Macrófagos infectados com Leishmania são protegidos de apoptose, entretanto não se conhece o mecanismo de transdução de sinal intracelular que interfere neste processo de morte. Neste trabalho, células J 774 em cultura, com privação de nutrientes, sofrem apoptose, a qual aumenta na presença dos indutores camptotecina (CPT) ou fator de necrose tumoral recombinante (rTNF). Estas células quando infectadas com amastigotas ou promastigotas de Leishmania (L.) chagasi (5 parasitos/uma célula) são protegidas de apoptose. Avaliando as possíveis vias intracelulares envolvidas nesse processo, observamos que a privação de nutrientes altera o potencial de membrana da mitocôndria, havendo reversão com a infecção tanto com promastigotas e amastigotas, entretanto a reversão da alteração do potencial de membrana induzida por rTNF só foi observada com infecção com promastigotas. Tanto a atividade de caspase 3, como a detecção de caspase 3 clivada induzidas por H202 são revertidas com a infecção com promastigotas ou amastigotas. Quando analisamos a expressão de poli (ADP ribose) polimerase (PARP), em relação às células sem indução, a indução por CPT não levou ao aumento da PARP de 116 kDa, mas, aumento da banda de 24 kDA. Por outro lado, a infecção por amastigota de Leishmania (L.) chagasi em células J774 levou à diminuição da expressão de PARP de 116 kDa, mas aumento da de 24 kDa. Nas células infectadas por promastigotas de Leishmania (L.) chagasi, observamos uma diminuição da banda de 116 kDa, aparecimento de uma molécula de 89kDa e diminuição da expressão da de 24 kDa. Nas células sob indução por CPT, a infecção levou a resultados similares, exceto a diminuição da molécula de 24 kDa quando infectado por amastigota. Avaliando-se a influência da proteína do choque térmico de 83 kDa de Leishmania infantum, como possível fator que interferiria no processo de apoptose, observamos que a fagocitose de bactérias Escherichia coli (M15) contendo plasmídio com gene de HSP83 expressando essa proteína, leva a diminuição da apoptose nessas células, mesmo quando induzidas por CPT ou rTNF. Nossos dados mostram que a infecção de macrófagos J774 in vitro por Leishmania (L.) chagasi, interfere no processo de apoptose afetando diversas vias de sinalização intracelular de apoptose, tanto extrínsecas quanto intrínsecas, sendo que promastigota é mais efetiva em inibir apoptose nesta linhagem macrofágica. / Macrophages infected by Leishmania are protected from apoptosis, however the mechanism of intracellular signal transduction that interferes in this death process remains unknown. In this work, J774 cells in culture, under nutrient deprivation undergo apoptosis, which is increased in the presence of inducers: camptothecin (CPT) or recombinant tumoral necrosis factor (rTNF). These cells infected by amastigotes or promastigotes of Leishmania (L.) chagasi (5 parasites per cell) are protected from apoptosis. Evaluating the possible intracellular pathways involved in this process, we observed nutrient deprivation alters the mitochondrial membrane potential, reversed by both amastigote and promastigote infection, in contrast, mitochondrial membrane potential was altered by rTNF and it was reversed only by promastigotes. Both caspase 3 activity and caspase 3 cleavage detection induced by H2O2 are reversed with amastigote or promastigote infection. When we analysed the expression of poly (ADP-ribose) polymerase, related to no induced cells . CPT induction didnLt increase 116 kDa PARP, but increased a 24 kDa fragment. Otherwise, Leishmania (L.) chagasi amastigote infection in J774 cells decreased 116 kDa PARP, but increased a 24 kDa fragment. Incells infected by Leishmania (L.) chagasi , we observed a decrease of 116 kDa fragment, appearance of a 89 kDa fragment and a decreasing of a 24 kDa fragment. In the cells under CPT induction similar results were found, except a decreasing of a 24 kDa when infected by amastigote. Evaluating the Leishmania (L.) infantum Heat Shock Protein of 83 kDa, as a possible factor that interferes in the apoptosis process, we observed that a phagocytosis of Escherichia coli (M15) bacteria with a HSP83 gene within a plasmid expressing this protein induced by isopropyl β - D- tiogalactopiranosideo (IPTG), considerably diminished apoptosis in these cells even when induced by CPT or rTNF. Our data show that Leishmania (L.) chagasi infection in J774 macrophages in vitro notoriously interferes in the apoptosis process affecting several intracellular pathways involved in both extrinsic and intrinsic pathways, more prominently with promastigote in this macrophage cell lineage.

