Les rétinopathies ischémiques prolifératives telles que la rétinopathie diabétique proliférative et la rétinopathie du prématuré sont les principales causes de la perte de la vision dans la population active et pédiatrique des pays industrialisés. Malgré le fait que les événements initiateurs sont différents et propres à chacune des pathologies, les rétinopathies ischémiques prolifératives sont le résultat d’un processus biphasique. On a d’abord une phase initiale de dégénérescence microvasculaire suivie d’une néovascularisation excessive et pathologique de la rétine hypoxique qui tente de réinstaurer l’apport en nutriments et en énergie. Mais au lieu d’aller revasculariser les zones avasculaires de la rétine, ces nouveaux vaisseaux sanguins sont mal orientés et se dirigent plutôt vers le vitré normalement avasculaire. Ceci provoque des tensions physiques dans la rétine et mène à long terme à son détachement et une perte de vision conséquente. Les traitements actuels ne viennent pas sans effets secondaires majeurs. Par exemple, la formation de la cataracte et l’augmentation de la pression intraoculaire avec l’utilisation des corticostéroïdes ou la perte de la vision partielle dans le cas du traitement au laser sont fréquemment observées. De même, la thérapie anti-VEGF (Vascular endothelial growth factor) apporte aussi son lot de complications, telles que la thromboembolie veineuse et l’augmentation de la neurotoxicité après un long usage, vu les propriétés neuro- et vaso-protectrices du VEGF. Le développement d’une nouvelle approche thérapeutique pour les rétinopathies ischémiques prolifératives est donc nécessaire afin de contrer ces limitations thérapeutiques. Dans notre première étude, nous mettons en évidence un nouveau mécanisme par lequel les cellules neuronales sous stress diabétique sont à l’origine d’une forte inflammation oculaire. Nos résultats démontrent que le co-récepteur multi-ligand Neuropiline-1, le VEGF et la Sémaphorine-3A agissent de concert afin d’attirer une sous-population particulière de phagocytes mononucléaires susceptibles d’activer le processus de croissance vasculaire pathologique dans la rétine diabétique. De plus, notre étude propose une base pour de futures recherches sur l’impact des phagocytes mononucléaires exprimant Neuropiline-1 dans les pathologies du système nerveux central caractérisées par une inflammation excessive. Nos résultats permettent aussi de mettre en lumière le caractère anti-inflammatoire potentiel des thérapies actuelles anti-VEGF (à cause du rôle de VEGF dans la mobilisation des phagocytes mononucléaires via Neuropiline-1) au niveau oculaire. Dans notre deuxième étude, nous mettons en évidence l’activation du facteur HIF1α dans les phagocytes mononucléaires présents dans la rétine hypoxique. L’utilisation d’une approche protéomique non biaisée de spectrométrie de masse en tandem nous a permis d’identifier les partenaires interagissant avec HIF1α dans un milieu déficient en oxygène. Nous avons pu ainsi déterminer pour la première fois l’association entre la voie d’HIF1α et celle d’IRE1α (un des trois senseurs de la voie de l’UPR « unfolded protein response ») dans le processus d’adaptation à l’oxygène des phagocytes mononucléaires. Nos résultats révèlent ensuite l’importance d’IRE1α (plus principalement son activité kinase) dans la production d’HIF1α. Nous démontrons finalement que la synergie entre les signalisations d’IRE1α et HIF1α pourrait être responsable du comportement pathogénique des phagocytes mononucléaires via leur libération de cytokines inflammatoires; ce qui participerait ainsi à la progression des rétinopathies. Collectivement, nos travaux ont permis d’identifier d’importants régulateurs de l’activité pathogénique des phagocytes mononucléaires. Nous montrons : 1) le rôle de Neuropiline-1 dans l’infiltration des phagocytes mononucléaires au niveau des zones endommagées de la rétine et 2) l’impact du mécanisme convergent entre les voies d’IRE1α et HIF1α sur leur sécrétion de facteurs pro-inflammatoires durant les rétinopathies. Nos résultats offrent une base pour le développement de nouvelles stratégies thérapeutiques (ciblant Neuropiline-1, IRE1α et HIF1α) dans le traitement de maladies oculaires et d’autres pathologies caractérisées par une inflammation excessive. / Proliferative ischemic retinopathies such as proliferative diabetic retinopathy (PDR) and retinopathy of prematurity (ROP) are the principal causes of vision loss in working age and pediatric populations of industrialized countries. Although they display different initial triggers, proliferative ischemic retinopathies are biphasic ocular diseases that affect retinal vessels. There is an initial degeneration of the microvasculature, followed by a hypoxic stress on the retina. This triggers a second phase of deregulated and destructive blood vessel growth within the retina. Given this sequence of events and prominent clinical features, the currently most widely