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Cytokines Induce Deoxyribonucleic Acid Strand Breaks and Apoptosis in Human Pancreatic Islet Cells¹

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden; Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium

Address all correspondence and requests for reprints to: D. L. Eizirik, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103 B-1090 Brussels, Belgium. E-mail: deizirik{at}mebo.vub.ac.be

	Abstract

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We have previously observed that a 6-day exposure of human pancreatic islets to a combination of cytokines (interleukin-1ß 50 U/ml + tumour necrosis factor- 1000 U/ml + interferon- 1000 U/ml) severely impairs ß-cell functions. In the present study, we examined whether this condition affects DNA integrity and viability of human islet cells. Cells were studied after 3, 6, and 9 days of cytokine treatment by both single cell gel electrophoresis (the "comet assay," a sensitive method for detection of DNA strand breaks) and by a cytotoxicity assay using the DNA binding dyes Hoechst 33342 and propidium iodide as indices for the number of viable, necrotic, and apoptotic cells. Cytokine treatment for 6 and 9 days resulted in a 50% increase in comet length (P < 0.01 vs. controls), indicating DNA strand breaks, as well as in a significant increase in the number of apoptotic cells (P < 0.02 vs. controls), but not in the number of necrotic cells. The arginine analogs N^G-nitro-L-arginine and N^G-monomethyl-L-arginine prevented nitric oxide formation by the cytokines but did not interfere with cytokine-induced DNA strand breaks and apoptosis. The present data suggest that prolonged (6–9 days) exposure of human pancreatic islets to a mixture of cytokines induces DNA strand breaks and cell death by apoptosis. These deleterious effects of cytokines appear to be independent of nitric oxide generation.

	Introduction

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CLINICAL onset of insulin-dependent diabetes mellitus (IDDM) is probably preceded by a progressive decrease in ß-cell mass. Intercellular differences in susceptibility to the pathogenic effects are thought to influence the disease course (reviewed in 1 . Potential mediators of ß-cell dysfunction and destruction in this prediabetic period include macrophages, T lymphocytes and/or their inflammatory products, such as cytokines and free radicals (reviewed in Refs. 2–4).

Among the cytokines, interleukin- 1 (IL-1) seems to be the main mediator of ß-cell dysfunction (4). Indeed, IL-1ß, alone or in combination with TNF- or IFN-, decreases rat islet insulin biosynthesis and release (2, 4), an effect largely mediated by induction of the enzyme nitric oxide synthase (iNOS) and subsequent synthesis of the radical nitric oxide (NO) (reviewed in 5 . Cytokine-induced NO production can impair rat ß-cell function trough two main mechanisms, namely blockage of the enzyme aconitase (6, 7) and induction of DNA strand breaks (8). DNA damage can lead to cell death by apoptosis (9, 10). Rat ß-cells possess a constitutive apoptotic program, which is activated when protein synthesis is suppressed (11). There is also evidence that exposure of rat ß-cells to cytokines can lead to apoptosis (12, 13).

In human islet preparations, IL-1ß alone does not impair ß-cell functions (14), but combinations of cytokines suppress insulin release (15, 16), and decrease cell viability (17, 18, 19). However, it remains unclear whether human islet cell death occurs by necrosis or apoptosis, and whether NO plays a role in this damaging process. Indeed, while one study suggested that NO is the main mediator of cytokine-induced human ß-cell dysfunction (15), other studies showed dissociation between NO generation and the deleterious effects of cytokines (16). In this context, it is noteworthy that human islets are more resistant than rat or mouse islets to several ß-cell toxins (20) but show similar sensitivity to peroxynitrite, a radical formed by the reaction between NO and superoxide (21).

In the present study, we investigated whether prolonged exposure of human pancreatic islets to a combination of cytokines, i.e. IL-1ß + TNF- + IFN- (16, 19) induce DNA strand breaks and cell death (either by necrosis or apoptosis), and whether NO plays a role in these effects.

	Material and Methods

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Materials
Chemicals were purchased from the following sources: culture medium RPMI-1640, FCS, penicillin and streptomycin, HBSS, trypsin, BSA, agarose, N^G-nitro-L-arginine (L-NA) and L^G-monomethyl-L-arginine (L-MA) from Sigma Chemical Co. (St. Louis, MO); collagenase from Clostridium hystolyticum from Boheringer-Mannheim (Mannheim, Germany). The DNA binding dye Hoechst 33342 (HO 342) was purchased from Calbiochem, La Jolla, CA, and propidium iodide (PI) from Sigma. All other chemicals of analytical grade were obtained from E. Merck (Darmstadt, Germany) or Sigma.

