RESUMO
Lung cancer is the first leading cause of cancer-related deaths in the world. Aberrant glycosylation in lung tumors leads to the expression of tumor-associated carbohydrate structures, such as the Tn antigen, consisting of N-acetyl-galactosamine (GalNAc) linked to a serine or threonine residue in proteins (α-GalNAc-O-Ser/Thr). The Tn antigen can be recognized by the Macrophage Galactose/GalNAc lectin (MGL), which mediates various immune regulatory and tolerogenic functions, mainly by reprogramming the maturation of function of dendritic cells (DCs). In this work, we generated two different Tn-expressing variants from the Lewis-type lung murine cancer cell line LL/2, which showed different alterations in the O-glycosylation pathways that influenced the interaction with mouse MGL2 and the immunomodulatory properties of DCs. Thus, the identification of the biological programs triggered by Tn+ cancer cells might contribute to an improved understanding of the molecular mechanisms elicited by MGL-dependent immune regulatory circuits.
Assuntos
Galactose , Neoplasias Pulmonares , Animais , Antígenos Glicosídicos Associados a Tumores/química , Galactosamina , Lectinas , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Camundongos , Serina , TreoninaRESUMO
In sandflies, males and females feed on carbohydrates but females must get a blood meal for egg maturation. Using artificial blood meals, this study aimed to understand how galactosamine interferes with sandfly digestive physiology. We also used galactosamine to manipulate the digestive physiology of Lutzomyia longipalpis to investigate its influence on sandfly digestion and Leishmania development within their insect vectors. Galactosamine was capable to reduce Lu. longipalpis trypsinolytic activity in a dose-dependent manner. This effect was specific to galactosamine as other similar sugars were not able to affect sandfly trypsin production. An excess of amino acids supplemented with the blood meal and 15 mM galactosamine was able to abrogate the reduction of the trypsinolytic activity caused by galactosamine, suggesting this phenomenon may be related to an impairment of amino acid detection by sandfly enterocytes. The TOR inhibitor rapamycin reduces trypsin activity in the L. longipalpis midgut. Galactosamine reduces the phosphorylation of the TOR pathway repressor 4EBP, downregulating TOR activity in the gut of L. longipalpis. Galactosamine reduces sandfly oviposition, causes an impact on sandfly longevity and specifically reduces sandfly gut proteases whereas increasing α-glycosidase activity. The administration of 15 and 30 mM galactosamine increased the number of promastigote forms of Le. mexicana and Le. infantum in galactosamine-treated L. longipalpis. Our results showed that galactosamine influences amino acid sensing, reduces sandfly gut protease activity through TOR downregulation, and benefits Leishmania growth within the Lu. longipalpis gut.
Assuntos
Galactosamina/administração & dosagem , Proteínas de Insetos/metabolismo , Leishmania/fisiologia , Peptídeo Hidrolases/metabolismo , Psychodidae/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Regulação para Baixo , Feminino , Galactosamina/farmacologia , Trato Gastrointestinal/parasitologia , Trato Gastrointestinal/fisiologia , Psychodidae/enzimologia , Psychodidae/parasitologiaRESUMO
INTRODUCTION AND OBJECTIVES: Necroptosis and endoplasmic reticulum (ER) stress has been implicated in acute and chronic liver injury. Activated eukaryotic initiation factor 2 alpha (eIF2α) attenuates protein synthesis and relieves the load of protein folding in the ER. In this study, we aimed to analyze the impact of eIF2α phosphorylation on hepatocyte necroptosis in acute liver injury. MATERIALS AND METHODS: Male BALB/c mice were injected with tunicamycin or d-galactosamine, and LO2 cells were incubated with tunicamycin to induce acute liver injury. 4-Phenylbutyric acid (PBA) and salubrinal were used to inhibit ER stress and eIF2α dephosphorylation, respectively. We analyzed the eIF2α phosphorylation, ER stress, and hepatocyte necroptosis in mice and cells model. RESULTS: Tunicamycin or d-galactosamine significantly induced ER stress and necroptosis, as well as eIF2α phosphorylation, in mice and LO2 cells (p<0.05). ER stress aggravated tunicamycin-induced hepatocyte necroptosis in mice and LO2 cells (p<0.05). Elevated eIF2α phosphorylation significantly mitigated hepatocyte ER stress (p<0.05) and hepatocyte necroptosis in mice (34.37±3.39% vs 22.53±2.18%; p<0.05) and LO2 cells (1±0.11 vs 0.33±0.05; p<0.05). Interestingly, tumor necrosis factor receptor (TNFR) 1 protein levels were not completely synchronized with necroptosis. TNFR1 expression was reduced in d-galactosamine-treated mice (p<0.05) and cells incubated with tunicamycin for 12 and 24h (p<0.05). ER stress partially restored TNFR1 expression and increased necroptosis in tunicamycin-incubated cells (p<0.05). CONCLUSIONS: These results imply that ER stress can mediate hepatocyte necroptosis independent of TNFR1 signaling and elevated eIF2α phosphorylation can mitigate ER stress during acute liver injury.