RESUMO

Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9 nM) and MTV-LRP1 (Kd 399 ± 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.

Assuntos

Anti-Inflamatórios/farmacologia , Endotoxinas/antagonistas & inibidores , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Peptídeos/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Barreira Hematoencefálica/metabolismo , Células CHO , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Expressão Gênica , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Modelos Biológicos , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas/métodos , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Fator A de Crescimento do Endotélio Vascular/metabolismo

RESUMO

The neutralization of tumor necrosis factor alpha (TNFα) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNFα-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNFα and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNFα-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNFα was assessed. By microscale thermophoresis, the KD value for the interaction was 4.17 ± 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNFα-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNFα-antagonist agent.

Assuntos

Anti-Inflamatórios/farmacologia , Colágeno/genética , Endotoxinas/antagonistas & inibidores , Etanercepte/farmacologia , Receptores Tipo II do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Colágeno/metabolismo , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Etanercepte/química , Etanercepte/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodos , Multimerização Proteica , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/toxicidade

RESUMO

AIM: Type 1 diabetes mellitus (T1D) is an autoimmune disease characterized by the progressive destruction of ß cells, mediated by the interaction between T cells and several cytokines. The pathogenesis of T1D has established its possible relationship with miRNAs. In this study, we analyze the expression profile of miR-15a and miR-16 in peripheral blood mononuclear cells (PBMCs) and their possible association with apoptosis, inflammation, or autoimmunity markers. PATIENTS AND METHODOLOGY: 38 T1D patients and 41 control subjects were recruited. mRNAs were analyzed by means of qPCR and TaqMan probes. PBMCs were treated with different concentrations of glucose (baseline, 11 and 25 mM) with or without an inflammatory stimulus as TNF-α (10 ng/ml). RESULTS: A decrease in the levels of the miR-15a expression in basal conditions is observed in T1D patients compared to healthy control subjects (relative units 0.5 vs. 1.8, p < 0.05). This change in miR-15a and miR-16 is not affected by the addition of TNF-α. No association is observed with inflammatory markers (IL-6, TNF-α, vCAM) or apoptosis (bcl2 expression). The relationship with immunological markers shows an interaction effect between miR16 and IA-2 (p < 0.03). CONCLUSION: TNF-α does not affect the expression profile of miR-15a and miR16 in PBMCs. A weak correlation is observed between miR-16 and with the autoimmunity profile (IA-2 autoantibody).

Assuntos

Apoptose/fisiologia , Autoimunidade/fisiologia , Diabetes Mellitus Tipo 1/metabolismo , Mediadores da Inflamação/metabolismo , MicroRNAs/biossíntese , Adolescente , Adulto , Apoptose/efeitos dos fármacos , Autoimunidade/efeitos dos fármacos , Biomarcadores/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Chile/epidemiologia , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/imunologia , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Fator de Necrose Tumoral alfa/toxicidade , Adulto Jovem