For the experiments performed in Uppsala (determination of DNA strand breaks by the "comet assay" - see below) recombinant murine TNF- and human IFN- (bioactivity of 10 and 6.7 U/ng, respectively), were purchased from AMS Biotechnology (Sollentuna, Sweden) and IL-1ß (50 U/ng) was a generous gift from Dr K. Bendtzen (Laboratory of Medical Immunology, Rigshospitalet, Copenhagen, Denmark). For the experiments performed in Brussels (determinations of necrosis and apoptosis - see below) recombinant human IL-1ß (200 U/ng) and recombinant human IFN- (48 U/ng) were obtained from Genzyme (Cambridge, MA) and recombinant murine TNF- (220 U/ng) was purchased from Innogenetics (Gent, Belgium).

Islet isolation, culture, and cytokine treatment
Islets from 17 human heart beating donors were isolated at the Central Unit of the ß-Cell Transplant (Vrije Universiteit Brussel, Brussels, Belgium). The mean age of the donors was 31 ± 3 yr. Aliquots of the islet-enriched fraction were examined routinely by electron microscopy, which indicated less than 2% exocrine cells in all preparations. Light microscopical examination of immunocytochemically stained islets (22) was carried out routinely on all preparations used in this study, and indicated the prevalence of insulin-and glucagon-positive cells to be respectively 50.0 ± 3.5% and 12.2 ± 1.9%. The isolation and initial culture conditions for human pancreatic islets have been previously described (23). The islets were subsequently either used for experiments in Brussels (see below) or sent by air to Uppsala, where they were cultured in medium RPMI 1640 containing 10% FCS and 5.6 mM glucose (24). After 5–6 days in culture in Uppsala, groups of islets were exposed to cytokines (IL-1ß 50 U/ml plus TNF and IFN at 1000 U/ml) for 3, 6, or 9 days. The concentrations of cytokines used were derived from our previous studies (16, 19). After each time point islets were retrieved, dissociated, and used for the measurement of DNA damage, as described below. Culture medium was collected for the determination of nitrite accumulation by the Griess reaction (25, 26). In some experiments, human islets were exposed to cytokines in the presence of 5 mM L-NA, a treatment that significantly decreases cytokine-induced islet nitrite production (16).

Discrimination between necrosis and apoptosis was carried out in Brussels on ß-cell enriched preparations. After culture for 8–18 days in HAM’s F-10 medium supplemented with 1% BSA and containing 6 mM glucose (23), the islets were dispersed into single cells by 10–20 min discontinuous pipetting in dissociation medium (22) supplemented with 100 µg/ml trypsin. The single cells were further cultured for 2–3 days attached to the bottom of 25 cm² T-flasks (Falcon, NJ), and then recovered in dissociation medium supplemented with 10 µg/ml propidium iodine (Sigma). Sorting through a FACStar flow cytometer (Beckton Dickinson, San Jose, CA) was used to prepare viable single cell preparations enriched in ß-cells (82 ± 3% ß-cells; n = 5) (22). These cells were then cultured as single cells in polylysine coated microtiter cups (96-well plates, Falcon; 3000 cells/cup), filled with 200 µl HAM’s F-10 supplemented with 0.5% BSA, 50 µM IBMX and 7.5 mM glucose and exposed for 3, 6, and 9 days to cytokines (IL-1ß, 50 U/ml + IFN-, 1000 U/ml + TNF-, 1000 U/ml). In some experiments, the cells were also exposed to 1 mM L-MA, a treatment that completely suppresses cytokine-induced nitrite production by human islets (Pavlovic et al., unpublished data). After 3, 6, and 9 days the percent of living, necrotic and apoptotic cells was determined as described below.