The glycophosphatidylinositol(GPI)-linked membrane protein ART2 is an antigenic determinant for T lymphocytes that regulate the expression of diabetes in the BB/W rat model. Though little is understood of the physiologic role of ART2 on T lymphocytes, ART2 is a member of the mono-ADP-ribosyl transferase subgroup ofthe ADP-ribosyl transferase (ART) protein family. The ART protein family, which traditionally has been divided into mono-ADP-ribosyl transferases (mono-ARTs), poly(ADP)-ribose polymerases (PARPs), and ADP-ribosyl cyclases, influences various aspects of cellular physiology including: apoptosis, DNA damage repair, chromatin remodeling, telomere replication, cellular transport, immune regulation, neuronal function, and bacterial virulence. A structural alignment of ART2.2 with chicken PARP indicated the potential for ART2.2 to catalyze ADP-ribose polymers in an activity thought to be specific to the PARP subgroup and important for their regulation of nuclear processes. Kinetic studies determined that the auto-ADP-ribosyl transferase activity of ART2.2 is multitmeric and heterogeneous in nature. Hydroxylamine-cleaved ADP-ribose moieties from the ART2.2 multimers ran as polymers on a modified sequencing gel, and digestion of the polymers with snake-venom phosphodiesterase produced AMP and the poly(ADP)ribose-specific product, PR-AMP, which was resolved by analytical HPLC and structurally confirmed by ESI-MS. The ratio of AMP to PR-AMP was higher than that of PARP raising the possibility that the ART2.2 polymers had a different branching structure than those of PARP. This alternative branching was confirmed by the presence of ribose phosphate polymers in the snake venom phophodiesterase treated samples. The site of the auto-poly(ADP)-ribose modification was determined to be R185, a residue previously proposed to influence the level of auto-ADP ribosylation of ART2.2 by mutational analysis. These data provide the first demonstration of a hybrid between mono-ARTs and PARPs and are the earliest indication that PARP-like enzymes can exist outside the nucleus and on the cell surface.

Macrófagos infectados com Leishmania são protegidos de apoptose, entretanto não se conhece o mecanismo de transdução de sinal intracelular que interfere neste processo de morte. Neste trabalho, células J 774 em cultura, com privação de nutrientes, sofrem apoptose, a qual aumenta na presença dos indutores camptotecina (CPT) ou fator de necrose tumoral recombinante (rTNF). Estas células quando infectadas com amastigotas ou promastigotas de Leishmania (L.) chagasi (5 parasitos/uma célula) são protegidas de apoptose. Avaliando as possíveis vias intracelulares envolvidas nesse processo, observamos que a privação de nutrientes altera o potencial de membrana da mitocôndria, havendo reversão com a infecção tanto com promastigotas e amastigotas, entretanto a reversão da alteração do potencial de membrana induzida por rTNF só foi observada com infecção com promastigotas. Tanto a atividade de caspase 3, como a detecção de caspase 3 clivada induzidas por H202 são revertidas com a infecção com promastigotas ou amastigotas. Quando analisamos a expressão de poli (ADP ribose) polimerase (PARP), em relação às células sem indução, a indução por CPT não levou ao aumento da PARP de 116 kDa, mas, aumento da banda de 24 kDA. Por outro lado, a infecção por