RESUMO
INTRODUCTION: Periapical lesions arise as a result of the activation and interaction of the host immune responses against root canal infection. Recently identified Toll-like receptors (TLR) seem to be involved in the recognition and development of immune responses against a myriad of microorganisms. However, very little information is available on the role of TLR in the induction of periapical lesions. METHOD: The role of TLR-2 and TLR-4 in the activation of murine macrophages stimulated using Fusobacterium nucleatum and Peptostreptococcus anaerobius was investigated. The production of nitric oxide (NO) and reactive oxygen species (ROS) was assessed. RESULTS: The results demonstrate that TLR-2 and TLR-4 are involved in the production of ROS by activated macrophages. The microorganisms induced similar levels of NO production by TLR-2-competent and TLR-2-deficient macrophages, regardless of the addition of interferon-gamma (IFN-gamma), ruling out a role for TLR-2 in the NO production induced by these bacteria. Only P. anaerobius induced NO production by TLR-4-competent macrophages without the addition of IFN-gamma. However, after IFN-gamma addition, F. nucleatum induced macrophage NO production. Therefore, NO production stimulated by IFN-gamma and these microorganisms seems to be TLR-4-independent. CONCLUSION: TLR-2 seems to be involved in the induction of ROS production by macrophages in response to prevalent root canal bacteria, while only F. nucleatum induced ROS production by TLR-4-competent macrophages. Both microorganisms significantly induced large amounts of NO independent of TLR-2 and TLR-4. We conclude that microorganisms may participate in the induction and progression of periapical lesions through NO and ROS production by activated macrophages.
Assuntos
Cavidade Pulpar/microbiologia , Sequestradores de Radicais Livres/imunologia , Macrófagos/imunologia , Óxido Nítrico/imunologia , Espécies Reativas de Oxigênio/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Células Cultivadas , Feminino , Fusobacterium nucleatum/imunologia , Interferon gama/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Peptostreptococcus/imunologiaRESUMO
Phase objects can become visible by slightly defocusing an optical microscope, a technique seldom used as a useful tool. We revisited the theory of defocusing and apply it to our optical microscope with optics corrected at infinity. In our approximation, we obtain that the image contrast is proportional to the two-dimensional (2D) Laplacian of the phase difference introduced by the phase object. If the index of refraction of the phase object is uniform the image obtained from defocusing microscopy is the image of curvature (Laplacian of the local thickness) of the phase object, while standard phase-contrast microscopy gives information about the thickness of the object. We made artificial phase objects and measured image contrasts with defocusing microscopy. Measured contrasts are in excellent agreement with our theoretical model. We use defocusing microscopy to study curvature fluctuations (ruffles) on the surface of macrophages (cell of the innate immune system), and try to correlate mechanical properties of macrophage surface and phagocytosis. We observe large coherent propagating structures: Their shape, speed, density are measured and curvature energy estimated. Inhomogeneities of cytoskeleton refractive index, curvature modulations due to thermal fluctuations and/or periodic changes in cytoskeleton-membrane interactions cause random fluctuations in image contrast. From the temporal and spatial contrast correlation functions, we obtain the decay time and correlation length of such fluctuations that are related to their size and the viscoelastic properties of the cytoskeleton. In order to associate the dynamics of cytoskeleton with the process of phagocytosis, we use an optical tweezers to grab a zymosan particle and put it into contact with the macrophage. We then measure the time for a single phagocytosis event. We add the drug cytochalasin D that depolymerizes the cytoskeleton F-actin network: It inhibits the large propagating coherent fluctuations on the cell surface, increases the relaxation time of cytoskeleton fluctuations, and increases the phagocytosis time. Our results suggest that the methods developed in this work can be of utility to assess the importance of cytoskeleton motility in the dynamics of cellular processes such as phagocytosis exhibited by macrophages.
