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1.
Heliyon ; 7(10): e08111, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34693048

RESUMO

BACKGROUND: We have previously reported that spinal cord respiration (cellular mitochondrial oxygen consumption) and ATP content are conserved in the studied model of experimental autoimmune encephalomyelitis (EAE), foreseeing a recovery of the diseased rats. This exemplary lesion of multiple sclerosis is used here to measure spinal cord bioenergetics in C57BL6 mice. Our hypothesis is that, despite the well-known focal axonal mitochondrial pathology, bioenergetics of the CNS is reasonably preserved in this disease. METHODS: EAE was induced with an immunodominant myelin oligodendrocyte glycoprotein epitope in complete Freund's adjuvant, appended by injections of pertussis toxin. A low- and high-dose of the encephalitogen, administered into base of tail or hind-flank, were investigated. Control mice received only the incomplete adjuvant into tail. Oxygen measurements were based on quenching the phosphorescence of Pd(II) meso-tetra (sulfophenyl) tetrabenzoporphyrin by molecular oxygen. Cellular ATP was measured using the luciferin/luciferase system. RESULTS: The kinetics of spinal cord oxygen consumption was zero-order (linear with time) and inhibited by cyanide, confirming oxygen was reduced by cytochrome oxidase. The rate of respiration (in µM O2.min-1.mg-1; measured on Days 13-28) in control mice was (mean ± SD) 0.086 ± 0.024 (n = 8) and in immunized mice was 0.079 ± 0.020 (n = 15, P = 0.265, Mann-Whitney test). Consistently, cellular ATP (in µmol mg-1 dry pellet weight; measured on Days 13-28) in control mice was 0.068 ± 0.079 (n = 11) and in immunized mice was 0.063 ± 0.061 (n = 24, P = 0.887, Mann-Whitney U test). CONCLUSIONS: In vitro measurements of spinal cord bioenergetics show conservation of the mitochondrial function in mice with EAE. These results suggest the previously documented reduced mitochondrial electrochemical potential in this disease is alterable, and likely reflects the adverse events of neuroinflammation.

2.
Heliyon ; 7(6): e07219, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34159274

RESUMO

BACKGROUND: We have previously reported on the use of the phosphorescence oxygen analyzer for measuring spinal cord cellular respiration. This analytical tool is used here to investigate the effects of two inhibitors of NADH:ubiquinone oxidoreductase, rotenone and 1-methyl-4-phenylpyridinium, on cellular respiration in striatal tissue. Both neurotoxins can induce Parkinson's disease-like symptoms, and have been used to study this disease in animals. Our hypothesis is that striatal cellular respiration is a sensitive biomarker for the adverse effects of toxins, and the phosphorescence oxygen analyzer can be used as a screening tool for this purpose. METHODS: Striatal fragments were collected from C57BL6 mice and immersed in Pd phosphor solution [phosphate-buffered saline, 3.0 µM 'Pd(II)-meso-tetra (sulfophenyl) tetrabenzoporphyrin' and 0.5% fat-free albumin, with and without 5.0 mM glucose]. The sample was transferred to a glass vial containing 2-mL Pd phosphor solution. The vial was sealed from air and placed in the instrument that measures dissolved oxygen as function of time. Immunoblots of the studied tissue were positive for the dopamine neuronal cell biomarker tyrosine hydroxylase. RESULTS: Striatal oxygen consumption was linear with time, exhibiting zero-order kinetics of oxygen reduction by cytochrome oxidase. Cyanide sensitive respiration was ≥90%, confirming oxygen was reduced by cytochrome oxidase. The rate of respiration increased by ~2-fold in the presence of glucose. Striatal oxygen consumption in the presence of rotenone or 1-methyl-4-phenylpyridinium was exponential, demonstrating impaired respiration. CONCLUSION: Striatal cellular mitochondrial oxygen consumption was impaired by the studied inhibitors of complex I of the respiratory chain. This effect is expected to deplete NAD+ (oxidized nicotinamide adenine dinucleotide), a principle driver of glycolysis. In vivo studies are required to determine if these toxin-induced metabolic derangements contribute to the development of sporadic Parkinson's disease. This analytic tool can be used to screen environmental toxins for their in vitro effects on the striatum.

