RESUMO
Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an indispensable component used in the treatment of acute lymphoblastic leukemia (ALL) and certain lymphoma entities. Native Erwinia chrysanthemi-derived ASNase (n-crisantaspase) has been approved as a second-line drug for treating patients exhibiting allergy syndromes to native and pegylated Escherichia coli-derived ASNase (EC-ASNase). However, it still induces hypersensitivity in at least 17 % of treated patients. In the present study, we investigated the pharmacological activity, immunogenicity and anti-leukemic activity of a new pegylated recombinant crisantaspase (PEG-r-crisantaspase). The results demonstrate that when compared to n-crisantaspase in vivo, PEG-r-crisantaspase maintains a complete depletion of plasma Asn for up to 72 h with a 50-fold lower dose. In mice receiving PEG-r-crisantaspase, specific antibodies against the enzyme were undetectable, indicating a lower immunogenicity of the pegylated enzyme. In vitro, PEG-r-crisantaspase exhibits similar cytotoxic effects (EC50 < 5 × 10(-4) U/mL for the most sensitive cell lines) to n-crisantaspase on various leukemia and lymphoma cells and was shown to be more efficient than EC-ASNase. Three repeated PEG-r-crisantaspase injections (2-20 U/Kg) prevented leukemia development in leukemia-bearing mice for 17 days and significantly prolonged animal survival to 7-12 days. Therefore, PEG-r-crisantaspase appears to be a promising drug candidate for ALL treatment and should be further explored in experimental and clinical trials.
Assuntos
Antineoplásicos , Asparaginase , Animais , Anticorpos/sangue , Antineoplásicos/química , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Asparaginase/química , Asparaginase/imunologia , Asparaginase/farmacologia , Asparaginase/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dickeya chrysanthemi , Feminino , Humanos , Camundongos , Polietilenoglicóis/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In its vertebrate host, Leishmania encounters cells that express TLRs. Using genetically resistant C57BL/6 mice deficient in either TLR2, 4, or 9, we show in this study that only TLR9-deficient mice are more susceptible to infection with Leishmania major. TLR9-deficient mice resolved their lesions and controlled parasites growth with much lower efficiency than wild-type C57BL/6 mice. The absence of TLR9 also transiently inhibited the development of curative Th1 response. In an attempt to analyze the possible basis for such aberrant response in TLR9(-/-) mice, we have studied the importance of TLR9 for the activation of dendritic cells (DCs) by L. major. Results show that DCs in the draining lymph nodes are activated following infection with L. major. Furthermore, bone marrow-derived DCs as well as DCs freshly isolated from the spleen of C57BL/6 mice can be activated by either heat-killed or live L. major in vitro. In sharp contrast, L. major failed to activate DCs from TLR9(-/-) mice. Noteworthily, activation of DCs was abolished either following treatment of the parasites with DNase or after acidification of the endosomal compartment of DCs by chloroquine, pinpointing the DNA of L. major as the possible ligand of TLR9 leading to the activation of DCs. Results showed that DNA purified from L. major was indeed capable of activating DCs in a strictly TLR9-dependent manner. Moreover we showed that the L. major DNA-induced TLR9 signaling in DCs condition these cells to promote IFN-gamma production by CD4(+) T cells.
Assuntos
Diferenciação Celular/imunologia , DNA de Protozoário/imunologia , Células Dendríticas/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Células Th1/imunologia , Receptor Toll-Like 9/fisiologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/imunologia , Diferenciação Celular/genética , Células Dendríticas/metabolismo , Células Dendríticas/parasitologia , Feminino , Predisposição Genética para Doença , Imunofenotipagem , Leishmania major/genética , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Dados de Sequência Molecular , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Th1/metabolismo , Células Th1/parasitologia , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genéticaAssuntos
Linfoma de Zona Marginal Tipo Células B/genética , MicroRNAs/biossíntese , Neoplasias Esplênicas/genética , Perfilação da Expressão Gênica , Humanos , Linfoma de Zona Marginal Tipo Células B/metabolismo , Linfoma de Zona Marginal Tipo Células B/patologia , MicroRNAs/genética , Neoplasias Esplênicas/metabolismo , Neoplasias Esplênicas/patologiaAssuntos
Leucemia Linfocítica Crônica de Células B/genética , Transdução Genética , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Técnicas In Vitro , Lentivirus/genética , Linfoma de Zona Marginal Tipo Células B/genética , Proteínas Recombinantes/genética , Fator de Transcrição STAT3/genéticaRESUMO
Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an important drug for treating patients with acute lymphoblastic leukaemia (ALL) and natural killer (NK) cell lymphoma. Although different native or pegylated ASNase-based chemotherapy are efficient, disease relapse is frequently observed, especially in adult patients. The neo-synthesis of Asn by asparagine synthetase (AsnS) following ASNase treatment, which involves the amino acid response and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways, is believed to be the basis of ASNase-resistance mechanisms. However, AsnS expression has not emerged as an accurate predictive factor for ASNase susceptibility. The aim of this study was to identify possible ASNase sensitivity/resistance-related genes or pathways using a new asparaginase, namely a pegylated r-crisantaspase, with a focus on classic Asn-compensatory responses and cell death under conditions of Asn/L-glutamine limitation. We show that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes (especially NFκB-related genes) are associated with ASNase susceptibility. The response of malignant NK cell lines to ASNase may depend on Asn-compensatory mechanisms and other cellular processes such as cleavage of BCL2A1, a prosurvival member of the Bcl-2 protein family. These results suggest that according to cellular context, factors other than AsnS can influence ASNase susceptibility.