Measurement of DNA strand breaks
DNA strand breakage was quantified using the alkaline version of the comet assay (single cell gel electrophoresis), performed as previously described for islet tissue (8, 21), with the modifications described below. Groups of 150 islets were rinsed in HBSS and then gently dispersed into single cells with trypsin. Islet single cells ( 2 x 10⁴ per slide) were embedded in duplicate, on top of a 0.6% low melting point agar base layer (on slides precoated with 0.6% agar) and placed in an incubator at 37 C for 1 h, to allow repair of putative trypsin-induced damage. Slides were placed in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Na sarcosinate, 10% DMSO, and 1% Triton) for 1 h to remove nonnuclear cell components and then placed in electrophoresis buffer (0.3 M NaOH, 1 mM EDTA) for 40 min. This procedure allows the DNA containing strand breaks to unwind, and the DNA fragments to move towards the anode, forming a comet tail during electrophoresis (carried out for 24 min at 20 V). The slides are neutralized and stained with ethidium bromide (20 µg/ml). The length and the intensity of the fragmented DNA in the tail region is proportional to the extent or severity of DNA damage (27). In the present study, DNA damage was evaluated using the overall comet length, which is the diameter of the head region/intact DNA and the tail length - the distance damaged DNA has migrated following electrophoresis. DNA damage was directly quantified using a fluorescent image analysis system (28), with an average of 20–40 nucleoids examined in each experiment. Briefly, the slides were examined at 500x magnification in a fluorescence microscope (excitation filter: 515–560 nm; barrier filter: 590 nm) attached to a black and white CCD video camera (Model ICD-42E, type F/L. Ikegami Tsushinki Co., Tokyo, Japan), connected to a computer-based image analysis system. The image analysis program Aequitas (IA version 1.3. DDL Ltd., Cambridge, UK), with its special application for the comet assay AutoCell (version 8A, Reppalon AB. Hägersten, Sweden), was used when evaluating the degree of DNA damage in individual cells. Details on the formulas used for defining DNA damage, the program and the performance of the image analysis system were described previously (28).

Assessment of viable, necrotic, and apoptotic islet cells
The percentages of viable, apoptotic, and necrotic cells were assessed in the single cell preparations using a recently described method (11). Briefly, after 3, 6, or 9 days exposure to cytokines, the cells were incubated for 15 min with the DNA binding dyes HO 342 (20 µg/ml) and PI (10 µg/ml). HO freely crosses the plasma membrane, entering both cells with damaged and intact membranes and leading to a blue stain of DNA. PI is a highly polar dye, which can only penetrate cells with damaged membranes, staining their nuclei in red. After the 15-min incubation, the cells were examined in an inverted fluorescence microscope with UV excitation at 340–380 nm. Viable cells are identified by their intact nuclei with blue fluorescence (HO 342), necrotic cells by their intact nuclei with yellow fluorescence (HO 342 plus PI), apoptotic cells by their fragmented nuclei, exhibiting either a blue (HO 342; early apoptosis) or yellow (HO 343 plus PI; late apoptosis) fluorescence. This fluorescence assay has been validated by electron microscopy, and offers the advantage of being quantitative (11). In each experimental condition, a minimum of 500 cells were counted. The necrosis and apoptosis indices were calculated as (% necrotic or apoptotic cells in experimental condition - % necrotic or apoptotic cells in control/100 - % dead cells in control) x 100 (20, 29).

Statistical analysis
Data are presented as means ± SEM, and statistical differences between groups was determined using Student’s paired or unpaired t test. When multiple comparisons were performed, the data was analyzed by ANOVA. In all experiments, each islet preparation (islets obtained from one human donor) was considered as one individual observation, even when experiments were performed in several replicates.

	Results

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In an initial series of experiments, whole human islets were exposed for 3, 6, or 9 days to combinations of cytokines. The total medium nitrite (pmol/islet x 9 days; n = 4) was: control, 4.91 ± 0.61; cytokines, 16.3 ± 2.7 (P < 0.01 vs. control; t test). The mean of the overall comet lengths was significantly increased after 6 and 9 days exposure to cytokines (Fig. 1), suggesting the occurrence of DNA strand breaks.

View larger version (30K): [in this window] [in a new window]   Figure 1. DNA damage following treatment of human islets with and without a combination of three cytokines (IL-1ß, 50 U/ml + TNF-, 1000 U/ml + IFN-, 1000 U/ml) for 3, 6, or 9 days. Results represent the overall comet length (intact head diameter and tail length) ± SEM from five to six independent experiments (total of 1100 nucleoid analyzed). *, P < 0.001 when compared to respective controls; unpaired t test.

View larger version (36K): [in this window] [in a new window]   Figure 2. DNA damage following treatment of human islets with three cytokines (IL-1ß, 50 U/ml + TNF-, 1000 U/ml + IFN-, 1000 U/ml) and/or L-NA (5 mM) for 6 or 9 days. Results represent the overall comet length (intact head diameter and tail length) ± SEM from three to four independent experiments (total of 1008 nucleoids analysed). *P < 0.01 when compared with the respective controls (Control or L-NA); ANOVA.