Assuntos
Membrana Celular/patologia , Microscopia/métodos , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Citocalasina D/farmacologia , Citoesqueleto/metabolismo , Vidro , Processamento de Imagem Assistida por Computador , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Contraste de Fase/métodos , Microscopia de Vídeo , Modelos Estatísticos , Distribuição Normal , Fagocitose , Fatores de Tempo , Zimosan/farmacologiaRESUMO
Vaccinia virus (VV) triggers a mitogenic signal at an early stage of infection. VV-induced proto-oncogene c-fos mRNA with kinetics paralleling that stimulated by serum. The VV virokine, or vaccinia virus growth factor (VGF), was not crucial for c-fos induction because it was observed upon infection with the virokine-minus mutant VV (VGF(-)). Furthermore, c-fos expression did not require infectious virus particles, as it occurred even with UV-inactivated VV and was equally induced by the different multiplicities of infection, i.e. 1.0, 5.0, and 25.0. c-fos expression was preceded by VV-induced DNA binding activity and was mediated via the cis-acting elements serum response element (SRE), activating protein-1 (AP-1), and cAMP-response element (CRE). VV activated the protein kinases p42MAPK/ERK2 and p44MAPK/ERK1 and the transcription factor ATF1 in a time-dependent manner with kinetics that paralleled those of VV-stimulated DNA-protein complex formation. The mitogenic signal transmission pathways leading to c-fos activation upon VV infection were apparently mediated by the protein kinases MEK, ERK, and PKA. This assumption was based on the findings that: 1) c-fos transcript was down-regulated; 2) the SRE, AP-1, and CRE binding activities were significantly reduced; and 3) the activation of p42MAPK/ERK2, p44MAPK/ERK1, and ATF1 were drastically affected when the viral infections were carried out in the presence of specific protein kinase inhibitor. Moreover, the mutant VV (VGF(-)) was also able to activate ERK1/2. It is noteworthy that virus multiplication was equally affected by the same kinase inhibitors. Taken together, our data provide evidence that the early mitogenic signal triggered upon VV infection relies upon the activation of the protein kinases MEK, ERK, and PKA, which are needed for both signal transduction and virus multiplication.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/metabolismo , Vaccinia virus/metabolismo , Vaccinia virus/patogenicidade , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA/metabolismo , DNA Complementar/metabolismo , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Cinética , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Modelos Biológicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismo , Elemento de Resposta Sérica/genética , Transdução de Sinais , Fatores de Tempo , Transcrição GênicaRESUMO
In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified from Trypanosoma cruzi trypomastigotes (tGPI-mucin) to trigger phosphorylation of different mitogen-activated protein kinases (MAPKs) and related transcription factors in inflammatory macrophages. Kinetic experiments show that the peak of extracellular signal-related kinase (ERK)-1/ERK-2, stress-activated protein kinase (SAPK) kinase-1/mitogen-activated protein kinase (MAPK) kinase-4, and p38/SAPK-2, phosphorylation occurs between 15 and 30 min after macrophage stimulation with tGPI-mucin or GPI anchors highly purified from tGPI-mucins (tGPI). The use of the specific inhibitors of ERK-1/ERK-2 (PD 98059) and p38/SAPK-2 (SB 203580) phosphorylation also indicates the role of MAPKs, with possible involvement of cAMP response element binding protein, in triggering TNF-alpha and IL-12 synthesis by IFN-gamma-primed-macrophages exposed to tGPI or tGPI-mucin. In addition, tGPI-mucin and tGPI were able to induce phosphorylation of I kappa B, and the use of SN50 peptide, an inhibitor of NF-kappa B translocation, resulted in 70% of TNF-alpha synthesis by macrophages exposed to tGPI-mucin. Finally, the similarity of patterns of MAPK and I kappa B phosphorylation, the concentration of drugs required to inhibit cytokine synthesis, as well as cross-tolerization exhibited by macrophages exposed to tGPI, tGPI-mucin, or bacterial LPS, suggest that receptors with the same functional properties are triggered by these different microbial glycoconjugates.
