Detection of GAD65 autoreactive T-cells by HLA class I tetramers in type 1 diabetic patients.

Type 1 diabetes (T1D) is an autoimmune disease, in which pancreatic beta 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 beta 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.

Identification and characterization of adult stem/progenitor cells in the human bladder (bladder spheroids): perspectives of application in pediatric surgery.

There is evidence that tissue-specific stem cells reside in certain adult tissues. Their specific properties remain elusive, because they are rare in parent tissues and heterogeneous; furthermore, technical difficulties have been encountered in their identification and the characterization of their progeny. The aim of this study was to isolate stem/progenitor cells from the human bladder. We have devised a method for isolating stem/progenitor cells from the human bladder. This is based on the enzymatic digestion of fresh surgical bladder specimens, followed by culture of cells in the presence of EGF and bFGF. We also used markers that identified and finally characterized these cells. Spheroids with self-replicative potential were obtained from all bladder specimens. The isolated population contained a subset of CD34+ CD45- cells. These spheroids represent a predominant functional type of stem/progenitor cells within the human bladder. This envisage their potential use for the treatment of animal models in pediatric surgery.

Notch3 and pre-TCR interaction unveils distinct NF-kappaB pathways in T-cell development and leukemia.

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 pTalpha/pre-TCR and display constitutive activation of NF-kappaB, providing survival signals for immature thymocytes. We provide genetic and biochemical evidence that Notch3 triggers multiple NF-kappaB activation pathways. A pre-TCR-dependent pathway preferentially activates NF-kappaB via IKKbeta/IKKalpha/NIK complex, resulting in p50/p65 heterodimer nuclear entry and recruitment onto promoters of Cyclin D1, Bcl2-A1 and IL7-receptor-alpha genes. In contrast, upon pTalpha deletion, Notch3 binds IKKalpha and maintains NF-kappaB activation through an alternative pathway, depending on an NIK-independent IKKalpha homodimer activity. The consequent NF-kappaB2/p100 processing allows nuclear translocation of p52/RelB heterodimers, which only trigger transcription from Bcl2-A1 and IL7-receptor-alpha genes. Our data suggest that a finely tuned interplay between Notch3 and pre-TCR pathways converges on regulation of NF-kappaB activity, leading to differential NF-kappaB subunit dimerization that regulates distinct gene clusters involved in either cell differentiation or proliferation/leukemogenesis.

PKC theta mediates pre-TCR signaling and contributes to Notch3-induced T-cell leukemia.

Protein kinase (PK)C theta is a critical regulator of mature T-cell activation and proliferation, being implicated in TCR-triggered nuclear factor (NF)-kappa B activation and providing important survival signals to leukemic T cells. We previously showed that overexpression of pT alpha/pre-TCR and constitutive activation of NF-kappa B characterize the T-cell leukemia/lymphoma developing in Notch3-IC transgenic mice. We report here that PKC theta is a downstream target of Notch3 signaling and that its activation and membrane translocation require a functional pre-TCR in order to trigger NF-kappa B activation in thymocytes and lymphoma cells of transgenic mice. Furthermore, deletion of PKC theta in Notch3-IC transgenic mice reduces the incidence of leukemia, correlating with decreased NF-kappa B activation. This paper therefore suggests that PKC theta mediates the activation of NF-kappa B by pre-TCR in immature thymocytes and contributes to the development of Notch3-dependent T-cell lymphoma.