L. (L.) amazonensis (La) e L. (V.) braziliensis (Lb) podem causar um espectro de manifestações clínicas e imunopatológicas no homem, sendo La responsável pela forma anérgica difusa e Lb pela forma mucocutânea da doença, formas polares e de elevada gravidade. Neste sentido, o objetivo do presente estudo foi avaliar os aspectos da resposta imune celular no ponto de inoculação e no linfonodo de drenagem de camundongos BALB/c inoculados no coxim plantar com 106 promastigotas de La e Lb. A evolução da lesão foi avaliada semanalmente sendo que na 4ª e 8ª semana PI biópsias do ponto de inoculação foram coletadas para determinação da densidade de células dendríticas (CD207+ e CD11c+), linfócitos T CD4+ e CD8+ e células iNOS+ por imunoistoquímica, e o linfonodo de drenagem para caracterização de subpopulações de células dendríticas e de linfócitos T CD4 e CD8 por citometria de fluxo. Células de linfonodo de drenagem foram cultivadas, com estímulo homólogo, para quantificação de citocinas (IL-4, IL- 10 e IFN-g) e nitrito nos sobrenadantes. A infecção por La levou à progressão da doença, com aumento do tamanho da lesão e da carga parasitária tanto na pele quanto no linfonodo de drenagem, enquanto que a infecção por Lb mostrou um discreto aumento da lesão entre a 6ª e 7ª semana PI com posterior regressão e redução da carga parasitária na pele e no linfonodo de drenagem. Aumento do número de células dendríticas dérmicas e de Langerhans foi observado na pele de camundongos inoculados com La na 4ª semana PI, juntamente com o aumento no número de células de Langerhans nos linfonodos de drenagem. Resposta imune celular preferencial de células T CD4+ foi observada tanto na pele quanto no linfonodo de camundongos inoculados com La, que mostrou ser predominantemente do tipo Th2 com a produção aumentada de IL-10 e IL-4. Já a infecção por Lb levou ao aumento na expressão de células dendríticas dérmicas e Langerhans na pele dos animais inoculados com Lb somente na 8ª semana PI, assim como aumento do número de células dendríticas dérmicas no linfonodo. A resposta imune celular foi caracterizada por células T CD4+ e CD8+ em pele e linfonodo dos animais infectados com Lb, vinculada a um perfil Th1 com a produção preferencial de IFN-g e altos níveis de NO. Aumento no número de células T regulatórias foi observado na infecção por Lb, que mostrou correlação direta com o número de linfócitos T CD4+ produtores de IL-10. Assim, a infecção por La foi relacionada à suscetibilidade, enquanto que a infecção por Lb foi relacionada à resistência do hospedeiro vertebrado. Estes resultados evidenciam não só o papel do parasita na modulação da resposta imune do hospedeiro como também das células dendríticas à infecção por Leishmania / L. (L.) amazonensis (La) and L. (V.) braziliensis (Lb) are responsible for a spectrum of clinical and immunopathological manifestations in humans, La is able to cause anergic diffuse leishmaniasis and Lb mucocutaneous leishmaniasis, polar forms with high severity. In this way, the aim of the present study was to evaluate aspects of the cellular immune response in the site of infection and in the draining lymph node of BALB/c mice inoculated in the hind footpad with 106 promastigotes of La and Lb. The evolution of the lesion size was evaluated weekly and in the 4th and 8th week PI biopsies from the site of infection were collected to determine the density of dendritic cells (CD207+ and CD11c+), CD4+ and CD8+ T cells and iNOS+ cells by immunohistochemistry, and the draining lymph node to characterize subsets of dendritic cells and CD4+ and CD8+ T cells by flow citometry. The draining lymph nodes cells were cultured with specific antigen to determine the cytokines (IL-4, IL-10 e IFN-g), and the nitric oxide in the supernatants. The infection caused by La led to the progression of disease with increase on lesion size and parasite load in the skin and draining lymph node, while Lb infection showed a discrete increase on the lesion size between 6th and 7th week PI with late regression and reduction in the skin as well as lymph node parasite load. An increase on the number of dermal dendritic and Langerhans cells were observed in the skin of BALB/c mice infected with La at 4th week PI together with an increase of Langerhans cells in the draining lymph node. The preferential CD4+ T cell immune response was observed in skin and lymph node of mice infected with La, which showed to be rather Th2 with an increase on the levels of IL-4 and IL-10. However, Lb infection led an increase of dermal dendritic cells and Langerhans cells in the skin only at 8th week PI, as well as an increase in the dermal dendritic cells in lymph node. The cellular immune response were characterized by CD4+ and CD8+ T cells in the skin and draining lymph node of mice infected with Lb, which was related to a Th1 immune response with production of high levels of IFN-g and nitric oxide. An increase of Regulatory T cells was observed in Lb infection, which showed the positive correlation with the IL-10 producing CD4+ T cells. So, the La infection was related to the susceptibility, while Lb infection was related to the resistance in the vertebrate host. These results emphasize the role of the parasite in the modulation of the host immune response to Leishmania infection

