S U M M A R Y Pancreatic ␤ -cells contain large amounts of zinc. We took advantage of this to try to localize, quantify, and isolate insulin-producing cells from islet preparations. Our study was designed to identify a non-toxic zinc-sensitive fluorescent probe able to selectively label labile zinc in viable ␤ -cells and to exhibit excitation and emission wavelengths in the visible spectrum, making this technique exploitable by most instruments. We tested Newport Green, a probe excitable at 485 nm with a dissociation constant in the micromolar range corresponding to a low affinity for zinc. The loading of the lipophilic esterified form of Newport Green was easy, rapid, specific, and non-toxic to cells. Confocal microscopy highlighted an intense fluorescence associated with secretory granules. Regression analyses showed a good relationship between zinc fluorescence and islet number ( r ϭ 0.98) and between zinc fluorescence and insulin content ( r ϭ 0.81). The determination of Zn fluorescence per DNA enabled us to assess the quality of the different islet preparations intended for islet allografting in terms of both purity and viability. Cell sorting of dissociated Newport Green-labeled cells resulted in a clear separation of ␤ -cells, as judged by insulin content per DNA and immunocytochemical analysis. This zinc probe, the first able to specifically label living cells in the visible spectrum, appears very promising for ␤ -cell experimentation, both clinically and for basic research. P ancreatic islets contain a much higher concentration of zinc than other tissues. Zinc plays an important role in packaging insulin because it is firmly established as an integral part of the insulin crystal as a 2-Zn-insulin hexamer. In addition, free ionized zinc is found in the extragranular space of ␤ -cells, where it may act as a reservoir for granular zinc (Figlewicz et al. 1984). These pools of free and loosely bound zinc can be visualized histochemically by most cytological stains, unlike zinc tightly complexed to proteins such as metalloproteins, including transcription factors and metalloenzymes. Our study was designed to search for a new non-toxic probe that was able to specifically stain living ␤ -cells. Our aim was threefold: (a) to visualize ␤ -cells in pancreatic islets by confocal microscopy or image analysis; (b) to isolate these ␤ -cells for physiological studies; and (c) to rapidly estimate, in allograft management, the amount of viable ␤ -cells in semipurified preparations containing contaminating exocrine cells. The latter pretransplantation evaluation of the quality of the graft has become a crucial prerequisite to ensure successful engraftment. Here we describe the use of Newport Green, a new zinc-sensitive probe with an excitation wavelength in the visible spectrum, which fulfills all the criteria required for efficient staining of human ␤ -cells in clinical and research applications. Materials and Methods Human Islet ProcessingHuman pancreata ( n ϭ 11; mean age 35 Ϯ 12 years) were harvested from adult brain-...

Enteroviruses may be linked to insulin-dependent diabetes mellitus (IDDM). The prevalence of enteroviral (EV) infection at onset of adult IDDM was investigated by detection of specific EV sequences in peripheral blood using a reverse transcription and a seminested polymerase chain reaction (seminested RT-PCR). EDTA-treated whole blood samples taken from 12 newly diagnosed IDDM patients with ketosis or ketoacidosis were examined. The comparison groups were 12 adult patients suffering from metabolic decompensation in the course of IDDM, 12 adult patients with decompensated non-IDDM, and 15 healthy adults without any presumed EV infection or metabolic disease. EV genome was detected in five of 12 (42%) newly diagnosed IDDM patients and in one of 12 (8%) patients in the course of IDDM. By contrast, none of the 12 non-IDDM patients and none of the 15 healthy adults had EV sequences in whole blood. Subsequent sequencing of the EV PCR products from the six positive patients showed a significant homology with Coxsackie B3 or B4 viruses, and some common patterns were observed among the sequences. The present study demonstrates that Coxsackie B virus RNA sequences can be detected in peripheral blood from patients at the onset or in the course of IDDM and provides evidence for a role for enteroviruses in adult type I diabetes.

Beta cell loss occurs at the onset of type 1 diabetes and after islet graft. It results from the dysfunction and destruction of beta cells mainly achieved by apoptosis. One of the mediators believed to be involved in beta cell apoptosis is Fas, a transmembrane cell surface receptor transducing an apoptotic death signal and contributing to the pathogenesis of several autoimmune diseases. Fas expression is particularly induced in beta cells by inflammatory cytokines secreted by islet-infiltrating mononuclear cells and makes cells susceptible to apoptosis by interaction with Fas-ligand expressing cells. We have previously demonstrated that 1,25(OH)2D3, the active metabolite of vitamin D, known to exhibit immunomodulatory properties and prevent the development of type 1 diabetes in NOD mice, is efficient against apoptosis induced by cytokines in human pancreatic islets in vitro. The effects were mainly mediated by the inactivation of NF-kappa-B. In this study we demonstrated that 1,25(OH)2D3 was also able to counteract cytokine-induced Fas expression in human islets both at the mRNA and protein levels. These results were reinforced by our microarray analysis highlighting the beneficial effects of 1,25(OH)2D3 on death signals induced by Fas activation. Our results provides additional evidence that 1,25(OH)2D3 may be an interesting tool to help prevent the onset of type 1 diabetes and improve islet graft survival.