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Hepatócitos/metabolismo , Necroptose/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Animais , Antibacterianos/toxicidade , Western Blotting , Linhagem Celular , Sobrevivência Celular , Doença Hepática Induzida por Substâncias e Drogas/patologia , Cinamatos/farmacologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Galactosamina/toxicidade , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Técnicas In Vitro , Camundongos , Necroptose/efeitos dos fármacos , Fenilbutiratos/farmacologia , Fosforilação , Tioureia/análogos & derivados , Tioureia/farmacologia , Tunicamicina/toxicidadeRESUMO
ABSTRACT PURPOSE: To describe an animal model for acute liver failure by intraperitoneal d-galactosamine injections in rats and to define when is the best time to intervene through King's College and Clichy´s criteria evaluation. METHODS: Sixty-one Wistar female rats were distributed into three groups: group 1 (11 rats received 1.4 g/kg of d-galactosamine intraperitoneally and were observed until they died); group 2 (44 rats received a dose of 1.4 g/kg of d-galactosamine and blood and histological samples were collected for analysis at 12 , 24, 48 , 72 and 120 hours after the injection); and the control group as well (6 rats) . RESULTS: Twelve hours after applying d-galactosamine, AST/ALT, bilirubin, factor V, PT and INR were already altered. The peak was reached at 48 hours. INR > 6.5 was found 12 hours after the injection and factor V < 30% after 24 hours. All the laboratory variables presented statistical differences, except urea (p = 0.758). There were statistical differences among all the histological variables analyzed. CONCLUSION: King's College and Clichy´s criteria were fulfilled 12 hours after the d-galactosamine injection and this time may represent the best time to intervene in this acute liver failure animal model.
Assuntos
Animais , Feminino , Ratos , Falência Hepática Aguda/induzido quimicamente , Galactosamina , Fatores de Tempo , Ratos Wistar , Falência Hepática Aguda/patologia , Falência Hepática Aguda/terapia , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Injeções Intraperitoneais , Fígado/patologiaRESUMO
PURPOSE:To describe an animal model for acute liver failure by intraperitoneal d-galactosamine injections in rats and to define when is the best time to intervene through King's College and Clichy´s criteria evaluation.METHODS:Sixty-one Wistar female rats were distributed into three groups: group 1 (11 rats received 1.4 g/kg of d-galactosamine intraperitoneally and were observed until they died); group 2 (44 rats received a dose of 1.4 g/kg of d-galactosamine and blood and histological samples were collected for analysis at 12 , 24, 48 , 72 and 120 hours after the injection); and the control group as well (6 rats) .RESULTS:Twelve hours after applying d-galactosamine, AST/ALT, bilirubin, factor V, PT and INR were already altered. The peak was reached at 48 hours. INR > 6.5 was found 12 hours after the injection and factor V < 30% after 24 hours. All the laboratory variables presented statistical differences, except urea (p = 0.758). There were statistical differences among all the histological variables analyzed.CONCLUSION:King's College and Clichy´s criteria were fulfilled 12 hours after the d-galactosamine injection and this time may represent the best time to intervene in this acute liver failure animal model.(AU)
Assuntos
Animais , Ratos , Falência Hepática Aguda/terapia , Falência Hepática Aguda/veterinária , Galactosamina/uso terapêutico , Ratos WistarRESUMO