In the first study, I aimed to compare the angiotensin II type 1 receptor (AT1R) expression levels of the isolated pancreatic islets from normal and mouse model of T2DM. In addition, 4-week-old diabetic mice were orally treated with AT1R antagonist losartan for 8 weeks. It is found that AT1R mRNA was upregulated markedly in diabetic islets and double-immunolabeling confirmed that AT1R was localized to beta-cells. Losartan selectively improved glucose-induced insulin release and (pro)insulin biosynthesis in diabetic islets. Oral losartan treatment delayed the onset of diabetes, and reduced hyperglycemia and glucose intolerance in diabetic mice. These data indicate that AT1R antagonism improves beta-cell function and glucose tolerance in young T2DM mice. / In the second study, I aimed to examine how the upregulated RAS could impair beta-cell function, where oxidative stress is the potential mediator. Meanwhile, T2DM results in oxidative stress-mediated activation of uncoupling protein 2 (UCP2), a negative regulator of islet function. Thus, it was postulated that some of the protective effects of AT1R antagonism might be mediated through interference with this pathway and tested this hypothesis in a mouse model of T2DM. In order to achieve this, losartan was given to 4-week-old diabetic mice for 8 weeks. UCP2-driven oxidative damage and apoptosis were analyzed in isolated islets. Results showed that losartan selectively inhibited oxidative stress via NADPH oxidase downregulation; this in turn suppressed UCP2 expression, thus improving beta-cell insulin secretion while decreasing apoptosis-induced beta-cell mass loss in diabetic mice islets. These data indicate that islet AT1R activation in young diabetic mice can lead to progressive islet beta-cell failure through UCP2-driven oxidative damage and apoptosis. / The mechanisms by which chronic hyperglycemia associated with glucotoxicity causes beta-cell dysfunction and apoptosis remain ambiguous. Voltage-gated outward potassium (Kv) current, which mediates beta-cell membrane potential and limits insulin secretion, could play a role in glucotoxicity. Meanwhile the RAS has been shown to be upregulated by prolonged exposure to high glucose. In the third part of my study, I therefore investigated the effects of prolonged exposure to high glucose and angiotensin II (Ang II) on the expression and activity of Kv channels in mouse pancreatic beta-cell. Dissociated mice beta-cells, incubated in 5.6 mM or 28 mM glucose for 3-5 days, were used for electrophysiological study; while isolated islets cultured for 1-7 days were proceeded for gene/protein expression analysis. Both Kv channel expression and current were markedly increased by prolonged glucose incubation. Simultaneously, Ang II reduced Kv current under normal glucose condition, while high glucose incubation abolished the effect of Ang II. Moreover, the ability of Ang II on Kv current reduction was eliminated by inhibiting AT2R but not AT1R. These data indicated that Ang II reduced Kv current via AT2R, which was abolished by prolonged high glucose incubation. On the other hand, high glucose increased Kv channel expression and current, which might alter the ability of insulin secretion in beta-cell. (Abstract shortened by UMI.) / Chu, Kwan Yi. / Adviser: P. S. Leung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3246. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 163-188). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