3.
BMC Neurosci ; 16: 37, 2015 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-26092157

RESUMO

BACKGROUND: Mitochondrial dysregulation is important in axonal damage and demyelination in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). There is however, no evidence in the literature of any study that has examined cellular bioenergetics of the central nervous system (CNS) during the early development and clinical course of EAE. EAE, a rodent model of relapsing/remitting MS, is a CD4(+) T cell-mediated disease of the CNS. We hypothesize that CNS bioenergetics might predict prognosis, and that preserved bioenergetics might underlie the remission from disease. The study aims therefore, to determine whether the clinical history of EAE is influenced by cellular respiration of the CNS in susceptible Dark Agouti (DA) and resistant Albino Oxford (AO) rats. METHODS: Experimental autoimmune encephalomyelitis was induced by myelin basic protein in complete Freud Adjuvant in the footpads of DA and AO rats. A phosphorescence analyzer that determines cellular respiration was used to monitor oxygen consumption and ATP concentration was measured using the Enliten ATP assay system. Disease pathology was demonstrated by H&E and Luxol fast blue staining of sections of the lumbar regions of the spinal cord. Mitochondrial size in relation to axonal size was determined by electron microscopy. Apoptosis was studied by HPLC measurement of intracellular caspase-3 activity and caspase immunohistochemistry. Role and source of caspase 1 was studied by double immunofluorescence with antibodies for caspase-1, microglia (anti-Iba1) and astrocytes (anti-GFAP). RESULTS: The cellular respiration of the CNS did not vary between diseased and normal rats. We also demonstrate here, that at the peak of disease, inflammation as shown by caspase-1, produced by activated microglia and infiltrating cells, was significant in susceptible DA rats. The mitochondrial:axonal size ratio did not vary in the different groups although mitochondria were smaller in spinal cords of diseased DA rats. Demyelination, observed only in areas of mononuclear infiltration of the spinal cord of diseased DA rats, was demonstrated by light microscopy and electron microscopy. CONCLUSION: We conclude that EAE at this early stage does not significantly affect CNS cellular respiration and this might underlie the reason for the recovery of diseased rats.


Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Medula Espinal/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/fisiologia , Astrócitos/metabolismo , Astrócitos/patologia , Axônios/metabolismo , Axônios/patologia , Caspase 1/metabolismo , Caspase 3/metabolismo , Encefalomielite Autoimune Experimental/patologia , Metabolismo Energético , Adjuvante de Freund , Vértebras Lombares , Masculino , Microglia/metabolismo , Microglia/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Tamanho Mitocondrial/fisiologia , Proteína Básica da Mielina , Ratos , Especificidade da Espécie , Medula Espinal/patologia
4.
Cell Physiol Biochem ; 34(2): 255-65, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25033832

RESUMO

BACKGROUND/AIMS: The use of engineered nanomaterials in the form of nanoparticles (NP) for various biomedical applications, as well as in consumer products, has raised concerns about their safety for human health. These NP are intended to be administered directly into the circulation following intravenous injection, or they may reach the circulation following other routes of administration such as oral or inhalation, and interact with circulating cells such as erythrocytes. However, little is known about the interaction of amorphous SiNP with erythrocytes. METHODS: We studied the interaction of amorphous silica nanoparticles (SiNP) at various concentrations (1, 5, 25 and 125 µg/ml) with mouse erythrocytes in vitro. RESULTS: Incubation of erythrocytes with SiNP caused a dose-dependent hemolytic effect. Likewise, the activity of lactate dehydrogenase was dose-dependently increased by SiNP. Transmission electron microscopy analysis revealed that SiNP are taken up by erythrocytes. Lipid erythrocyte susceptibility to in vitro peroxidation measured by malondialdehyde showed a significant and dose-dependent increase in erythrocytes. SiNP also enhanced the antioxidant activities of superoxide dismutase (SOD), catalase and reduced glutathione (GSH). Moreover, SiNP increased caspase 3, triggered annexin V-binding and caused a dose-dependent increase of cytosolic calcium concentration. CONCLUSION: It can be concluded that SiNP cause a dose-dependent hemolytic activity and are taken up by the erythrocytes. We also found that SiNP induce the occurrence of oxidative activity, apoptosis and increase cytosolic Ca(2+), which may explain their haemolytic activity. Our in vitro data suggest that SiNP may, plausibly, lead to anemia and circulatory disorders in vivo.