used local ocular therapeutic interventions directly target pathological blood vessel growth, yet present a number of non-desirable off-target effects such as the destruction of the retina itself. In fact, currently available treatments for proliferative ischemic retinopathies present non-negligible side effects, such as cataract formation with intravitreal use of corticosteroid or reduced visual field with laser-based photocoagulation surgery. Similarly, the anti-VEGF (Vascular endothelial growth factor) therapy may be associated with thromboembolic events, neuronal toxicity and atrophy when used as frequent long-term treatment given the fact that VEGF serves a vaso- and neuro-protective factor in the retina. Overcoming these therapeutic limitations and exploring novel pharmacological avenues are therefore required to ameliorate the safety profiles of current interventions. In our first study, we describe a novel mechanism by which severely stressed neuronal cells in the diabetic retina provoke destructive inflammation in the eye. We demonstrate that the multi-ligand co-receptor Neuropilin-1, VEGF and Semaphorin3A act as potent attractants for a specialized population of immune cells (mononuclear phagocytes) that later promote the exaggerated pathological vessel growth associated with the disease progression. Importantly, we provide evidence for a novel pharmacological intervention that reduces the inflammation associated with pathological retinal vessel growth. Our findings also suggest that current anti-VEGF therapies (a popular treatment for ocular vascular diseases) may in part be effective by reducing destructive ocular inflammation. In our second study, we provide evidence that those mononuclear phagocytes activate HIF1α in the hypoxic and inflamed retina. After using the unbiased proteomic approach of tandem mass spectrometry, we were able to identify HIF1α partners and found a novel link between HIF1α and the UPR (unfolded protein response) sensor IRE1α. Our data next established the crucial role of IRE1α (precisely via its kinase activity) in HIF1α production. We also suggested that the synergy between IRE1α and HIF1α pathways may be responsible of the pathogenic activity of the hypoxic mononuclear phagocytes via their secretion of inflammatory cytokines, thus contributing to the progression of the retinopathy. Collectively, our work identifies important regulators of the pathogenic activity of mononuclear phagocytes. We show that: 1) Neuropilin-1 promotes the infiltration of mononuclear phagocytes in the retina and 2) the convergent mechanism between IRE1α and HIF1α pathways is responsible for their release of pro-inflammatory factors during retinopathy. Our results could be used as a basis for the development of alternative therapeutic strategies (targeting Neuropilin-1, IRE1α and HIF1α) to treat ocular diseases or other pathologies characterized by an excessive inflammation.

A doença granulomatosa crônica é uma imunodeficiência primária dos fagócitos causada por mutações no sistema NADPH oxidase resultando em burst oxidativo ausente ou reduzido. Nosso objetivo foi realizar uma análise genética molecular do complexo NADPH oxidase em pacientes com diagnóstico clínico de DGC. Cinqüenta e quatro pacientes com diagnóstico clínico sugestivo da DGC foram incluídos em nosso estudo. As populações de neutrófilos e monócitos foram avaliadas pela capacidade de produzir peróxido de hidrogênio por meio do teste de DHR. Dezoito pacientes apresentaram defeito no burst oxidativo, enquanto trinta e oito apresentaram produção de peróxido normal. O DNA genômico dos dezoito pacientes com burst oxidativo diminuído foi extraído, os genes da cadeia beta polipeptídica do complexo citocromo b e o factor citoplasmático de neutrófilos, foram sequenciados. Sete pacientes apresentaram diferentes mutações, tanto no gene CYBB como no NCF1. Concluímos que a combinação do teste de DHR e o sequenciamento direto são métodos eficazes para o diagnóstico genético da DGC. / Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the phagocyte NADPH oxidase system resulting in absent or reduced oxidative burst. Our goal was to perform a molecular genetic analysis of complex NADPH oxidase in patients with clinical diagnosis of CGD. Fifty-four patients with a clinical diagnosis of CGD were included in our study. The populations of neutrophils and monocytes were evaluated for the ability to produce hydrogen peroxide through the DHR test. Eighteen patients had a defect in the oxidative burst, while thirty-eight had normal peroxide production. Genomic DNA of the eighteen patients with decreased oxidative burst was extracted, the genes the chain complex cytochrome beta polypeptide and the neutrophil cytoplasmic factor, were sequenced. Seven patients had different mutations, both in the CYBB gene as in NCF1. We conclude that the combination of direct sequencing and DHR test methods are effective for the genetic diagnosis of CGD.