BACKGROUND AND OBJECTIVES: Type 2 diabetes mellitus (T2DM) is a complex disease with genetic predisposition and histopathological characterization. Pancreatic islet amyloidosis, hyaline arteriolosclerosis, and diabetic glomerulopathy are histopathological hallmarks of T2DM at autopsy examination. The associations of genetic variants with diabetic amyloidosis, arteriosclerosis and glomerulopathy have not been fully elucidated. Several candidate genes including apolipoprotein E (ApoE), insulin degrading-enzyme (IDE) and glucose transporter-1 ( GLUT1) have been reported to increase risk of T2DM in human studies although results are not always consistent. Capitalizing on the pathological hallmarks of T2DM, I used autopsy specimens to investigate the risk associations of polymorphisms of ApoE (rs429358 and rs7412), IDE (rs6583813) and GLUT1 (rs710218) genes with clinical features and specific pathological changes in diabetic kidney and pancreas. I further explored the mechanisms of these associations by evaluating the histopathological changes and protein expression in pancreas and kidney. / CONCLUSIONS: These findings suggest that genetic factors have important effects in the development of tissue-specific changes and chronic complications in T2DM. Islet amyloidosis, arteriosclerosis and glomerulosclerosis in T2DM may share common pathogenetic processes as suggested by the coexistence of chaperone proteins, amyloid P and ApoE. Genetic--pathologic correlation studies are useful in advancing our understanding of the mechanisms of complex diseases such as T2DM. / METHODS AND MATERIALS: Genomic DNA was extracted from white blood cell-concentrated paraffin embedded formalin fixed spleen tissues. Genotyping for ApoE, IDE and GLUT1 polymorphisms was determined by polymerase chain reaction (PCR) and ligase detection reaction (LDR). The pathological changes were blindly assessed in pancreatic and kidney tissues of autopsy specimens. Protein expression of these genes was examined by immunostaining and quantified by using Metamorph image analysis system. / RESULTS: In a consecutive study population of 3693 autopsy specimens containing 328 T2DM and 209 control cases, the respective frequencies of genotypes were as follows: 1) TT of GLUT1 rs710218: 11.2% vs. 11.3%; 2) ApoE epsilon2: 19.4% vs. 10.9%; 3) ApoE epsilon4: 12.1% vs. 9.1% and 4) C carriers of IDE rs6583813: 51.2% vs. 47.9%. The key genotype-phenotype correlations were as follows. 1) In the T2DM cases, GLUT1 rs710218 IT genotype carriers (0% in TT genotype vs. 59.1% in AA genotype, P=0.0407) were less likely but ApoE epsilon 2 allele carriers (57.1% in epsilon2 allele carriers vs. 23.5% in epsilon3 allele carriers P=0.0382) were more likely to have diabetic glomerular hypertrophy than referential group. ApoE epsilon2 carriers showed increased glomerular ApoE protein expression with the immunoreactivity found mainly in the mesangial regions and nodular lesions. On the other hand, ApoE epsilon 3/epsilon4 cases had diffuse ApoE expression in glomerular capillaries. 2) ApoE epsilon4 carriers were more likely to have islet amyloidosis than non-carriers (62.5% in epsilon4 allele carriers vs. 23.6% in epsilon 3 allele carriers P=0.0232). There was immunolocalization of the chaperone proteins, amyloid P and ApoE in both islet amyloid deposits and arterial walls with hyaline arteriolosclerosis. 3) In T2DM cases, IDE rs6583813 C allele carriers had higher prevalence of vascular disorders [hypertension (67.4% vs. 43.6%, P=0.0332), death due to cardiovascular disease (58.1% vs. 25.6%, P=0.0479) and cerebral vascular accident (CVA) (20.9% vs. 2.4%, P=0.0412)1 than T allele carriers. / Guan, Jing. / Adviser: Chan Chung Ngor Juliana. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 175-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

A paracoccidioidomicose (PCM) é uma doença sistêmica causada pelos fungos Paracoccidioides brasiliensis e Paracoccidioideslutzii. O comprometimento da pele e mucosa oral são frequentes na PCM. As células dendríticas e queratinócitos do tegumento, devido à sua função como células apresentadoras de antígenos, atuam na resposta imune inata e adaptativa contra agentes patogênicos. Com o objetivo de verificar a interação do P. brasiliensis com essas células, estudamos 47 biopsias de mucosa oral e 52 de pele de lesões de doentes com diagnóstico comprovado de PCM. As biopsias foram submetidas à técnica de imuno-histoquímica de dupla-marcação com os anticorpos anti-fator XIIIa (marcador de dendrócitos dérmicos), anti-CD207 (marcador de células de Langerhans maduras), anti-pancitoqueratinas (AE1-AE3) e anti-P. brasiliensis. Fez-se também a reação de dupla marcação por técnica de imunofluorescência, com análise por microscopia confocal a laser, para a melhor visualização da interação entre queratinócitos e os fungos. Quarenta e dois por cento das amostras de mucosa oral exibiram formas fúngicas no citoplasma de dendrócitos dérmicos. As células de Langerhans, tanto nas biopsias de mucosa oral como de pele, não mostraram leveduras ou antígenos do fungo no seu citoplasma. Cinquenta e quatro por cento das biopsias de pele e sessenta por cento das amostras de mucosa exibiram