Macrophage migration inhibitory factor (MIF) mediates immunity against Toxoplasma gondii infection by inducing inflammatory cytokines required to control the parasite replication. However, the role of this inflammatory mediator in the cell-mediated immune response against this infection is still poorly understood. Here, we used T. gondii-infected WT and Mif (-/-) mice to analyze the role of MIF in the maturation of CD11b(+) and CD8α (+) dendritic cells (DCs). We found that MIF promotes maturation of CD11b(+) but not CD8α (+) DCs, by inducing IL-12p70 production and CD86 expression. Infected Mif (-/-) mice showed significantly lower numbers of TNF and inducible nitric oxide synthase- (iNOS-) producing DCs (TipDCs) compared to infected WT mice. The adoptive transfer of Ly6C(high) monocytes into infected WT or Mif (-/-) mice demonstrated that MIF participates in the differentiation of Ly6C(high) monocytes into TipDCs. In addition, infected Mif (-/-) mice display a lower percentage of IFN-γ-producing natural killer (NK) cells compared to WT mice, which is associated with reducing numbers of TipDCs in Mif (-/-) mice. Furthermore, administration of recombinant MIF (rMIF) into T. gondii-infected Mif (-/-) mice restored the numbers of TipDCs and reversed the susceptible phenotype of Mif (-/-) mice. Collectively, these results demonstrate an important role for MIF inducing cell-mediated immunity to T. gondii infection.
Assuntos
Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Monócitos/metabolismo , Toxoplasmose/metabolismo , Animais , Enterotoxinas/farmacologia , Feminino , Galactosamina/farmacologia , Imunidade Celular/imunologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Oxirredutases Intramoleculares/genética , Lipopolissacarídeos/farmacologia , Fatores Inibidores da Migração de Macrófagos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Monócitos/efeitos dos fármacos , Neutrófilos/microbiologia , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/patogenicidade , Toxoplasmose/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE:: To describe an animal model for acute liver failure by intraperitoneal d-galactosamine injections in rats and to define when is the best time to intervene through King's College and Clichy´s criteria evaluation. METHODS:: Sixty-one Wistar female rats were distributed into three groups: group 1 (11 rats received 1.4 g/kg of d-galactosamine intraperitoneally and were observed until they died); group 2 (44 rats received a dose of 1.4 g/kg of d-galactosamine and blood and histological samples were collected for analysis at 12 , 24, 48 , 72 and 120 hours after the injection); and the control group as well (6 rats) . RESULTS:: Twelve hours after applying d-galactosamine, AST/ALT, bilirubin, factor V, PT and INR were already altered. The peak was reached at 48 hours. INR > 6.5 was found 12 hours after the injection and factor V < 30% after 24 hours. All the laboratory variables presented statistical differences, except urea (p = 0.758). There were statistical differences among all the histological variables analyzed. CONCLUSION:: King's College and Clichy´s criteria were fulfilled 12 hours after the d-galactosamine injection and this time may represent the best time to intervene in this acute liver failure animal model.
Assuntos
Galactosamina , Falência Hepática Aguda/induzido quimicamente , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Injeções Intraperitoneais , Fígado/patologia , Falência Hepática Aguda/patologia , Falência Hepática Aguda/terapia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Studies have shown that edaravone may prevent liver injury. This study aimed to investigate the effects of edaravone on the liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in female BALB/c mice. Edaravone was injected into mice 30 min before and 4 h after GalN/LPS injection. The survival rate was determined within the first 24 h. Animals were killed 8 h after GalN/LPS injection, and liver injury was biochemically and histologically assessed. Hepatocyte apoptosis was measured by TUNEL staining; proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in the liver were assayed by ELISA; expression of caspase-8 and caspase-3 proteins was detected by Western blot assay; and caspase-3 activity was also determined. Results showed that GalN/LPS induced marked elevations in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Edaravone significantly inhibited elevation of serum AST and ALT, accompanied by an improvement in histological findings. Edaravone lowered the levels of TNF-α and IL-6 and reduced the number of TUNEL-positive cells. In addition, 24 h after edaravone treatment, caspase-3 activity and mortality were reduced. Edaravone may effectively ameliorate GalN/LPS-induced liver injury in mice by reducing proinflammatory cytokines and inhibiting apoptosis.
