thickening of anterior mitral valve leaflet. A severe mitral regurgitation with reduction in mitral inflow E-wave-to-A-wave ratio was observed in 32-week-old NAGLU-/- mice. Compared to WT mice, NAGLU-/- mice exhibited a significantly lower survival with increased mortality observed in particular after 25 weeks of age. Histopathological analysis revealed a significant increase of myocardial fiber vacuolization, accumulation of HS in the myocardial vacuoles, recruitment of inflammatory cells and collagen deposition within the myocardium, and an increase of LV fibrosis in NAGLU-/- mice compared to WT mice. Biochemical analysis of heart samples from affected mice showed increased expression levels of cardiac failure hallmarks such as calcium/calmodulin-dependent protein kinase II, connexin43, α-smooth muscle actin, α-actinin, atrial and brain natriuretic peptides, and myosin heavy polypeptide 7. Furthermore, heart samples from NAGLU-/- mice showed enhanced expression of the lysosome-associated membrane protein-2 (LAMP2), and the autophagic markers Beclin1 and LC3 isoform II (LC3-II). Overall, our findings demonstrate that NAGLU-/- mice develop heart disease, valvular abnormalities and cardiac failure associated with an impaired lysosomal autophagic flux.

The inhibitor of Bruton tyrosine kinase ␥ (IBtk␥) is a negative regulator of the Bruton tyrosine kinase (Btk), which plays a major role in B-cell differentiation; however, the mechanisms of IBtk␥-mediated regulation of Btk are unknown. Here we report that B-cell receptor (BCR) triggering caused serine-phosphorylation of IBtk␥ at protein kinase C consensus sites and dissociation from Btk. By liquid chromatography and mass-mass spectrometry and functional analysis, we identified IBtk␥-S87 and -S90 as the critical amino acid residues that regulate the IBtk␥ binding affinity to Btk. Consistently, the mutants IBtk␥ carrying S87A and S90A mutations bound constitutively to Btk and down-regulated Ca 2؉ IntroductionBruton tyrosine kinase (Btk) is a member of the Tec family of nonreceptor protein tyrosine kinases and is expressed in B cells, macrophages, and neutrophils. 1 Btk sustains the developmental program of pre-B cells by limiting the pre-B cell expansion and by promoting B-cell differentiation. 1,2 Consistently, mutations of BTK cause the human X-linked agammaglobulinemia and the murine X-linked immunodeficiency syndromes, which are characterized by increased susceptibility to recurrent bacterial infections as a consequence of the impaired generation of mature B cells and low production of immunoglobulin. 3,4 Btk is a crucial component of the immunoglobulin B-cell receptor (BCR) signaling pathway. Evidence from 5 indicates that Btk is required for a proper tyrosine phosphorylation of phospholipase C-␥ (PLC-␥), which in turn leads to inositol-3,4,5-triphosphate, a major mediator of [Ca 2ϩ ]i mobilization, and to diacylglycerol, an activator of protein kinase C (PKC). 6 These pathways activate specific transcription factors, including NF-B, 7,8 which regulate the gene transcription program required for B-cell survival and cell-cycle progression. Accordingly, DT40 Btk Ϫ/Ϫ chicken B cells show a drastic decrease in Ca 2ϩ signaling and NF-B activation on antigen stimulation, 9 and Btk Ϫ/Ϫ mice have a significant reduction of B cells. 10,11 Btk harbors the Pleckstrin homology domain (PH) and the Src homology domain 2 (SH2) and SH3, suggesting that its regulation occurs through protein-protein interaction. 12 The PH domain mediates the binding of Btk to phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3 ) resulting in the recruitment of Btk to the plasma membrane. 13 The kinase activity of Btk is up-regulated by the Src-mediated phosphorylation of Y551 at the activation loop of the Btk kinase domain and by autophosphorylation of Y223 in the Src homology domain 3, 14,15 whereas it is inactivated by the PKC␤-mediated phosphorylation of S180. 16 Genetic evidence indicates that Syk, BLNK, and Btk are all required for the full activation of PLC␥ and Ca 2ϩ signaling on BCR triggering, [17][18][19][20] which suggests the occurrence of a multimolecular complex for PLC␥ activation. 21,22 In this scenario, it was proposed that the BCR triggering causes the Syk-mediated tyrosine phosphorylation of the signaling adaptor BLNK, w...