Notch signaling plays a critical role in T-cell differentiation and leukemogenesis. We previously demonstrated that, while pre-TCR is required for thymocytes proliferation and leukemogenesis, it is dispensable for thymocyte differentiation in Notch3-transgenic mice. Notch3-transgenic premalignant thymocytes and T lymphoma cells overexpress pTa/pre-TCR and display constitutive activation of NF-jB, providing survival signals for immature thymocytes. We provide genetic and biochemical evidence that Notch3 triggers multiple NF-jB activation pathways. A pre-TCR-dependent pathway preferentially activates NF-jB via IKKb/IKKa/NIK complex, resulting in p50/p65 heterodimer nuclear entry and recruitment onto promoters of Cyclin D1, Bcl2-A1 and IL7-receptor-a genes. In contrast, upon pTa deletion, Notch3 binds IKKa and maintains NF-jB activation through an alternative pathway, depending on an NIK-independent IKKa homodimer activity. The consequent NF-jB2/p100 processing allows nuclear translocation of p52/RelB heterodimers, which only trigger transcription from Bcl2-A1 and IL7-receptor-a genes. Our data suggest that a finely tuned interplay between Notch3 and pre-TCR pathways converges on regulation of NF-jB activity, leading to differential NF-jB subunit dimerization that regulates distinct gene clusters involved in either cell differentiation or proliferation/ leukemogenesis.
The penton base of adenovirus mediates viral attachment to integrin receptors and particle internalisation, properties that can be exploited to reengineer prokaryotic viruses for the infection of mammalian cells. We report that filamentous phage displaying either the full-length penton base gene or a central region of 107 amino acids on their surface were able to bind, internalise, and transduce mammalian cells expressing integrin receptors. Both phage bound alphavbeta3, alphavbeta5, alpha3beta1, and alpha5beta1 integrin subtypes. Cell-binding was shown by electron microscopy; internalisation was investigated by immunofluorescence and confirmed by micropanning. As it has been described for adenovirus, pharmacologic disruption of phosphoinositide-30H kinase, but not of myosin light-chain kinase, inhibited phage internalisation. Recombinant phage encoding an eukaryotic expression cassette was able to mediate gene expression in mammalian cells. Taken together, these data open insights for the exploit of recombinant phage for integrin-targeted gene delivery.
Type 1 diabetes (T1D) is an autoimmune disease, in which pancreatic β cells are destroyed in genetically predisposed individuals. While the direct contribution of autoantibodies to the disease pathogenesis is controversial, it is generally recognised that the mechanism of β cell destruction is mediated by autoreactive T cells that had escaped the thymic selection. We aimed to design a method to detect circulating CD8+ T cells autoreactive against an epitope of the glutamic acid decarboxylase autoantigen, isoform 65 (GAD65) ex vivo in T1D patients by using HLA class I tetramers. Low frequencies of GAD65 peptide-specific CD8+ cytotoxic T lymphocytes were detected in peripheral blood lymphocytes (PBMC) of normal controls after GAD65 peptide-specific stimulation. Conversely, their frequencies were significantly higher than in controls in PBMC of T1D patients after GAD65 peptide stimulation. These preliminary data are encouraging in order to develop a reliable assay to be employed in large-scale screening studies.
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