Assuntos
Apoptose/efeitos dos fármacos , Asparaginase/toxicidade , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Aspartato-Amônia Ligase/toxicidade , Linhagem Celular Tumoral , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Linfoma/metabolismo , Linfoma/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , eIF-2 Quinase/metabolismoRESUMO
Toxoplasma gondii, an intracellular parasite infects the host through the oral route. Infection induces a cascade of immunological events that involve both the components of the innate and adaptative immune responses. Alteration of the homeostatic balance of infected intestine results in an acute inflammatory ileitis in certain strains of inbred mice. Both the infected enterocytes as well as the CD4 T cells from the lamina propria produce chemokines and cytokines that are necessary to clear the parasite whereas CD8 intraepithelial lymphocytes secrete transforming growth factor beta that reduces the inflammation. In this review, we describe the salient features of this complex network of interactions among the different components of the gut-associated lymphoid tissue cell population that are induced after oral infection with T. gondii.
Assuntos
Mucosa Intestinal/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Animais , Citocinas/imunologia , Homeostase/imunologia , Humanos , Imunidade nas Mucosas , CamundongosRESUMO
Research on B-cell non-Hodgkin lymphoma focuses mainly on oncogenic events occurring in lymphoma cells, but recently a new component has appeared that may be crucial in lymphomagenesis: the tumor microenvironment. Indeed, compelling evidence demonstrates the key role played by nonmalignant bystander cells in the establishment and proliferation of the tumor. Among these cells, stromal cells, monocytes/macrophages, and T cells in lymphoid organs have all been described as contributing to tumor progression. Interactions linked to cell-cell intimate contacts-but also mediated through soluble mediators such as cytokines and chemokines-do form a specific network. All these interrelations directed by the tumor create a friendly environment for lymphoma cells that permits them to proliferate. Blocking the cross-talk between the tumor microenvironment and lymphoma cells may thus represent a promising new strategy for treating B-cell malignancies.
Assuntos
Comunicação Celular/imunologia , Tecido Linfoide/imunologia , Linfoma de Células B/imunologia , Antineoplásicos/uso terapêutico , Efeito Espectador/efeitos dos fármacos , Efeito Espectador/imunologia , Comunicação Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Citocinas/metabolismo , Progressão da Doença , Humanos , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Linfoma de Células B/patologiaRESUMO
Mice from most inbred strains are resistant to infection with Leishmania major whereas mice from BALB strains are highly susceptible. Resistance and susceptibility result from the development of Th1 or Th2 cells, respectively. In this report, we document an IL-2 mRNA burst, preceding the reported early IL-4 response, in draining lymph nodes of susceptible mice infected with L. major. Neutralization of IL-2 during the first days of infection redirected Th1 cell maturation and resistance to L. major, through interference with the rapid IL-4 transcription in Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) cells. A burst of IL-2 transcripts also occurred in infected C57BL/6 mice that do not mount an early IL-4 response. However, although the LACK protein induced IL-2 transcripts in susceptible mice, it failed to trigger this response in resistant C57BL/6 mice. Reconstitution experiments using C.B.-17 SCID mice and LACK-reactive CD4(+) T cells from IL-2(-/-) BALB/c mice showed that triggering of the early IL-4 response required autocrine IL-2. Thus, in C57BL/6 mice, the inability of LACK-reactive CD4(+) T cells to express early IL-4 mRNA transcription, important for disease progression, appears due to an incapacity of these cells to produce IL-2.