Les nouveau-nés (enfants, ruminants) sont particulièrement sensibles à l’infection intestinale par le parasite Cryptosporidium parvum car leur système immunitaire est encore en cours de développement. Peu de solutions de contrôle existent à ce jour. Il n’existe pas de vaccin et seule une molécule l’Halocur™ possède une AMM pour les veaux mais l’utilisation du traitement est contraignante et il peut présenter une toxicité pour l’animal. Le développement de nouvelles alternatives immunoprophylactiques requiert de mieux comprendre les mécanismes immunitaires mis en jeux lors de l’infection. L’immunité innée joue un rôle prépondérant pour le contrôle de la phase aigüe de l’infection et nous avions montré au laboratoire que les phagocytes mononucléés CD11c+ sont des acteurs déterminant dans le processus protection. Lors de cette thèse nous avons confirmé le rôle des cellules dendritiques (DC) CD103+ en utilisant des souriceaux BatF3-/- chez qui le développement des deux sous-populations CD103+CD11b+ et CD103+CD11b- est altéré au niveau intestinal ce qui rend les animaux beaucoup plus sensibles à l’infection. / Newborns (children, ruminants) are particularly susceptible to intestinal infection by the parasite Cryptosporidium parvum because their immune system is still developing. To date, parasite control methods are limited. There is no vaccine and the only molecule which possess a marketing authorization for calves, Halocur ™, presents toxicity at 2 times the therapeutic dose. The development of new immunoprophylactic methods requires better understanding of the immune mechanisms occurring during infection. Innate immunity plays a major role in controlling the acute phase of infection and we previously demonstrated in the laboratory that intestinal mononuclear phagocytes CD11c+ are key players in the protection process. In this thesis, we confirmed the role of dendritic cells (DC) CD103+ using mice BatF3-/- in which the development of the two DC subsets CD103+CD11b+ and CD103+CD11b- is altered in the intestine making these animals more susceptible to infection. This high susceptibility can be partially mitigated by preventive administration of IL-12 to Batf3-/- neonatal mice. Batf3-/- adult mice which are only deficient for the CD103+CD11b- DC subset were transiently susceptible to infection in contrast to conventional mice that are highly resistant.

Thesis (Ph.D.)--Mississippi State University. College of Veterinary Medicine. / Title from title screen. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2001. / Härtill 5 uppsatser.