amastigota de Leishmania (L.) chagasi em células J774 levou à diminuição da expressão de PARP de 116 kDa, mas aumento da de 24 kDa. Nas células infectadas por promastigotas de Leishmania (L.) chagasi, observamos uma diminuição da banda de 116 kDa, aparecimento de uma molécula de 89kDa e diminuição da expressão da de 24 kDa. Nas células sob indução por CPT, a infecção levou a resultados similares, exceto a diminuição da molécula de 24 kDa quando infectado por amastigota. Avaliando-se a influência da proteína do choque térmico de 83 kDa de Leishmania infantum, como possível fator que interferiria no processo de apoptose, observamos que a fagocitose de bactérias Escherichia coli (M15) contendo plasmídio com gene de HSP83 expressando essa proteína, leva a diminuição da apoptose nessas células, mesmo quando induzidas por CPT ou rTNF. Nossos dados mostram que a infecção de macrófagos J774 in vitro por Leishmania (L.) chagasi, interfere no processo de apoptose afetando diversas vias de sinalização intracelular de apoptose, tanto extrínsecas quanto intrínsecas, sendo que promastigota é mais efetiva em inibir apoptose nesta linhagem macrofágica. / Macrophages infected by Leishmania are protected from apoptosis, however the mechanism of intracellular signal transduction that interferes in this death process remains unknown. In this work, J774 cells in culture, under nutrient deprivation undergo apoptosis, which is increased in the presence of inducers: camptothecin (CPT) or recombinant tumoral necrosis factor (rTNF). These cells infected by amastigotes or promastigotes of Leishmania (L.) chagasi (5 parasites per cell) are protected from apoptosis. Evaluating the possible intracellular pathways involved in this process, we observed nutrient deprivation alters the mitochondrial membrane potential, reversed by both amastigote and promastigote infection, in contrast, mitochondrial membrane potential was altered by rTNF and it was reversed only by promastigotes. Both caspase 3 activity and caspase 3 cleavage detection induced by H2O2 are reversed with amastigote or promastigote infection. When we analysed the expression of poly (ADP-ribose) polymerase, related to no induced cells . CPT induction didnLt increase 116 kDa PARP, but increased a 24 kDa fragment. Otherwise, Leishmania (L.) chagasi amastigote infection in J774 cells decreased 116 kDa PARP, but increased a 24 kDa fragment. Incells infected by Leishmania (L.) chagasi , we observed a decrease of 116 kDa fragment, appearance of a 89 kDa fragment and a decreasing of a 24 kDa fragment. In the cells under CPT induction similar results were found, except a decreasing of a 24 kDa when infected by amastigote. Evaluating the Leishmania (L.) infantum Heat Shock Protein of 83 kDa, as a possible factor that interferes in the apoptosis process, we observed that a phagocytosis of Escherichia coli (M15) bacteria with a HSP83 gene within a plasmid expressing this protein induced by isopropyl β - D- tiogalactopiranosideo (IPTG), considerably diminished apoptosis in these cells even when induced by CPT or rTNF. Our data show that Leishmania (L.) chagasi infection in J774 macrophages in vitro notoriously interferes in the apoptosis process affecting several intracellular pathways involved in both extrinsic and intrinsic pathways, more prominently with promastigote in this macrophage cell lineage.