View larger version (14K): [in this window] [in a new window]   Figure 3. Effects of cytokines (IL-1ß, 50 U/ml + TNF-, 1000 U/ml + IFN-, 1000 U/ml) on the human islet apoptosis and necrosis index, as detected by fluorescence microscopy, after 3, 6, or 9 days of exposure. Data represent means ± SEM of five independent experiments. *P < 0.001 vs. control; paired t test.

View larger version (86K): [in this window] [in a new window]   Figure 4. Presence of living, necrotic and apoptotic cells following exposure of human islet to control condition (A) or cytokines (B) for 9 days. The figure is representative of five independent experiments; experimental conditions as in Fig. 3. The control preparation consists mainly of living cells, as indicated by the intact nuclei with blue fluorescence (A), whereas exposure to cytokines leads to a large percentage of apoptotic cells, as indicated by fragmented nuclei and yellow (late phase of apoptosis) fluorescence of the nuclear fragments.

	Discussion

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Cellular death by apoptosis is an active process, depending on gene transcription and protein synthesis (9, 10, 30). Apoptosis may be triggered by signals mediating tissue involution or differentiation, as well as by immune interactions during the removal of infected or transformed cells. This form of cell death is also observed in autoimmune conditions, such as experimental allergic encephalitis (31) and T-cell mediated autoimmune diabetes in mice (32). We now report that cytokines can induce apoptosis in human pancreatic islet cells. This notion has been independently demonstrated by two separate laboratories using different methods, namely the comet assay for detection of DNA strand breaks (27), and a fluorescence microscopic assay for counting the percent of apoptotic cells (11). The second technique was performed on human preparations with more than 80% ß-cells, indicating that these cells are a major target for cytokine-induced apoptosis. To our knowledge, this is the first demonstration of cytokine-induced apoptosis in human ß-cells. However, the present observations do not exclude that cytokines also induce apoptosis in islet non-ß-cells. Moreover, it remains to be determined whether cytokine-induced apoptosis is dose-dependent, and whether individual cytokines, or combinations of two cytokines, may elicit the same effect.

Following the original description by Southern and co-workers (33), there have been several studies suggesting that NO is an important mediator of cytokine-induced rodent ß-cell dysfunction and damage (reviewed in Refs. 5 and 34). Moreover, it has recently been suggested that IL-1-induced apoptosis in rat islets is significantly reduced by iNOS blockers (13). However, in the case of human islets, most studies failed to show protective effects of iNOS inhibitors against cytokine-induced ß-cell dysfunction (16, 35, 36). The claim that NO mediates cytokine-induced inhibition of insulin secretion by human islets (15) was based on a limited number of experiments - for instance, the conclusion that L-MA prevents these inhibitory effects was derived from observations in only two different human preparations. Moreover, analysis of these data indicate that the reputed protection by L-MA was only partial. The present data indicate that cytokine-induced DNA damage and apoptosis in human islets can not be explained by NO production, as suggested by the observation that two different iNOS blockers (L-MA and L-NA) fail to prevent the effects of cytokines. Alternative mechanisms for cytokine-induced islet cell death include generation of free oxygen radicals and aldehyde production (36), synthesis of ceramide (37, 38), and induction of Fas expression (39). However, it remains to be clarified whether any of these mechanisms are responsible for the present observations.

Recent studies have noticed DNA strand breaks and cell death in human islets exposed to chemical NO donors, or to peroxynitrite (a product of NO reaction with superoxide) (21, 40). It is therefore unclear why NO, as generated by cytokines, appears unrelated to cytokine-induced DNA strand breaks in the present work. A possible explanation is that cytokines induce several other genes and proteins in parallel to iNOS (reviewed in 5 . Some of these proteins, such as heat shock protein 70, heme oxygenase and manganese superoxide dismutase, might be involved in ß-cell defence and/or repair (41, 42, 43, 44), thus preventing the damage which is caused by the relatively small amounts of NO generated in cytokine-treated human islets. However, when these cells are treated with NO or peroxynitrite donors, there is an acute exposure to high radical concentrations, without sufficient time for adequate "defense" responses. It must be kept in mind that there are other putative sources of NO generation in the context of insulitis, including activated macrophages (45, 46) and islet capillary endothelial cells (47). Thus, it is conceivable that under these conditions, the combined production of NO by invading mononuclear cells, by endothelial cells and by the ß-cells, may generate enough NO to overwhelm ß-cell defenses, and thus contribute to cell death.