Assuntos
Eritrócitos/química , Nanopartículas , Estresse Oxidativo , Dióxido de Silício/química , Animais , Anexina A5/metabolismo , Cálcio/metabolismo , Caspase 3/metabolismo , Catalase/metabolismo , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Glutationa/metabolismo , Técnicas In Vitro , L-Lactato Desidrogenase/metabolismo , Malondialdeído/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Superóxido Dismutase/metabolismo
5.
Sultan Qaboos Univ Med J ; 14(1): e65-71, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24516756

RESUMO

OBJECTIVES: Aflatoxin B1 (AFB1) is a naturally occurring carcinogenic and immunosuppressive compound. This study was designed to measure its toxic effects on human peripheral blood mononuclear cells (PBMC). METHODS: The study recruited 7 healthy volunteers. PBMC were isolated and cellular respiration was monitored using a phosphorescence oxygen analyser. The intracellular caspase activity was measured by the caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin. Phosphatidylserine exposure and membrane permeability to propidium iodide (PI) were measured by flow cytometry. RESULTS: Cellular oxygen consumption was inhibited by 2.5 µM and 25 µM of AFB1. Intracellular caspase activity was noted after two hours of incubation with 100 µM of AFB1. The number of Annexin V-positive cells increased as a function of AFB1 concentration and incubation time. At 50 µM, a significant number of cells became necrotic after 24 hours (Annexin V-positive and PI-positive). CONCLUSION: The results show AFB1 is toxic to human lymphocytes and that its cytotoxicity is mediated by apoptosis and necrosis.

6.
Clin Immunol ; 149(1): 86-96, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23899994

RESUMO

Toll like receptors are primary sensors of both innate and adaptive immune systems. They activate APCs and influence T-cell function in inflammatory autoimmune response. Studies have shown that TLR manipulation may lead to either tolerance or trigger autoimmunity. Using diabetogenic and subdiabetogenic multiple low doses of streptozotocin, we demonstrate here that Pam3 CYS-CK4 a TLR-2 agonist, enhances and promotes diabetes in C57BL/6 male mice following increased apoptosis of ß islet cells. FACS analysis of isolated pancreatic lymph node cells revealed significant increased number of macrophages, dendritic cells, CD4(+) TNF-α(+), CD4(+) IFN-γ(+) and most significantly, CD4(+) IL-17(+) and reduced number of CD25(+)Fox p3(+) T cells after Pam3CSK4 treatment. Genetic deletion of IFN-γ prevents whereas deletion of IL-17 reduced severity of Pam3CSK4-induced enhancement of diabetes. TLR-2 agonist-enhanced diabetogenesis is also influenced by enhanced influx of antigen presenting cells and suppression of regulatory T cell activity.


Assuntos
Diabetes Mellitus Experimental/imunologia , Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/imunologia , Interleucina-17/imunologia , Lipopeptídeos/farmacologia , Receptor 2 Toll-Like/agonistas , Animais , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/imunologia , Interferon gama/genética , Interleucina-17/genética , Linfonodos/citologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Necrose Tumoral alfa/imunologia
7.
BMC Gastroenterol ; 13: 6, 2013 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-23311450