Assuntos
Antígenos de Protozoários/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-2/imunologia , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Proteínas de Protozoários/imunologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/parasitologia , Suscetibilidade a Doenças , Feminino , Interleucina-2/biossíntese , Interleucina-2/genética , Interleucina-4/biossíntese , Interleucina-4/genética , Interleucina-4/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos EspecíficosRESUMO
Acute and lethal ileitis can be elicited in certain strains of inbred mice after oral infection with the intracellular protozoan parasite Toxoplasma gondii. The development of this inflammatory process is dependent upon the induction of a robust Th1 response, including overproduction of IFN-gamma, TNF-alpha, and NO, as has been reported in other experimental models of human inflammatory bowel disease. In this study we have investigated the role of CD4(+) T cells from the lamina propria (LP) in the early inflammatory events after T. gondii infection using isolated and primary cultured intestinal cells from infected mice and immortalized mouse mIC(cl2) intestinal epithelial cells. Primed LP CD4(+) T cells isolated from parasite-infected mice produce substantial quantities of both IFN-gamma and TNF-alpha. IFN-gamma- and TNF-alpha-producing LP CD4(+) T cells synergize with infected mIC(cl2) and enhance the production of several inflammatory chemokines including macrophage-inflammatory protein-2, monocyte chemoattractant protein-1, monocyte chemoattractant protein-3, macrophage-inflammatory protein-1alphabeta, and IFN-gamma-inducible protein-10. Furthermore, primed LP CD4(+) T cells cocultured with infected mIC(cl2) inhibited replication of the parasite in the intestinal epithelial cells. Thus, LP CD4(+) T cells can interact with parasite-infected intestinal epithelial cells and alter the expression of several proinflammatory products that have been associated with the development of intestinal inflammation. The interaction between these two components of the gut mucosal compartment (CD4(+) T cells and enterocytes) may play a role in the immunopathogenesis of this pathogen-driven experimental inflammatory bowel disease model.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Comunicação Celular/imunologia , Ileíte/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Líquido Intracelular/imunologia , Líquido Intracelular/parasitologia , Toxoplasma/imunologia , Doença Aguda , Adjuvantes Imunológicos/fisiologia , Administração Oral , Animais , Linfócitos T CD4-Positivos/parasitologia , Linhagem Celular Transformada , Separação Celular , Quimiocinas/biossíntese , Quimiocinas/genética , Técnicas de Cocultura , Citocinas/fisiologia , Feminino , Ileíte/parasitologia , Ileíte/prevenção & controle , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/biossíntese , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/imunologia , Toxoplasmose/parasitologia , Toxoplasmose/prevenção & controle , Regulação para Cima/imunologiaRESUMO
BACKGROUND & AIMS: The loss of homeostasis is a hallmark of inflammatory bowel disease. Oral infection of susceptible mice with Toxoplasma gondii results in an acute lethal ileitis characterized by increased interferon gamma, tumor necrosis factor alpha, and inducible nitric oxide synthase; homeostasis results from transforming growth factor beta production by intraepithelial lymphocytes. The synthetic oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a potent anti-inflammatory molecule previously shown in vitro to suppress the de novo synthesis of inducible nitric oxide synthase and to induce the transcription and activation of genes from the transforming growth factor beta signaling pathway. METHODS: We evaluated the immune response in the small intestine and by intraepithelial lymphocytes after a single intraperitoneal dose of CDDO at the time of T. gondii oral infection. We abrogated the homeostatic effects of CDDO by blocking transforming growth factor beta in vivo. RESULTS: CDDO acid prevented ileitis development through the global down-regulation of inflammatory cytokines and chemokines. Total transforming growth factor beta(1) production by the intraepithelial lymphocytes increased, as did Smad2 expression. Blocking transforming growth factor beta reversed CDDO-induced protection and prevented the up-regulation of Smad2 in the small intestine. CONCLUSIONS: CDDO acid is a novel anti-inflammatory molecule capable of preventing ileitis by activating the transforming growth factor beta signaling pathway in a pathogen-driven ileitis model. This could represent a new treatment of inflammatory bowel disease.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Ileíte/parasitologia , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/administração & dosagem , Toxoplasmose/complicações , Fator de Crescimento Transformador beta/efeitos dos fármacos , Animais , Citocinas/efeitos dos fármacos , Citocinas/imunologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Feminino , Ileíte/prevenção & controle , Injeções Intraperitoneais , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , Modelos Animais , Transdução de Sinais/imunologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Intraepithelial lymphocytes (IEL) play a key role in gut homeostasis and are critical effector cells preventing the inflammatory intestinal lesions induced in mice following oral infection with Toxoplasma gondii. In this intestinal inflammatory model, CD4(+) T lymphocytes from the lamina propria (LP) synergize with the infected enterocytes to secrete pro-inflammatory chemokines and cytokines. In this study, we assessed the mechanisms accounting for the ability of IEL to modulate the inflammatory activity of these cells. Adoptive transfer of IEL purified from wild-type mice, or CD154-,CD95L- or IL-10-deficient mice infected with T. gondii completely impairs the development of the lethal ileitis in recipient mice orally infected with T. gondii. Compared with unprimed IEL isolated from naive mice, the CD8 alpha beta TCR alpha beta subset of primed IEL, isolated from T. gondii-infected mice, secretes increased amount of TGF-beta. IEL interact with the LP CD4(+) T lymphocytes, down-regulate their production of inflammatory cytokines such as IFN-gamma and reduce their proliferative activity. These effects are linked to the secretion of TGF-beta and are correlated with a shift in the balance between Smad7/T-bet down-regulation and Smad2/Smad3 up-regulation in LP CD4(+) T lymphocytes.