leveduras no citoplasma de queratinócitos. Os resultados obtidos permitem concluir que o parasitismo de queratinócitos pode representar possível mecanismo de evasão do fungo aos mecanismos imunes locais. Os dendrócitos dérmicos fator XIIIa positivos e queratinócitos podem estar a atuar como células apresentadoras de antígenos para suprir a função, provavelmente prejudicada, das células de Langerhans nas lesões de pele e mucosa oral da PCM humana / Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis, which compromises various organs, mainly the lungs. The skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 biopsies of skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to double immune staining technique with anti-factor XIIIa (marker of dermal dendrocytes), anti- CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed to better visualization of the interaction between keratinocytes and the fungi. Factor XIIIa + dermal dendrocytes of 42% samples of oral mucosa displayed yeast forms in their cytoplasm. We did not observe yeast cells in the cytoplasm of Langerhans cells in both skin and oral mucosa samples. Fifty -four percent of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms, or even as a result of keratinocytes ability to antigen presentation in PCM. Factor XIIIa dendrocytes and keratinocytes may be acting as antigenpresenting cells to fulfill the function of Langerhans cells, probably impaired, in skin and mucosa of human PCM

Les cellules β des îlots de Langerhans jouent un rôle central dans l’homéostasie glucidique car elles seules sécrètent l’insuline, unique hormone hypoglycémiante de l’organisme. La cellule β est un détecteur du glucose qui couple sa réponse sécrétoire et son expression génique aux niveaux ambiants de glucose. Le couplage entre le métabolisme du glucose et l’exocytose des granules d’insuline implique la génération d’une activité électrique. Son étude est importante pour déchiffrer la façon dont la cellule β encode la demande en insuline de l’organisme. Afin de contourner les limites des approches électrophysiologiques classiques incompatibles avec les études à long-terme, les enregistrements extracellulaires par matrice de microélectrodes (MEA) ont été mis en place.L’objectif de ma thèse était de mieux comprendre les signaux complexes enregistrés par MEAs. Cette étude a révélé l’existence d’une nouvelle signature électrique des cellules des îlots, les slow potentials (SP), qui reflète la fonction de couplage des cellules β. Les SP jouent un rôle important dans l’homéostasie du glucose et représentent un biomarqueur de la fonction normale des îlots. La réponse en hystérèse des îlots au glucose suggère l’existence d’un algorithme d’encodage de la demande en insuline intégrée au niveau du micro-organe. De plus, ce nouveau signal a été exploité pour le phénotypage d’îlots de souris invalidées pour le gène GluK2, que nous avons utilisées comme modèle d’interaction entre les cellules α et β. La caractérisation de ce nouveau type de signal constitue aussi une avancée importante pour le développement d’un biocapteur destiné à être intégré dans le futur à un pancréas artificiel. / Pancreatic β cells are central to glucose homeostasis because they are the only cell that secretes insulin, the sole hypoglycemic hormone in the organism. The β cell is a glucose sensor that regulates its secretory response and gene expression according to ambient glucose levels. The coupling between glucose metabolism and insulin granule exocytosis involves the generation of electrical activity. An investigation of this activity is important to decipher how β cells encode the organism’s insulin demand. In order to overcome the limits of classically used electrophysiological approaches that are not compatible with long-term studies, extracellular recordings using multielectrode arrays (MEA) have been set-up.My thesis aim was to better understand the complex signals recorded with MEA. This study revealed the existence of a new electrical signature of islet cells: slow potentials (SP) that reflect the coupling function of β cells. SP play an important role in glucose homeostasis and represent a biomarker of normal functioning of islets. The observed hysteretic response of islets to glucose suggests the existence of an algorithm encoding the insulin demand embedded at the microorgan level. Moreover, this new signal was used for the phenotyping of GluK2 deficient mouse islets that were employed as an α-to-β cell interaction model. The characterization of this new signal is an important progress in the development of a biosensor intended to be integrated in an artificial pancreas in the future.