	Acknowledgments

 
The authors thank I.-B. Hallgren, E. Törnelius, A. Nordin (Uppsala) and the technical staff of the Diabetes Research Center at the Vrije Universiteit Brussel for expert assistance. We are also much grateful to Drs. H. Vaghef and B. Hellman for advice and help regarding the use of the fluorescence image analysis system for the comet assay.

	Footnotes

 
¹ This work was supported by grants from the Juvenile Diabetes Foundation International (JDF DIRP 95–97; JDF R.G. 195023), the Swedish Medical Research Council (12X-9886; 12X-109; 12X-9237), the Swedish Centrala Försöksdjursnämnden, the Belgian Fund for Scientific Research (3.0057.94), and from the Flemish Community (Concerted Action 93/019 Matching Fund). Human islets were prepared by the Central Unit of the ß-Cell Transplant, with financial support of a Shared Cost Action in Medical and Health Research of the European Community (BMH CT 95–1561).

² Juvenile Diabetes Foundation International Postdoctoral Fellowship

³ Research Fellow of the Belgian Fund for Scientific Research.

Received January 27, 1997.

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				W. D'Hertog, L. Overbergh, K. Lage, G. B. Ferreira, M. Maris, C. Gysemans, D. Flamez, A. K. Cardozo, G. Van den Bergh, L. Schoofs, et al. Proteomics Analysis of Cytokine-induced Dysfunction and Death in Insulin-producing INS-1E Cells: New Insights into the Pathways Involved Mol. Cell. Proteomics, December 1, 2007; 6(12): 2180 - 2199. [Abstract] [Full Text] [PDF]

				D. Su, N. Zhang, J. He, S. Qu, S. Slusher, R. Bottino, S. Bertera, J. Bromberg, and H. H. Dong Angiopoietin-1 Production in Islets Improves Islet Engraftment and Protects Islets From Cytokine-Induced Apoptosis Diabetes, September 1, 2007; 56(9): 2274 - 2283. [Abstract] [Full Text] [PDF]

				R. Granata, F. Settanni, L. Biancone, L. Trovato, R. Nano, F. Bertuzzi, S. Destefanis, M. Annunziata, M. Martinetti, F. Catapano, et al. Acylated and Unacylated Ghrelin Promote Proliferation and Inhibit Apoptosis of Pancreatic {beta}-Cells and Human Islets: Involvement of 3',5'-Cyclic Adenosine Monophosphate/Protein Kinase A, Extracellular Signal-Regulated Kinase 1/2, and Phosphatidyl Inositol 3-Kinase/Akt Signaling Endocrinology, February 1, 2007; 148(2): 512 - 529. [Abstract] [Full Text] [PDF]

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				S. A. Sarkar, R. Wong, S. I. Hackl, O. Moua, R. G. Gill, A. Wiseman, H. W. Davidson, and J. C. Hutton Induction of Indoleamine 2,3-Dioxygenase by Interferon-{gamma} in Human Islets Diabetes, January 1, 2007; 56(1): 72 - 79. [Abstract] [Full Text] [PDF]

				P. Pacher, J. S. Beckman, and L. Liaudet Nitric Oxide and Peroxynitrite in Health and Disease Physiol Rev, January 1, 2007; 87(1): 315 - 424. [Abstract] [Full Text] [PDF]

				F. Ortis, A. K. Cardozo, D. Crispim, J. Storling, T. Mandrup-Poulsen, and D. L. Eizirik Cytokine-Induced Proapoptotic Gene Expression in Insulin-Producing Cells Is Related to Rapid, Sustained, and Nonoscillatory Nuclear Factor-{kappa}B Activation Mol. Endocrinol., August 1, 2006; 20(8): 1867 - 1879. [Abstract] [Full Text] [PDF]

				M. Kitazawa, Y. Shibata, S. Hashimoto, Y. Ohizumi, and T. Yamakuni Proinsulin C-Peptide Stimulates a PKC/I{kappa}B/NF-{kappa}B Signaling Pathway to Activate COX-2 Gene Transcription in Swiss 3T3 Fibroblasts J. Biochem., June 1, 2006; 139(6): 1083 - 1088. [Abstract] [Full Text] [PDF]

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				R. Eldor, A. Yeffet, K. Baum, V. Doviner, D. Amar, Y. Ben-Neriah, G. Christofori, A. Peled, J. C. Carel, C. Boitard, et al. Conditional and specific NF-{kappa}B blockade protects pancreatic beta cells from diabetogenic agents PNAS, March 28, 2006; 103(13): 5072 - 5077. [Abstract] [Full Text] [PDF]