RESUMO

BACKGROUND: In conditions of immunosuppression, the central nervous sty 5ystem (CNS) is the main target tissue for the reactivation of infection by Trypanosoma cruzi, the causative agent of Chagas disease. In experimental T. cruzi infection, interferon gamma (IFNγ)+ microglial cells surround astrocytes harboring amastigote parasites. In vitro, IFNγ fuels astrocyte infection by T. cruzi, and IFNγ-stimulated infected astrocytes are implicated as potential sources of tumor necrosis factor (TNF). Pro-inflammatory cytokines trigger behavioral alterations. In T. cruzi-infected mice, administration of anti-TNF antibody hampers depressive-like behavior. Herein, we investigated the effects of TNF on astrocyte susceptibility to T. cruzi infection and the regulation of cytokine production. METHODS: Primary astrocyte cultures of neonatal C57BL/6 and C3H/He mice and the human U-87 MG astrocyte lineage were infected with the Colombian T. cruzi strain. Cytokine production, particularly TNF, and TNF receptor 1 (TNFR1/p55) expression were analyzed. Recombinant cytokines (rIFNγ and rTNF), the anti-TNF antibody infliximab, and the TNFR1 modulator pentoxifylline were used to assess the in vitro effects of TNF on astrocyte susceptibility to T. cruzi infection. To investigate the role of TNF on CNS colonization by T. cruzi, infected mice were submitted to anti-TNF therapy. RESULTS: rTNF priming of mouse and human astrocytes enhanced parasite/astrocyte interaction (i.e., the percentage of astrocytes invaded by trypomastigote parasites and the number of intracellular parasite forms/astrocyte). Furthermore, T. cruzi infection drove astrocytes to a pro-inflammatory profile with TNF and interleukin-6 production, which was amplified by rTNF treatment. Adding rTNF prior to infection fueled parasite growth and trypomastigote egression, in parallel with increased TNFR1 expression. Importantly, pentoxifylline inhibited the TNF-induced increase in astrocyte susceptibility to T. cruzi invasion. In T. cruzi-infected mice, anti-TNF therapy reduced the number of amastigote nests in the brain. CONCLUSIONS: Our data implicate TNF as a promoter of T. cruzi invasion of mouse and human astrocytes. Moreover, the TNF-enriched inflammatory milieu and enhanced TNFR1 expression may favor TNF signaling, astrocyte colonization by T. cruzi and egression of trypomastigotes. Therefore, in T. cruzi infection, a self-sustaining TNF-induced inflammatory circuit may perpetuate the parasite cycle in the CNS and ultimately promote cytokine-driven behavioral alterations.

Assuntos

Astrócitos/metabolismo , Doença de Chagas/metabolismo , Mediadores da Inflamação/metabolismo , Trypanosoma cruzi , Fator de Necrose Tumoral alfa/toxicidade , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Linhagem Celular Tumoral , Células Cultivadas , Doença de Chagas/patologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL

RESUMO

AIMS: We have investigated the antihyperalgesic effects of limonene in mice that received intrathecal injection of gp120. MAIN METHODS: Male Swiss mice received gp120, IL-1ß or TNF-α intrathecally or sterile saline as a control. A mechanical sensitivity test was performed at 2 and 3h after the injection. Spinal cord and blood samples were isolated for protein quantification. KEY FINDINGS: Intrathecal administration of gp120 increased mechanical sensitivity measured with an electronic Von Frey apparatus, at 2 and 3h after the injections. Limonene administered orally prior to gp120 administration significantly decreased this mechanical sensitivity at 3h after the gp120 injection. In addition, intrathecal injection of gp120 increased IL-1ß and IL-10 in serum, and limonene prevented the ability of gp120 to increase these cytokines. Limonene also inhibited TNF-α and IL-1ß-induced mechanical hyperalgesia. Western blot assay demonstrated limonene was capable of increasing SOD expression in the cytoplasm of cells from spinal cord at 4h after intrathecal IL-1ß injection. SIGNIFICANCE: These results demonstrate that gp120 causes mechanical hyperalgesia and a peripheral increase in IL-1ß and IL-10, and that prior administration of limonene inhibits these changes. Also limonene modulates the activation of SOD expression in the spinal cord after spinal IL-1ß application. The ability of limonene to inhibit the mechanical hyperalgesia induced by gp120, TNF-α and IL-1ß emphasizes the anti-inflammatory action of limonene, specifically its ability to inhibit cytokine production and its consequences.

Assuntos

Anti-Inflamatórios não Esteroides/farmacologia , Cicloexenos/farmacologia , Proteína gp120 do Envelope de HIV/toxicidade , Hiperalgesia/prevenção & controle , Interleucina-1beta/toxicidade , Medula Espinal/efeitos dos fármacos , Terpenos/farmacologia , Fator de Necrose Tumoral alfa/toxicidade , Administração Oral , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Western Blotting , Cicloexenos/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Injeções Espinhais , Limoneno , Masculino , Camundongos , Medula Espinal/metabolismo , Terpenos/administração & dosagem