Islet transplantation has emerged as a promising cell-based therapy for the treatment of diabetes, but its clinical efficacy remains limited by deleterious host responses that underlie islet destruction. In this dissertation, we describe the assembly of cell surface-supported thin films that confer molecular-level control over the composition and biophysicochemical properties of the islet surface with implications for improving islet engraftment. Specifically, the process of layer-by-layer (LbL) polymer self assembly was employed to generate nanothin films of diverse architecture with tunable properties directly on the extracellular surface of individual islets. Importantly, these studies are the first to report in vivo survival and function of nanoencapsulated cells, and have helped establish a conceptual framework for translating the diverse applications of LbL films to cellular interfaces. Additionally, through proper design of film constituents, coatings displaying ligands and bioorthogonally reactive handles may be generated, providing a modular strategy for incorporating exogenously derived regulators of host responses alongside native constituents of the islet surface. Towards this end, a strategy was developed to tether thrombomodulin to the islet surface in a site-specific manner, thereby facilitating local generation of the powerful anti-inflammatory agent, activated protein C. Collectively, this work offers novel biomolecular strategies for cell surface engineering with broad biomedical and biotechnological applications in cell-based therapeutics and beyond.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 146-172). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Les cellules dendritiques jouent un rôle primordial dans le système immunitaire. En effet, ces cellules sont à l'interface entre l'immunité innée et adaptative par leur capacité de reconnaissance, d'internalisation et de dégradation de pathogènes afin de présenter des antigènes aux lymphocytes. La capacité de reconnaissance est engendrée par l'expression de différents récepteurs à la surface de ces cellules. Parmi ces récepteurs, deux grandes familles permettent la reconnaissance d'un large panel de différents pathogènes, comme les TLRs (« Toll-Like Receptors) et les lectines de type-C. Ces récepteurs sont utilisés comme marqueurs des différents sous-types de cellules dendritiques. Par exemple, parmi les lectines de type-C, DC-SIGN est majoritairement exprimée dans les cellules dendritiques dermiques alors que la Langérine est, quand à elle, fortement exprimée par les cellules dendritiques épidermiques, les cellules de Langerhans. Ces deux sous-types de cellules dendritiques divergent par leur réponse à l'infection par le VIH (« virus d'immunodéficience humain »). En effet, le virus utilise DC-SIGN pour détourner le rôle de ces cellules afin d'infecter les lymphocytes T alors que la reconnaissance du VIH par la Langérine, dans les cellules de Langerhans, conduit à la clairance de virus par son internalisation dans le granule de Birbeck. Cet organite est spécifique des cellules de Langerhans et nécessite l'expression de la Langérine. Ce travail de thèse s'est donc focalisé sur la caractérisation structurale et fonctionnelle de la Langérine. Il a permis de mettre en évidence l'importance de la structure tertiaire du domaine CRD et de la structure quaternaire de la protéine pour la formation et la bonne structuration du granule de Birbeck. Ensuite, l'étude fonctionnelle de cette lectine, notamment par résonance plasmonique de surface, nous a conduit à identifier une nouvelle spécificité de reconnaissance de la Langérine pour les glycosaminoglycanes dans un site d'interaction différent du site canonique. Enfin, nous avons caractérisé une spécificité de reconnaissance du site canonique pour les