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				E Anastasi, C Santangelo, A Bulotta, F Dotta, B Argenti, C Mincione, A Gulino, M Maroder, R Perfetti, and U Di Mario The acquisition of an insulin-secreting phenotype by HGF-treated rat pancreatic ductal cells (ARIP) is associated with the development of susceptibility to cytokine-induced apoptosis J. Mol. Endocrinol., April 1, 2005; 34(2): 367 - 376. [Abstract] [Full Text] [PDF]

				C. A. Gysemans, A. K. Cardozo, H. Callewaert, A. Giulietti, L. Hulshagen, R. Bouillon, D. L. Eizirik, and C. Mathieu 1,25-Dihydroxyvitamin D3 Modulates Expression of Chemokines and Cytokines in Pancreatic Islets: Implications for Prevention of Diabetes in Nonobese Diabetic Mice Endocrinology, April 1, 2005; 146(4): 1956 - 1964. [Abstract] [Full Text] [PDF]

				C. Santangelo, A. Scipioni, L. Marselli, P. Marchetti, and F. Dotta Suppressor of cytokine signaling gene expression in human pancreatic islets: modulation by cytokines Eur. J. Endocrinol., March 1, 2005; 152(3): 485 - 489. [Abstract] [Full Text] [PDF]

				A. K. Cardozo, F. Ortis, J. Storling, Y.-M. Feng, J. Rasschaert, M. Tonnesen, F. Van Eylen, T. Mandrup-Poulsen, A. Herchuelz, and D. L. Eizirik Cytokines Downregulate the Sarcoendoplasmic Reticulum Pump Ca2+ ATPase 2b and Deplete Endoplasmic Reticulum Ca2+, Leading to Induction of Endoplasmic Reticulum Stress in Pancreatic {beta}-Cells Diabetes, February 1, 2005; 54(2): 452 - 461. [Abstract] [Full Text] [PDF]

				B. KUTLU, N. NAAMANE, L. BERTHOU, and D. L. EIZIRIK New Approaches for in Silico Identification of Cytokine-Modified {beta} Cell Gene Networks Ann. N.Y. Acad. Sci., December 1, 2004; 1037(1): 41 - 58. [Abstract] [Full Text] [PDF]

				I. Kharroubi, L. Ladriere, A. K. Cardozo, Z. Dogusan, M. Cnop, and D. L. Eizirik Free Fatty Acids and Cytokines Induce Pancreatic {beta}-Cell Apoptosis by Different Mechanisms: Role of Nuclear Factor-{kappa}B and Endoplasmic Reticulum Stress Endocrinology, November 1, 2004; 145(11): 5087 - 5096. [Abstract] [Full Text] [PDF]

				J M Mellado-Gil and M Aguilar-Diosdado High glucose potentiates cytokine- and streptozotocin-induced apoptosis of rat islet cells: effect on apoptosis-related genes J. Endocrinol., October 1, 2004; 183(1): 155 - 162. [Abstract] [Full Text] [PDF]

				W. H. Schott, B. D. Haskell, H. M. Tse, M. J. Milton, J. D. Piganelli, C. M. Choisy-Rossi, P. C. Reifsnyder, A. V. Chervonsky, and E. H. Leiter Caspase-1 Is Not Required for Type 1 Diabetes in the NOD Mouse Diabetes, January 1, 2004; 53(1): 99 - 104. [Abstract] [Full Text] [PDF]

				N. Allaman-Pillet, J. Storling, A. Oberson, R. Roduit, S. Negri, C. Sauser, P. Nicod, J. S. Beckmann, D. F. Schorderet, T. Mandrup-Poulsen, et al. Calcium- and Proteasome-dependent Degradation of the JNK Scaffold Protein Islet-brain 1 J. Biol. Chem., December 5, 2003; 278(49): 48720 - 48726. [Abstract] [Full Text] [PDF]

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				B. Kutlu, A. K. Cardozo, M. I. Darville, M. Kruhoffer, N. Magnusson, T. Orntoft, and D. L. Eizirik Discovery of Gene Networks Regulating Cytokine-Induced Dysfunction and Apoptosis in Insulin-Producing INS-1 Cells Diabetes, November 1, 2003; 52(11): 2701 - 2719. [Abstract] [Full Text] [PDF]

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