Changes to the composition and function of the gut mononuclear phagocyte (MNP) compartment are associated with the development of intestinal inflammation. Much work has focused on the role of MNPs in gut-associated lymphoid tissue in maintaining homeostasis, however little is known regarding the roles of MNPs during colitis. We have investigated MNPs in the large intestinal lamina propria during the steady state and inflammation. One of our primary aims was to determine the contribution of MNP subsets to intestinal pathology. For our studies of inflammation, we focused mainly on the Helicobacter hepaticus infection + anti-IL-10R model, which induces inflammation of the colon and caecum (typhlocolitis). We defined the composition of the MNP compartment alongside intestinal pathology scores throughout Hh + anti-IL-10R typhlocolitis. Peak pathology, 2-3 weeks after induction of colitis, coincided with peak frequencies of CX₃CR1^int Ly6C^+ MNPs. Having observed the accumulation of CX₃CR1^int CD64^+ monocyte/macrophage MNPs in the inflamed lamina propria, we conducted comparative whole genome microarray analysis of these cells isolated from the large intestine three weeks after Hh + anti-IL-10R treatment. CX₃CR1^int CD64^+ MNPs selectively expressed a variety of pro- and anti-inflammatory genes, including a number of genes which individually can both promote and negatively regulate inflammation. IL-23 is essential for Hh + anti-IL-10R-induced intestinal pathology. We investigated the role of MNPs as a source of IL-23 which drives Hh + anti-IL-10R colitis. Unexpectedly, our results indicate that normally hyporesponsive CX₃CR1^hi macrophages may act as the initial source of IL-23, which induces development of colitis. Recruitment of Ly6C^+ MHCII^+ MNPs to the lamina propria was IL-23-dependent, and these cells also expressed IL-23, which may establish a positive feedback loop of immune cell recruitment, activation and IL-23 production. Finally, we also examined how MNPs might be recruited to the colonic lamina propria during inflammation. Our studies support the conclusion that CCR6 is not required for accumulation of monocyte-derived populations in the inflamed intestine. We cannot rule out a role for CCR2, however preliminary data from the Hh + anti-IL-10R colitis model suggest a potential role for CCR1 or its close relation CCRL2. Such pathways could represent new therapeutic targets in inflammatory bowel disease.

Orientador: Flávio Henrique Caetano / Banca: Hércules Menezes / Banca: Ricardo José Gomes / Resumo: Quando um tecido ou órgão é alvo de algum tipo de injúria, dá-se início ao processo de inflamação tecidual. As reações celulares e humorais que tomam parte desta reação são evocadas a fim de exterminar ou neutralizar o agente infeccioso e seus produtos, permitindo que o reparo tecidual se dê da melhor forma possível. Contudo, algumas vezes o resultado desta ação pode ser prejudicial, levando ao comprometimento ou a perda das funções do tecido ou órgão em questão, com conseqüências que podem afetar a saúde do indivíduo como um todo. Em contrapartida, a atividade física moderada tem sido apontada como um grande aliado no combate à várias doenças, potencializando a ação do sistema imune e reduzindo os possíveis danos e minimizando os efeitos nocivos das respostas inflamatórias. Desta forma, nos propomos a verificar os efeitos da atividade física moderada no processo inflamatório e de reparação tecidual, comparando 10 ratos Wistar que realizaram exercícios físicos moderados com 10 sedentários, por meio da análise numérica (microscopia de luz) e ultra-estrutural (microscopia eletrônica de transmissão) de mastócitos, macrófagos e fibroblastos, células muito importantes para tais fenômenos. O modelo de estudo escolhido foi o implante subcutâneo de esponjas de PVC, que mimetiza situações clínicas importantes como, por exemplo, implantes de marcapasso. Nossos resultados indicam que as células do grupo treinado respondem de maneira mais ágil (precoce) e efetiva à injúria, que o número de cada tipo celular foi significativamente maior neste grupo e ainda que, para quaisquer dos grupos, os tipos celulares em estudo agem estreitamente interrelacionados...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: When a tissue or organ is target of some offense, it feels beginning to the process of tissue inflammation. Cellular and humoral reactions that participate of this reaction are evoked to exterminate or to neutralize the infectious agent and your products, allowing the tissue repair to feel in the best possible way. However, the result of this action can sometimes be harmful, taking to the compromising or the loss of the functions of the tissue or organ in subject, with consequences that can affect the individual's health as a completely. On the other hand, the moderate physical activity has been pointed as a great ally in the combat to several diseases, improving the action of the immune system and reducing the possible damages and minimizing the noxious effects of the inflammatory answers. Thus, we intend