monosaccharides sulfatés de type glucosamine en utilisant la résonance plasmonique de surface et la cristallographie. / Dendritic cells play a crucial role in the immune system. Indeed, these cells are at the interface between innate and acquired immunity by their capacities of recognition, internalisation and pathogen degradation to present antigens to T lymphocytes. The recognition capacity is generated by the expression of diverse receptors onto the cell surface. Among these receptors, two large families allow the recognition of a large panel of different pathogens, as TLRs (“Toll-Like Receptor) and C-type lectins. These receptors are used as markers of different dendritic cells subtypes. For example, and among the C-type lectins, DC-SIGN is mainly expressed onto dermic dendritic cells contrary to langerin, which is highly expressed onto epidermic dendritic cells, called Langerhans cells. These two subtypes of dendritic cells differ in their response of HIV infection. Indeed, the virus recognition by DC-SIGN enables hijacking the dendritic cell to infect T lymphocyte contrary to langerin recognition, in Langerhans cells, which allows the clearance of the virus by its internalisation into Birbeck granules. This organite is specific of Langerhans cells and requires langerin expression. This work is focused on structural and functional characterisation of langerin. It highlights the importance of the CRD tertiary structure and the quaternary structure of the protein for the formation and the structure of Birbeck granules. Then, functional study by surface plasmon resonance enabled us to identify a new binding site of langerin for glycosaminoglycans. Finally, we have characterised a recognition specificity of langerin for sulphated monosaccharide of glucosamine type using surface plasmon resonance and crystallography.

O estresse oxidativo e a enzima NADPH oxidase-2 (NOX2) estão associados com a diminuição da massa funcional de células-β em pacientes com diabetes do tipo 2 (DT2). Neste estudo, testamos o papel da NOX2 sobre a glicotoxicidade em células-β. Ilhotas de camundongo C57BL/6J nocautes ou não para NOX2 (NOX2-KO e WT, respectivamente) foram isoladas e cultivadas por até 3 semanas em 10 ou 30 mmol/l de glucose (G10 e G30, respectivamente). A secreção de insulina foi maior nas ilhotas NOX2-KO vs. WT sem apresentar diferenças metabólicas ou do potencial redox da glutationa citosólica (EGSH). O cultivo de ilhotas em G30 aumenta a concentração de H2O2 e a oxidação de tióis no compartimento citosólico, seguido por aumento de apoptose de células-β, mas, preservando a reposta máxima secretória. Estas respostas foram quase idênticas em ambos os tipos de ilhotas. Em conclusão, a NOX2 regula negativamente a secreção de insulina em ilhotas de camundongos C57BL/6J, mas não é um componente crítico para a sobrevivência de células β em um modelo in vitro de glicotoxicidade. / Oxidative stress and NADPH oxidase-2 (NOX2) enzyme are associated to the decline of the functional β-cell mass in type 2 diabetes (T2D). Here, we tested the role of NOX2 on β-cell glucotoxicity. NOX2 knockout (NOX2 KO) and wild type (WT) C57BL/6J mice islets were isolated and cultured up to 3 weeks at 10 or 30 mmol/l glucose concentrations (G10 and G30, respectively). The insulin secretion was higher in NOX2-KO vs. WT islets despite similar metabolic and cytosolic glutathione-redox potential (EGSH) changes. The prolonged culture at G30 increases the H2O2 concentration and cytosolic thiol oxidation, followed by increased βcell apoptosis but preserving maximal secretory response. These responses were almost identical in both types of islets. In conclusion, NOX2 is a negative regulator of insulin secretion in C57BL/6J mouse islets, but is not a critical component for β-cell survival in a model of glucotoxicity in vitro.