Assuntos
Citocinas/biossíntese , Glicosilfosfatidilinositóis/metabolismo , Proteínas I-kappa B/metabolismo , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase 4 , Macrófagos/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Superfície Celular/fisiologia , Trypanosoma cruzi/imunologia , Fator 2 Ativador da Transcrição , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Imidazóis/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Inflamação/imunologia , Interleucina-12/biossíntese , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Mucinas/imunologia , NF-kappa B/fisiologia , Óxido Nítrico/biossíntese , Fosforilação/efeitos dos fármacos , Proteínas de Protozoários/imunologia , Piridinas/farmacologia , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Glycosylphosphatidylinositol (GPI) anchor and glycoinositolphospholipid (GIPL) are abundant molecules present in the membrane of parasitic protozoa that are common etiologic agents of medical and veterinary diseases. Recent studies have documented the immunostimulatory/regulatory activity of protozoan-derived GPI-anchors and related structures. Among the bioactivity displayed by the protozoan-derived GPI-anchor is the ability to elicit the synthesis of pro-inflammatory cytokines as well as nitric oxide by host macrophages. In contrast, at high concentrations GIPL and lipophosphoglycan (LPG) derived from protozoan parasites suppress several functions of the host immune system. Additionally, the protozoan-derived GPI-anchor and GIPL have been shown to serve as targets for both specific B and NK-T lymphocyte responses. This information extends our knowledge about parasite molecules that stimulate/regulate the host immune system during protozoan infection. The identification of receptor(s) and signaling pathways triggered by these GPI-related glycolipids may provide new insights for the development of therapies that inhibit detrimental immune responses or potentiate beneficial immune responses observed during infection with protozoan parasites.
Assuntos
Eucariotos/imunologia , Glicolipídeos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Fosfolipídeos/metabolismo , Infecções por Protozoários/imunologia , Transdução de Sinais , Animais , Eucariotos/química , Eucariotos/metabolismo , Eucariotos/patogenicidade , Glicolipídeos/química , Glicosilfosfatidilinositóis/química , Humanos , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , Fosfolipídeos/química , Infecções por Protozoários/parasitologia , VirulênciaRESUMO
To stuck the effect of chemotherapy on parasite-macrophage interaction we used the wild-type Y strain (drug-susceptible) of Trypanosoma cruzi and a drug-resistant parasite population derived from the same strain. Trypomastigotes isolated from untreated infected mice, as well as, 3 h after treatment with BZ were incubated with inflammatory macrophages and used to study phagocytosis, parasite destruction, cytokine release and reactive nitrogen intermediates (RN!) synthesis. Phagocytosis and destruction of the drug-susceptible parasites were significant/v enhanced by drug treatment. These enhancements were accompanied by an increase in cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)alpha] and RNI release by murine inflammatory macrophages primed with IFN-gamma. In contrast, BZ treatment of mice infected with drug-resistant T. cruzi population showed no effect whatsoever. The synthesis of IFN-gamma and RNI by splenocytes of mice infected with either susceptible and drug-resistant parasite populations, before and after treatment with BZ were also studied. On/v the splenocytes from mice infected with the drug-susceptible parasites treated with BZ produced high levels of IFN-gamma and RNI. Our findings indicate that BZ acts on the drug-susceptible T. cruzi parasites by enhancing the phagocytosis and the production of cytokines and RN!, thus, favouring the destruction of the intracellular parasites by the cellular compartment of the immune system.
Assuntos
Doença de Chagas/tratamento farmacológico , Citocinas/biossíntese , Macrófagos/efeitos dos fármacos , Nitroimidazóis/uso terapêutico , Parasitemia/tratamento farmacológico , Fagocitose/efeitos dos fármacos , Tripanossomicidas/uso terapêutico , Animais , Doença de Chagas/imunologia , Resistência a Medicamentos , Interleucina-12/biossíntese , Macrófagos/imunologia , Masculino , Camundongos , Óxido Nítrico/biossíntese , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Gene therapy is an active field that has progressed rapidly into clinical trials in a relatively short time. The key to success for any gene therapy strategy is to design a vector able to serve as a safe and efficient gene delivery vehicle. This has encouraged the development of nonviral DNA-mediated gene transfer techniques such as liposomes. Many liposome-based DNA delivery systems have been described, including molecular components for targeting given cell surface receptors or for escaping from the lysosomal compartment. Another recent technology using cationic lipids has been evaluated and has generated substantial interest in this approach to gene transfer.
Assuntos
Terapia Genética , Lipossomos , Transfecção , CátionsRESUMO
Gene therapy is an active field that has progressed rapidly into clinical trials in a relatively short time. The key to success for any gene therapy strategy is to design a vector able to serve as a safe and efficient gene delivery vehicle. This has encouraged the development of nonviral DNA-mediated gene transfer techniques such as liposomes. Many liposome-based DNA delivery systems have been described, including molecular components for targeting given cell surface receptors or for escaping from the lysosomal compartment. Another recent technology using cationic lipids has been evaluated and has generated substantial interest in this approach to gene transfer