to verify the effects of the moderate physical activity in the inflammatory and tissue repair process, comparing 10 Wistar rats that had carried through moderate physical exercises with 10 sedentary ones, through the numeric analysis (light microscopy) and ultrastructural (transmission electronic microscopy) of mast cells, macrophages and fibroblasts, which are very important cells for such phenomena. The model of chosen study was the subcutaneous implantation of PVC sponges, that simulates important clinical situations as, for instance, implant of pacemaker. Our results indicate that the cells of the trained group answer in a more agile (precocious) and effective way to the offense, that the number of each cellular type was significantly larger in this group and although, for any of the groups, the cellular types in study act narrowly interrelated. Therefore, we ended in our work that the moderate physical activity can contribute positive and significantly for the inflammation and tissue repair process, making possible that the homeostasis is reestablished more easily and, like this, avoiding larger damages to the individual. / Mestre

Made available in DSpace on 2014-06-11T19:22:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-04-20Bitstream added on 2014-06-13T19:49:27Z : No. of bitstreams: 1 rodrigues_rf_me_rcla.pdf: 5396854 bytes, checksum: 53dc5df0dd553ffb8f11bf6074d163cc (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Quando um tecido ou órgão é alvo de algum tipo de injúria, dá-se início ao processo de inflamação tecidual. As reações celulares e humorais que tomam parte desta reação são evocadas a fim de exterminar ou neutralizar o agente infeccioso e seus produtos, permitindo que o reparo tecidual se dê da melhor forma possível. Contudo, algumas vezes o resultado desta ação pode ser prejudicial, levando ao comprometimento ou a perda das funções do tecido ou órgão em questão, com conseqüências que podem afetar a saúde do indivíduo como um todo. Em contrapartida, a atividade física moderada tem sido apontada como um grande aliado no combate à várias doenças, potencializando a ação do sistema imune e reduzindo os possíveis danos e minimizando os efeitos nocivos das respostas inflamatórias. Desta forma, nos propomos a verificar os efeitos da atividade física moderada no processo inflamatório e de reparação tecidual, comparando 10 ratos Wistar que realizaram exercícios físicos moderados com 10 sedentários, por meio da análise numérica (microscopia de luz) e ultra-estrutural (microscopia eletrônica de transmissão) de mastócitos, macrófagos e fibroblastos, células muito importantes para tais fenômenos. O modelo de estudo escolhido foi o implante subcutâneo de esponjas de PVC, que mimetiza situações clínicas importantes como, por exemplo, implantes de marcapasso. Nossos resultados indicam que as células do grupo treinado respondem de maneira mais ágil (precoce) e efetiva à injúria, que o número de cada tipo celular foi significativamente maior neste grupo e ainda que, para quaisquer dos grupos, os tipos celulares em estudo agem estreitamente interrelacionados... / When a tissue or organ is target of some offense, it feels beginning to the process of tissue inflammation. Cellular and humoral reactions that participate of this reaction are evoked to exterminate or to neutralize the infectious agent and your products, allowing the tissue repair to feel in the best possible way. However, the result of this action can sometimes be harmful, taking to the compromising or the loss of the functions of the tissue or organ in subject, with consequences that can affect the individual's health as a completely. On the other hand, the moderate physical activity has been pointed as a great ally in the combat to several diseases, improving the action of the immune system and reducing the possible damages and minimizing the noxious effects of the inflammatory answers. Thus, we intend to verify the effects of the moderate physical activity in the inflammatory and tissue repair process, comparing 10 Wistar rats that had carried through moderate physical exercises with 10 sedentary ones, through the numeric analysis (light microscopy) and ultrastructural (transmission electronic microscopy) of mast cells, macrophages and fibroblasts, which are very important cells for such phenomena. The model of chosen study was the subcutaneous implantation of PVC sponges, that simulates important clinical situations as, for instance, implant of pacemaker. Our results indicate that the cells of the trained group answer in a more agile (precocious) and effective way to the offense, that the number of each cellular type was significantly larger in this group and although, for any of the groups, the cellular types in study act narrowly interrelated. Therefore, we ended in our work that the moderate physical activity can contribute positive and significantly for the inflammation and tissue repair process, making possible that the homeostasis is reestablished more easily and, like this, avoiding larger damages to the individual.