L’ostéonécrose (ON) et les fractures (FR) sont des complications qui prennent de plus en plus place dans le traitement pédiatrique de la leucémie aiguë lymphoblastique (LAL). L’ON peut être causée par différents facteurs, dont principalement l’utilisation de glucocorticoïdes. Les glucocorticoïdes sont administrés lors du traitement de la leucémie dans le but d’initier l’apoptose des cellules malignes tout en ayant un effet anti-inflammatoire. Cependant, l’utilisation de ces corticostéroïdes comprend des effets secondaires sérieux, notamment le développement d’ostéonécrose. Des variantes génétiques peuvent mettre certains patients plus à risque que d’autres. Plusieurs gènes ont déjà été signalés comme régulés par les actions glucocorticoïdes (GC). Les variations génétiques présentes dans les régions régulatrices de ces gènes peuvent affecter leur fonctionnement normal et, en fin de compte, de déterminer un risque accru de développer l’ON associé au traitement contre la leucémie. Pour cette raison, plusieurs polymorphismes ont été identifiés et étudiés dans la cohorte QcALL de Ste-Justine, concernant les gènes suivants : ABCB1, ACP1, BCL2L11, NFKB1, PARP1, et SHMT1. Ces gènes jouent majoritairement un rôle dans les mécanismes d’action des glucocorticoïdes, mais quelques-uns ont plutôt un effet direct sur le développement d’ostéonécrose. Nos recherches ont démontré une corrélation entre ces polymorphismes et l’apparition d’ostéonécrose chez les patients de la cohorte QcALL, traités aux glucocorticoïdes. L'incidence cumulative de l'ostéonécrose a été évaluée rétrospectivement chez 305 enfants atteints de la leucémie qui ont subi un traitement à l’hôpital Ste-Justine selon les protocoles DFCI de Boston (87-01, 91-01, 95-01 et 2000-01). Parmi les huit polymorphismes de BCL2L11 étudiés, les 891T> G (rs2241843) et 29201C> T (rs724710) ont été significativement associés à ON (p = 0.01 et p = 0.03, respectivement). L'association du polymorphisme 891T> G a été modulée par le type de corticostéroïde (CS), l’âge, le sexe et le groupe à risque (p ≤ 0,05). Le polymorphisme 29201C> T était particulièrement apparent chez les patients à haut risque (p = 0,003). La même étude était conduite en parallèle sur des patients de la cohorte DFCI de Boston (N = 192), et montrait des résultats significatifs pour les polymorphismes étudiés. En conclusion, les résultats de cette étude permettront de confirmer l’association de ces polymorphismes au développement d’ON chez les patients de LLA traités aux GC. / Osteonecrosis (ON) and fractures (FR) are complications that take place in the treatment of children acute lymphoblastic leukemia (cALL). They can be caused by various factors, mainly using glucocorticoids. The corticosteroids, dexamethasone (DXM) and prednisone (PDN) are administered during the treatment of leukemia to initiate apoptosis of malignant cells; while having an anti-inflammatory effect. However, the use of these corticosteroids has severe side effects, including the development of osteonecrosis. Moreover, some patients develop resistance to treatment, and are at risk of developing side effects. The genetic variants predispose some patients at higher risk than others. Several genes have been previously reported as up- or down regulated by the GCs actions. The genetic variations present in gene coding or regulatory regions can affect their function and ultimately determine an increased risk of developing ON associated to ALL therapy. Therefore, we investigated the association between several single nucleotide polymorphisms (SNPs) in six candidate genes: BCL2L11, NFKB1, PARP1, ABCB1, ACP1, and SHMT1. These genes play a role in the mechanisms of action of glucocorticoids, but some have more of a direct effect on the development of osteonecrosis. Our research has shown a correlation between these polymorphisms and the occurrence of osteonecrosis in patients in the QCALL cohort, treated with glucocorticoids. Cumulative incidence of osteonecrosis was assessed retrospectively in 305 children with ALL who underwent treatment with DFCI protocols (87-01, 91-01, 95-01 and 2000-01) in childhood ALL cohort from Quebec (QcALL). Among the eight tag BCL2L11 polymorphisms studied the 891T>G (rs2241843) and 29201C>T (rs724710) were significantly associated with ON (p = 0.01 and p = 0.03, respectively). Association of 891T>G polymorphism was modulated by type of corticosteroid (CS), age, sex and risk group (p ≤ 0.05 and that of 29201C>T was particularly apparent among high risk (p = 0.003) patients. These polymorphisms have shown significant ON association in several QcALL risk groups, mainly in corticosteroid groups, age < 10 years, and high risk (HR) group. Furthermore, the same study was conducted in parallel with patients in the replication (DFCI) cohort (N = 192), and we showed significant genetic association results for all studied polymorphisms. In conclusion, this study identifies that some ALL children have a high incidence of ON during the treatment that is highly associated with polymorphisms in different genes regulated by corticosteroids and ALL prognostic factors.