RESUMO

BACKGROUND: A novel in vitro system was employed to investigate liver tissue respiration (mitochondrial O2 consumption) in mice treated with concanavalin A (Con A). This study aimed to investigate hepatocyte bioenergetics in this well-studied hepatitis model. METHODS: C57Bl/6 and C57Bl/6 IFN-γ-/- mice were injected intravenously with 12 mg ConA/kg. Liver specimens were collected at various timepoints after injection and analyzed for cellular respiration and caspase activation. Serum was analyzed for interferon-gamma (IFN-γ) and aminotransferases. Fluorescence activated cell sorting analysis was used to determine the phenotype of infiltrating cells, and light and electron microscopy were used to monitor morphological changes. Phosphorescence analyzer that measured dissolved O2 as function of time was used to evaluate respiration. RESULTS: In sealed vials, O2 concentrations in solutions containing liver specimen and glucose declined linearly with time, confirming zero-order kinetics of hepatocyte respiration. O2 consumption was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. Enhanced liver respiration (by ≈68%, p<0.02) was noted 3 hr after ConA treatment, and occurred in conjunction with limited cellular infiltrations around the blood vessels. Diminished respiration (by ≈30%, p=0.005) was noted 12 hr after ConA treatment, and occurred in conjunction with deranged mitochondria, areas of necrosis, and prominent infiltrations with immune cells, most significantly, CD3+NKT+ cells. Increases in intracellular caspase activity and serum IFN-γ and aminotransferase levels were noted 3 hr after ConA treatment and progressed with time. The above-noted changes were less pronounced in C57Bl/6 IFN-γ-/- mice treated with ConA. CONCLUSIONS: Based on these results, liver tissue bioenergetics is increased 3 hr after ConA exposure. This effect is driven by the pathogenesis of the disease, in which IFN-γ and other cytokines contribute to. Subsequent declines in liver bioenergetics appear to be a result of necrosis and active caspases targeting the mitochondria within hepatocytes.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Concanavalina A/efeitos adversos , Concanavalina A/farmacologia , Metabolismo Energético/efeitos dos fármacos , Fígado/metabolismo , Animais , Caspases/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Doença Hepática Induzida por Substâncias e Drogas/fisiopatologia , Modelos Animais de Doenças , Metabolismo Energético/fisiologia , Interferon gama/sangue , Interferon gama/deficiência , Interferon gama/genética , Fígado/patologia , Fígado/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Transaminases/sangue
8.
J Neuroimmunol ; 246(1-2): 18-26, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22445739

RESUMO

Previous studies have shown that interferon-gamma (IFN-γ) is a proinflammatory cytokine that contributes to the pathogenesis of Guillain-Barré syndrome and its animal model, experimental autoimmune neuritis (EAN). Treatments with anti-IFN-γ antibodies improve clinical outcome in GBS patients and EAN animals and administration of IFN-γ markedly worsens EAN. Paradoxically, the mice deficient in IFN-γ remain susceptible to experimental autoimmune encephalomyelitis, an analogous disease in the central nervous system. These observations raise a question whether IFN-γ might be protective in autoimmune demyelinating diseases. To clarify the role of IFN-γ in the pathogenesis of autoimmune demyelinating diseases, we used P0 protein peptide 180-199 to induce EAN in IFN-γ knockout (KO) mice. After the acute phase of EAN, the clinical signs of IFN-γ KO mice were significantly more severe than those of wild type (WT) controls. After antigenic stimulation, the proliferation of splenic mononuclear cells was significantly higher in IFN-γ KO than in WT mice with EAN. At the peak of EAN, the proportion of interleukin (IL)-17A expressing cells in cauda equina (CE) infiltrating cells, and the levels of IL-17A in sera were elevated in IFN-γ KO mice when compared with their WT counterparts. The proportions of major histocompatibility complex (MHC) II, macrosialin, and IL-12/IL-23p40 expressing cells, relative to total CE infiltrating cells were correspondingly higher in IFN-γ KO than in WT mice with EAN. However, IFN-γ deficiency reduced the production of NO by cultured macrophages in response to proinflammatory stimuli and induced a systemic Th2-oriented immune response. In conclusion, IFN-γ deficiency exacerbates EAN via upregulating Th17 cells despite a mitigated systemic Th1 immune response.


Assuntos
Interferon gama/deficiência , Neurite Autoimune Experimental/imunologia , Neurite Autoimune Experimental/patologia , Células Th1/imunologia , Células Th1/patologia , Animais , Movimento Celular/imunologia , Proliferação de Células , Células Cultivadas , Predisposição Genética para Doença , Interferon gama/genética , Interferon gama/fisiologia , Camundongos , Camundongos Knockout , Neurite Autoimune Experimental/genética , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Células Th17/patologia , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/patologia , Regulação para Cima/genética , Regulação para Cima/imunologia
9.
J Cell Biochem ; 112(4): 1138-53, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21308747