Assuntos
Animais , Feminino , Antipirina/análogos & derivados , Apoptose/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Sequestradores de Radicais Livres/farmacologia , Alanina Transaminase/sangue , Antipirina/farmacologia , Aspartato Aminotransferases/sangue , /análise , /metabolismo , /análise , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Ensaio de Imunoadsorção Enzimática , Endotoxinas/toxicidade , Galactosamina/toxicidade , Hepatócitos/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , /análise , Lipopolissacarídeos/toxicidade , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Fator de Necrose Tumoral alfa/análiseRESUMO
Studies have shown that edaravone may prevent liver injury. This study aimed to investigate the effects of edaravone on the liver injury induced by D-galactosamine (GalN) and lipopolysaccharide (LPS) in female BALB/c mice. Edaravone was injected into mice 30 min before and 4 h after GalN/LPS injection. The survival rate was determined within the first 24 h. Animals were killed 8 h after GalN/LPS injection, and liver injury was biochemically and histologically assessed. Hepatocyte apoptosis was measured by TUNEL staining; proinflammatory cytokines [tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)] in the liver were assayed by ELISA; expression of caspase-8 and caspase-3 proteins was detected by Western blot assay; and caspase-3 activity was also determined. Results showed that GalN/LPS induced marked elevations in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Edaravone significantly inhibited elevation of serum AST and ALT, accompanied by an improvement in histological findings. Edaravone lowered the levels of TNF-α and IL-6 and reduced the number of TUNEL-positive cells. In addition, 24 h after edaravone treatment, caspase-3 activity and mortality were reduced. Edaravone may effectively ameliorate GalN/LPS-induced liver injury in mice by reducing proinflammatory cytokines and inhibiting apoptosis.
Assuntos
Antipirina/análogos & derivados , Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Citocinas/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Alanina Transaminase/sangue , Animais , Antipirina/farmacologia , Aspartato Aminotransferases/sangue , Caspase 3/análise , Caspase 3/metabolismo , Caspase 8/análise , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Edaravone , Endotoxinas/toxicidade , Ensaio de Imunoadsorção Enzimática , Feminino , Galactosamina/toxicidade , Hepatócitos/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Interleucina-6/análise , Lipopolissacarídeos/toxicidade , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Fator de Necrose Tumoral alfa/análiseRESUMO
Apple snail perivitellins are emerging as ecologically important reproductive proteins. To elucidate if the protective functions of the egg proteins of Pomacea canaliculata (Caenogastropoda, Ampullariidae), involved in embryo defenses, are present in other Pomacea species we studied scalarin (PsSC), the major perivitellin of Pomacea scalaris. Using small angle X-ray scattering, fluorescence and absorption spectroscopy and biochemical methods, we analyzed PsSC structural stability, agglutinating activity, sugar specificity and protease resistance. PsSC aggluttinated rabbit, and, to a lesser extent, human B and A erythrocytes independently of divalent metals Ca(2+) and Mg(2+) were strongly inhibited by galactosamine and glucosamine. The protein was structurally stable between pH 2.0 to 10.0, though agglutination occurred only between pH 4.0 to 8.0 (maximum activity at pH 7.0). The agglutinating activity was conserved up to 60 °C and completely lost above 80 °C, in agreement with the structural thermal stability of the protein (up to 60 °C). PsSC was able to withstand in vitro gastrointestinal digestion, and showed no trypsin inhibition activity. The presence of lectin activity has been reported in eggs of other Pomacea snails, but here we link for the first time, this activity to an apple snail multifunctional perivitellin. This novel role for a snail egg storage protein is different from closely related P.canaliculata defensive proteins.