Assuntos
Proteínas de Ligação a DNA/imunologia , Ileíte/imunologia , Inflamação/imunologia , Linfócitos T/imunologia , Transativadores/imunologia , Transferência Adotiva , Animais , Feminino , Ileíte/parasitologia , Immunoblotting , Inflamação/parasitologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/parasitologia , Intestinos/imunologia , Intestinos/parasitologia , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Proteínas Smad , Linfócitos T/parasitologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Fator de Crescimento Transformador beta/imunologiaRESUMO
BACKGROUND & AIMS: Toxoplasma gondii, an obligate intracellular parasite, can invade intestinal epithelial cells and elicit a robust Th1 immune response. In this model of intestinal inflammation, CD8(+) intraepithelial lymphocytes (IELs) secrete transforming growth factor (TGF)-beta, which appears necessary for the maintenance of homeostasis in the intestine. However, the mechanism responsible for the IEL migration to the inflamed intestine is still unclear. METHODS: An in vitro coculture cell system was used to quantify the IEL attraction by an infected intestinal epithelial cell line (m-IC(cl2)). We used CCR5-deficient mice to determine which chemokine receptor-chemokine interaction could be responsible for the recruitment of antigen-specific CD8(+) IELs to the small intestine for the promotion of parasite clearance and host recovery. RESULTS: We observed increased expression of several chemokine receptors (CCR1, CCR2, CCR5, CXCR3) in the infected ileum. In particular, CCR5 expression was markedly increased in antigen-primed CD8(+) IELs. Experiments using recombinant chemokines as well as blocking antibodies showed that macrophage inflammatory protein (MIP)-1alpha and MIP-1beta were critical for their homing. CD8(+) IELs isolated from CCR5-deficient mice (CCR5-/-), despite their high production of TGF-beta and overexpression of activation markers, were impaired in their ability to migrate in vitro to the m-IC(cl2) monolayer or in vivo to the inflamed intestine after adoptive transfer. CONCLUSIONS: Our data emphasize the biologic role of CCR5 as an important component in the migration of intraepithelial CD8(+) T cells and the regulation of the inflammatory response following parasite infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Enterite/imunologia , Mucosa Intestinal/imunologia , Receptores CCR5/fisiologia , Toxoplasma/imunologia , Animais , Movimento Celular , Quimiocinas/genética , Quimiotaxia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Receptores CCR5/genética , Receptores de Quimiocinas/genética , Toxoplasmose Animal/imunologiaRESUMO
The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood. Oral infection of susceptible C57BL/6 mice with Toxoplasma gondii results in a lethal ileitis within 7-9 days postinfection. An immunodominant Ag of T. gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficient parasite (Deltasag1) engineered. We investigated the ability of Deltasag1 parasite to induce a lethal intestinal inflammatory response in susceptible mice. C57BL/6 mice orally infected with Deltasag1 parasites failed to develop ileitis. In vitro, the mutant parasites replicate in both enterocytes and dendritic cells. In vivo, infection with the mutant parasites was associated with a decrease in the chemokine and cytokine production within several compartments of the gut-associated cell population. RAG-deficient (RAG1(-/-)) mice are resistant to the development of the ileitis after T. gondii infection. Adoptive transfer of Ag-specific CD4(+) effector T lymphocytes isolated from C57BL/6-infected mice into RAG(-/-) mice conferred susceptibility to the development of the intestinal disease. In contrast, CD4(+) effector T lymphocytes from mice infected with the mutant Deltasag1 strain failed to transfer the pathology. In addition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T. gondii were rendered susceptible following intranasal presensitization with the SAG1 protein. This process was associated with a shift toward a Th1 response. These findings demonstrate that a single Ag (SAG1) of T. gondii can elicit a lethal inflammatory process in this experimental model of pathogen-driven ileitis.