RESUMO

Sphingosine (SPH) is an important bioactive lipid involved in mediating a variety of cell functions including apoptosis. However, the signaling mechanism of SPH-induced apoptosis remains unclear. We have investigated whether SPH inhibits survival signaling in cells by inhibiting Akt kinase activity. This study demonstrates that treatment of Jurkat cells with SPH leads to Akt dephosphorylation as early as 15 min, and the cells undergo apoptosis after 6 h. This Akt dephosphorylation is not mediated through deactivation of upstream kinases, since SPH does not inhibit the upstream phosphoinositide-dependent kinase 1 (PDK1) phosphorylation. Rather, sensitivity to the Ser/Thr protein phosphatase inhibitors (calyculin A, phosphatidic acid, tautomycin, and okadaic acid) indicates an important role for protein phosphatase 1 (PP1) in this process. In vitro phosphatase assay, using Akt immunoprecipitate following treatment with SPH, reveals an increase in Akt-PP1 association as determined by immunoprecipitation analysis. Moreover, SPH-induced dephosphorylation of Akt at Ser(473) subsequently leads to the activation of GSK-3ß, caspase 3, PARP cleavage, and ultimately apoptosis. Pre-treatment with caspase 3 inhibitor z-VAD-fmk and Ser/Thr phosphatase inhibitor abrogates the effect of SPH on facilitating apoptosis. Altogether, these results demonstrate that PP1-mediated inhibition of the key anti-apoptotic protein, Akt, plays an important role in SPH-mediated apoptosis in Jurkat cells.


Assuntos
Apoptose/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Esfingosina/farmacologia , Clorometilcetonas de Aminoácidos/farmacologia , Western Blotting , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Imunoprecipitação , Células Jurkat , Toxinas Marinhas , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Fosfatase 1/antagonistas & inibidores , Piranos/farmacologia , Serina/metabolismo , Compostos de Espiro/farmacologia
10.
J Neuroimmunol ; 232(1-2): 68-74, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21109309

RESUMO

Experimental allergic encephalomyelitis (EAE) is characterized by inflammatory infiltrates of myelin antigen(s) specific T cells and consecutive demyelination. Injection of encephalitogen into the footpads induces disease in genetically susceptible Dark Agouti rats (DA) but not in Albino Oxford (AO) rats although mild inflammatory infiltrates are observed in both strains early after disease induction. In addition, only DA rats develop disease when cells from (AO×DA) F(1) hybrids are passively transferred into sub-lethally radiated AO and DA parent hosts. The aim of the study was therefore to examine the participation of accessory cells, macrophages, dendritic cells and microglia in EAE development at the level of the target tissue in these two strains using specific membrane markers. We demonstrate here that in the induction phase of EAE in DA rats, macrophages (CD68(+); CD45(hi)CD11b(+)) are the first detectable infiltrating cells in the subpial regions of the spinal cord but were not found in AO rats. During the same period, resident microglial cells which are of the ramified variety are observed in both DA and AO rats. In DA rats at the peak of disease, when profuse influx of T cells is seen, macrophages and dendritic cells appear in the parenchyma of the CNS. In addition, at that time, microglial cells are activated. FACS analyses also reveal a significant increase in CD45(hi)CD11c(+) dendritic cells and CD45(hi)D11b(+) macrophages compared with levels in naïve and immunized AO rats. During resolution of disease in DA rats, the expression of microglia and macrophage markers is comparable with those in naïve non-immunized DA and immunized AO rats. We conclude that an initial influx of macrophages is indispensible for the development of EAE in DA rats. The presence of dendritic cells and myeloid dendritic cells at the peak of disease supports the role of these cells in EAE especially in relapses and chronicity. The activation pattern of microglia in DA rats does not indicate their role as antigen presenting cells in disease induction since they are ramified at the induction phase and only become activated after the overwhelming influx of T cells.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Macrófagos/imunologia , Animais , Separação Celular , Células Dendríticas/imunologia , Suscetibilidade a Doenças/imunologia , Encefalomielite Autoimune Experimental/patologia , Citometria de Fluxo , Imuno-Histoquímica , Masculino , Ratos , Medula Espinal/imunologia , Medula Espinal/patologia
11.
Biochem Biophys Res Commun ; 383(4): 426-32, 2009 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-19376088

RESUMO

Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser(15). Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not prevent Dubca cell apoptosis, suggesting that p53(Ser-15) phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.