A doença granulomatosa crônica é uma imunodeficiência primária dos fagócitos causada por mutações no sistema NADPH oxidase resultando em burst oxidativo ausente ou reduzido. Nosso objetivo foi realizar uma análise genética molecular do complexo NADPH oxidase em pacientes com diagnóstico clínico de DGC. Cinqüenta e quatro pacientes com diagnóstico clínico sugestivo da DGC foram incluídos em nosso estudo. As populações de neutrófilos e monócitos foram avaliadas pela capacidade de produzir peróxido de hidrogênio por meio do teste de DHR. Dezoito pacientes apresentaram defeito no burst oxidativo, enquanto trinta e oito apresentaram produção de peróxido normal. O DNA genômico dos dezoito pacientes com burst oxidativo diminuído foi extraído, os genes da cadeia beta polipeptídica do complexo citocromo b e o factor citoplasmático de neutrófilos, foram sequenciados. Sete pacientes apresentaram diferentes mutações, tanto no gene CYBB como no NCF1. Concluímos que a combinação do teste de DHR e o sequenciamento direto são métodos eficazes para o diagnóstico genético da DGC. / Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the phagocyte NADPH oxidase system resulting in absent or reduced oxidative burst. Our goal was to perform a molecular genetic analysis of complex NADPH oxidase in patients with clinical diagnosis of CGD. Fifty-four patients with a clinical diagnosis of CGD were included in our study. The populations of neutrophils and monocytes were evaluated for the ability to produce hydrogen peroxide through the DHR test. Eighteen patients had a defect in the oxidative burst, while thirty-eight had normal peroxide production. Genomic DNA of the eighteen patients with decreased oxidative burst was extracted, the genes the chain complex cytochrome beta polypeptide and the neutrophil cytoplasmic factor, were sequenced. Seven patients had different mutations, both in the CYBB gene as in NCF1. We conclude that the combination of direct sequencing and DHR test methods are effective for the genetic diagnosis of CGD.

INTRODUÇÃO: A ativação da NADPH oxidase de neutrófilos requer o acoplamento das subunidades citosólicas p47phox, p67phox, p40phox e Rac2 ao componente de membrana citocromo b558. Em trabalhos anteriores, nós mostramos que as isoformas vasculares da oxidase são reguladas pela proteína dissulfeto isomerase (PDI), uma chaperona redox. Neste trabalho, nós utilizamos um sistema cellfree semirecombinante como ferramenta para investigar o papel da PDI na ativação da NADPH oxidase do neutrófilo. RESULTADOS: Inibidores da PDI, scrambled RNAse (100g/mL) ou bacitracina (1mM), praticamente suprimiram a geração de superóxido. Para avaliação dos efeitos do estado redox da PDI sobre a atividade da oxidase, amostras de PDI foram previamente oxidadas (H2O2; 0,5 mM) ou reduzidas (DTT; 0,5 mM). A PDI oxidada (100 nM) aumentou a produção de superóxido em aproximadamente 30%, enquanto a mesma concentração de PDI reduzida promoveu efeito inverso, inibindo a atividade do complexo. A adição de um peptídeo contendo a seqüência peptídica do sítio ativo da PDI inibiu a produção de superóxido em 70%. Dados de imunolocalização e colocalização demonstraram que a interação da PDI com a subunidade p47phox parece ser intensificada pelo estímulo com PMA e envolvem modificações do estado redox de ambas as proteínas. CONCLUSÕES: Nossos dados confirmam a associação física e funcional entre a PDI e o complexo NADPH oxidase. Além disso, sugerem que a PDI exerça um importante papel como fator de regulação redox da ativação da oxidase. / Activation of the leukocyte NADPH oxidase requires the assembly of the cytosolic subunits p47phox, p67phox and p40phox and Rac2 with the membranebound cytochrome b558. We have previously shown that the vascular oxidase is regulated by the redox chaperone protein disulfide isomerase (PDI). Taking advantage of the semirecombinant cellfree system, we sought to investigate the role of PDI in the activation of neutrophil NADPH oxidase. The PDI thiol inhibitors scrambled RNase (100g/mL) or bacitracin (1mM), almost suppressed superoxide generation. In order to investigate if the redox status of PDI thiols could modulate superoxide generation, PDI was oxidized or reduced by treatment with H2O2 (0.5mM) or DTT (1mM), respectively. Oxidized PDI increased by 30% superoxide production, while reduced PDI diminished superoxide generation also in 30%. The addition of a peptide (1M) containing PDI´s exact active site sequence inhibited superoxide production by 70 %. Immuno and colocalization data demonstrated the interaction of PDI with the subunit p47phox to be intensified by PMA stimulation and to involve redox status exchange of both proteins. Our data confirm the physical and functional association between PDI and the oxidase complex. Moreover, we show a relevant role for PDI as a redoxdependent supportive factor for NADPH oxidase activation.