Assuntos
Apoptose , MAP Quinase Quinase 4/metabolismo , Serina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Animais , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular , Ativação Enzimática , Fosforilação
12.
Mol Immunol ; 47(1): 28-36, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19356801

RESUMO

Several peripheral mechanisms appear to be operational in limiting autoimmune damage of the islets of Langerhans and organ-specific T cell-mediated autoimmunity in general. These include cyclophosphamide sensitive T regulatory cells (Treg cells) and Th2 derived cytokine downregulation. We used the model of multiple low doses of streptozotocin (MLD-STZ) induced diabetes in susceptible C57BL/6 mice and resistant BALB/c mice to study these regulatory mechanisms. We show that low dose cyclophosphamide (CY) sensitive CD4(+)CD25(+)FoxP3(+) Treg cell-dependent mechanisms can be demonstrated in C57Bl/6 mice susceptible to MLD-STZ diabetes induction. CY pretreatment decreased Foxp3(+) cell count, glycemia, glycosuria and insulitis. In contrast, CY did not overcome resistance to diabetes induction in BALB/c mice. However, in BALB/c mice, deletion of ST2, an orphan member of the IL-1R family responsible for Th2 cell signaling leads to enhanced susceptibility to diabetes induction as evaluated by level of glycemia and glycosuria, number of infiltrating cells and beta cell loss. RT-PCR analysis of mRNA transcripts of diabetogenic cytokines revealed that the expression of TNF-alpha, and IFN-gamma was significantly enhanced in pancreatic lymph nodes by day 10 after diabetes induction in ST2-deficient mice in comparison with wild type BALB/c mice while IL-17 was detected only in ST2(-/-) mice by day 21. Our results are compatible with the notion that Treg cells are involved in MLD-STZ diabetes in susceptible mice and demonstrate that ST2-mediated signaling may also be involved in limiting Th1/Th17-mediated autoimmune pathology in diabetes resistant strain.


Assuntos
Diabetes Mellitus Experimental/imunologia , Receptores de Interleucina/fisiologia , Linfócitos T Reguladores/imunologia , Animais , Autoimunidade , Ciclofosfamida/farmacologia , Citocinas/biossíntese , Citocinas/genética , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/etiologia , Regulação para Baixo , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-17/análise , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina/deficiência , Transdução de Sinais , Estreptozocina , Linfócitos T Auxiliares-Indutores/imunologia
13.
J Immunol ; 182(2): 1167-73, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19124760

RESUMO

Galectin-3 (Gal-3) is a member of the beta-galactoside-binding lectin family and plays an important role in inflammation. However, the precise role of Gal-3 in autoimmune diseases remains obscure. We have investigated the functional role of Gal-3 in experimental autoimmune encephalomyelitis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. Gal-3 deficient (Gal-3-/-) mice developed significantly milder EAE and markedly reduced leukocyte infiltration in the CNS compared with similarly treated wild-type (WT) mice. Gal-3-/- mice also contained fewer monocytes and macrophages but more apoptotic cells in the CNS than did WT mice. Following Ag stimulation in vitro, lymph node cells from the immunized Gal-3-/- mice produced less IL-17 and IFN-gamma than did those of the WT mice. In contrast, Gal-3-/- mice produced more serum IL-10, IL-5, and IL-13 and contained higher frequency of Foxp3+ regulatory T cells in the CNS than did the WT mice. Furthermore, bone marrow-derived dendritic cells from Gal-3-/- mice produced more IL-10 in response to LPS or bacterial lipoprotein than did WT marrow-derived dendritic cells. Moreover, Gal-3-/- dendritic cells induced Ag-specific T cells to produce more IL-10, IL-5, and IL-12, but less IL-17, than did WT dendritic cells. Taken together, our data demonstrate that Gal-3 plays an important disease-exacerbating role in EAE through its multifunctional roles in preventing cell apoptosis and increasing IL-17 and IFN-gamma synthesis, but decreasing IL-10 production.


Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/terapia , Galectina 3/deficiência , Galectina 3/genética , Índice de Gravidade de Doença , Animais , Apoptose/genética , Apoptose/imunologia , Células Cultivadas , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Galectina 3/fisiologia , Glicoproteínas/administração & dosagem , Glicoproteínas/imunologia , Inibidores do Crescimento/deficiência , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Interleucina-10/antagonistas & inibidores , Interleucina-10/biossíntese , Interleucina-17/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Regulação para Cima/imunologia
14.
Mol Immunol ; 44(7): 1623-30, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17034862

RESUMO

Helminth parasites are known to alter host immune responses and the responsible molecules are a potential source of biological immunoadjuvants. Previously, we have reported strong Th-2 type immunomodulatory properties of Taenia crassiceps glycans. In this study, we report interferon-gamma (IFN-gamma) stimulatory activity of fractionated Taenia glycans and Lewis sugars with comparable glycan composition. Our data show that Taenia glycans and Lewis X pentasaccharide are potent stimulators of the Th-1 type cytokine IFN-gamma. We postulate that the terminal beta-(1-4)-galactose residue in Lewis X is associated with IFN-gamma stimulation from naive BALB/c mouse spleen and peritoneal exudate cells. Antibodies to toll-like receptors (TLRs) inhibited the Lewis X-induced IFN-gamma secretion. Lewis X up-regulated the expression of NF-kappaB p65 from naive spleen cells and IFN-gamma transcription in peritoneal exudate cells. These data demonstrate the ability of Lewis type helminth glycans to modulate host responses in a Th-1 direction via NF-kappaB p65, IFN-gamma and macrophage TLRs.


Assuntos
Líquido Ascítico/imunologia , Interferon gama/metabolismo , Antígenos CD15/imunologia , Polissacarídeos/imunologia , Baço/imunologia , Taenia/imunologia , Animais , Anticorpos/farmacologia , Líquido Ascítico/citologia , Líquido Ascítico/efeitos dos fármacos , Sequência de Carboidratos , Fracionamento Celular , Células Cultivadas , Regulação da Expressão Gênica , Interferon gama/antagonistas & inibidores , Interferon gama/genética , Antígenos CD15/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Dados de Sequência Molecular , Peritônio/citologia , Peritônio/efeitos dos fármacos , Peritônio/imunologia , Polissacarídeos/farmacologia , Baço/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Receptores Toll-Like/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Transcrição Gênica/efeitos dos fármacos
15.
Eur J Immunol ; 36(1): 216-23, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16358360

RESUMO

IL-23, a proximal regulator of IL-17, may be a major driving force in the induction of autoimmune inflammation. We have used a model of subdiabetogenic treatment with multiple low doses of streptozotocin (MLD-STZ; 4 x 40 mg/kg body weight) in male C57BL/6 mice to study the effect of IL-23 on immune-mediated beta cell damage and the development of diabetes, as evaluated by blood glucose, quantitative histology, immunohistochemistry and expression of relevant cytokines in the islets. Ten daily injections of 400 ng IL-23, starting on the first day of MLD-STZ administration led to significant and sustained hyperglycemia along with weight loss compared with controls (no IL-23), and a significant increase in the number of infiltrating cells, a lower insulin content, enhanced apoptosis, expression of IFN-gamma and IL-17 (not seen in the controls) and a significant increase in the expression of TNF-alpha and IL-18 in the pancreatic islets. IL-23 treatment started 5 days prior to MLD-STZ administration had no effect on diabetogenesis or cytokines expression in the pancreatic islets. We provide the first evidence in an animal model that IL-23 is involved in the development of type-1 diabetes, by inducing IL-17 and possibly IFN-gamma production in the target tissue.


Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Diabetes Mellitus Experimental/etiologia , Interleucinas/farmacologia , Estreptozocina/administração & dosagem , Animais , Antibióticos Antineoplásicos/efeitos adversos , Apoptose , Citocinas/biossíntese , Citocinas/efeitos dos fármacos , Diabetes Mellitus Experimental/imunologia , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Interleucina-23 , Subunidade p19 da Interleucina-23 , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/efeitos dos fármacos , Baço/imunologia , Estreptozocina/efeitos adversos
16.
Mol Immunol ; 42(12): 1495-502, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15950742

RESUMO

Complex glycans derived from lipid, nucleic acid and protein free extracts of Taenia crassiceps metacestode larvae were found to have adjuvant effect against Leishmania mexicana antigens in BALB/c mice experimentally infected with L. mexicana. A single intraperitoneal or subcutaneous injection of Taenia glycans altered the Th-1/Th-2 balance in experimentally infected mice as determined by Western blot analysis of IgG 1 and IgG 2a antibodies to L. mexicana antigens. Leishmania antigens which were immunogenic in Taenia glycan vaccinated mice were different from those of non-vaccinated mice. Vaccination induced leishmania antigen specific IFN-gamma expression in vitro culture by spleen cells from Taenia glycan vaccinated-leishmania infected mice and not from mock vaccinated-leishmania infected BALB/c mice. We conclude that T. crassiceps glycans have immunoadjuvant effects against leishmania and may be developed as adjuvants in anti-leishmania vaccines.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Leishmania mexicana/imunologia , Taenia/imunologia , Vacinas/imunologia , Adjuvantes Imunológicos , Animais , Quimiocina CCL5/biossíntese , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interleucina-6/biossíntese , Leishmaniose/imunologia , Leishmaniose/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Taenia/metabolismo
17.
Mol Immunol ; 41(4): 391-8, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15163536

RESUMO

In murine infections of Schistosoma mansoni and Taenia crassiceps, a characteristic polarization of the immune response from a Th-1 type to a Th-2 type occurs with progression of infection. In S. mansoni, egg glycoprotein carbohydrates are said to be responsible for this immunomodulatory activity. We have previously shown that in vitro systems, T. crassiceps carbohydrates (TCHO) up-regulate the expression of interleukin 6 (IL-6) in naïve spleen cells, while conventional Th-2 cytokines were not detected. In this report, we show that peritoneal macrophages are the source of this early IL-6 and that these cells recognize T. crassiceps carbohydrates via Toll-like receptors (TLRs). Co-stimulation experiments with synthetic sugars showed that the most likely active moieties in TCHO are Lewis X analogues. To our knowledge, this is the first report on native carbohydrates of a helminth parasite stimulating mammalian innate immune cells to produce a Th-2 polarizing cytokine (IL-6) via a Toll-like receptor. It is hypothesized this is a general mechanism of Th-2 immunodominance in helminth infections in mammalian hosts.


Assuntos
Antígenos de Helmintos/imunologia , Carboidratos/imunologia , Regulação da Expressão Gênica/imunologia , Interleucina-6/biossíntese , Macrófagos Peritoneais/imunologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Taenia/imunologia , Células Th2/efeitos dos fármacos , Animais , Células Cultivadas/imunologia , Células Cultivadas/metabolismo , Proteínas do Ovo/imunologia , Imunidade Inata , Epitopos Imunodominantes/imunologia , Interleucina-6/genética , Interleucina-6/metabolismo , Antígenos CD15/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Oligossacarídeos/síntese química , Oligossacarídeos/imunologia , Schistosoma mansoni/imunologia , Baço/citologia , Células Th2/imunologia , Células Th2/metabolismo , Receptores Toll-Like
18.
Immunology ; 107(4): 411-9, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12460185

RESUMO

T helper type 2 (Th2) -polarized immune responses are characteristically dominant in helminth infections. Two murine models that show a Th1 to Th2 polarization with infection progression are those of Schistosoma mansoni and Taenia crassiceps. In both, an early Th1 response is replaced by a late Th2 response. We report that the nucleic acid-, protein- and lipid-free carbohydrate fraction of T. crassiceps metacestodes (denoted T-CHO) possesses Th2-like immunomodulatory activity. Immunization of two strains of rats (Dark Agouti and Albino Oxford) and BALB/c mice with chicken albumin in the presence of T-CHO resulted in selective enhancement of immunoglobulin G1 (IgG1) antibodies, considered to be associated with Th2 responses in both rats and mice. Interleukin-6 (IL-6) followed by IL-10 were the dominant cytokines detected in in vitro cultures of mouse spleen cells stimulated with T-CHO. IL-4 and IL-5 were not detected in these culture supernates. Furthermore, Taenia carbohydrates were mitogenic to spleen cells, activated serine phosphorylation of proteins and up-regulated the expression of the anti-apoptotic protein, Bcl-2. When mouse spleen cells were cultured in the presence of Taenia carbohydrates, a concentration-dependent down-regulation of IL-2 and an overlapping up-regulation of IL-6 secretion were seen.


Assuntos
Carboidratos/imunologia , Imunoglobulina G/imunologia , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Teníase/imunologia , Adjuvantes Imunológicos , Animais , Formação de Anticorpos/imunologia , Western Blotting , Células Cultivadas/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Endogâmicos , Baço/imunologia , Taenia/imunologia
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