RESUMO

The aim of this study was to observe the influence of gap junction (GJ) functional changes on the hepatotoxicity of TNF-α. Three different methods were employed to study functional effects of the GJ inhibition: 1) pretreatment with a GJ inhibitor; 2) inoculation of cells at high and low densities; and 3) inhibition of the expression of connexin 32 (Cx32) by small inhibitory RNA transfection. We then observed the influence of these treatments on hepatotoxicity following treatment with different concentrations of TNF-α for various duration. The hepatotoxicity of TNF-α was observed to occur in a dose- and time-dependent manner; after pretreatment inhibition, the hepatotoxicity of TNF-α was significantly reduced (P < 0.01). The hepatotoxicity of TNF-α was also found to be remarkably lower in cells that had been inoculated at low density (as measured by the amount of GJ formation among cells) than in those inoculated at density (P < 0.01). In addition, following Cx32 inhibition, the hepatotoxicity of TNF-α was significantly decreased (P < 0.01) as well. Together, these results suggest that inhibition of GJ function or of its component Cx32 significantly decreases the hepatotoxicity of TNF-α, and that the expression of Cx32 plays an important role in the hepatotoxicity of TNF-α.

Assuntos

Conexinas/metabolismo , Junções Comunicantes/metabolismo , Hepatócitos/metabolismo , Animais , Linhagem Celular , Conexinas/genética , Hepatócitos/efeitos dos fármacos , Ratos , Fator de Necrose Tumoral alfa/toxicidade , Proteína beta-1 de Junções Comunicantes

RESUMO

Agmatine, an endogenous cationic amine, has been shown to exert antidepressant-like effects. This study investigated the ability of agmatine administered orally to abolish the depressive-like behavior induced by the administration of the pro-inflammatory cytokine, tumor necrosis factor (TNF-α) in mice. In control animals, agmatine (0.001, 0.01, 0.1, and 1 mg/kg) reduced the immobility time in the tail suspension test (TST). Acute administration of TNF-α (0.001 fg/mouse, i.c.v.) increased immobility time in the TST, indicative of a depressive-like behavior, and agmatine (0.0001, 0.1, and 1 mg/kg) prevented this effect. Additionally, we examined the effects of the combined administration of sub-effective doses of agmatine with antidepressants, the NMDA receptor antagonist MK-801 and the neuronal nitric oxide synthase inhibitor 7-nitroindazole (7-NI) in mice exposed to either TNF-α or saline. In control mice, administration of a sub-effective dose of agmatine (0.0001 mg/kg) combined with sub-effective doses of either fluoxetine (5 mg/kg, p.o.), imipramine (0.1 mg/kg, p.o.), bupropion (1 mg/kg, p.o.), MK-801 (0.001 mg/kg, p.o.) or 7-NI (25 mg/kg, i.p.) produced a synergistic antidepressant-like effect in the TST. All these administrations prevented the increased immobility time induced by TNF-α. The effect of agmatine in the TNF-α model of depression appears to be associated, at least partially, with an activation of the monoaminergic systems and inhibition of NMDA receptors and nitric oxide synthesis, although converging signal transduction pathways that may underlie the effect of agmatine should be further investigated. This set of results indicates that agmatine may constitute a new therapeutic alternative for the treatment of depression associated with inflammation.

Assuntos

Agmatina/uso terapêutico , Antidepressivos/uso terapêutico , Depressão/induzido quimicamente , Depressão/tratamento farmacológico , Fator de Necrose Tumoral alfa/toxicidade , Análise de Variância , Animais , Modelos Animais de Doenças , Maleato de Dizocilpina/uso terapêutico , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Feminino , Elevação dos Membros Posteriores , Resposta de Imobilidade Tônica/efeitos dos fármacos , Indazóis/uso terapêutico , Camundongos

RESUMO

Taking into account that pro-inflammatory cytokines and oxidative and nitrosative stress are implicated in the pathogenesis of depression and that α-tocopherol has antidepressant, anti-inflammatory and antioxidant properties, this study investigated the ability of α-tocopherol to abolish the depressive-like behavior induced by i.c.v. administration of TNF-α in the mouse TST. Additionally, we investigated the occurrence of changes in the levels of Bcl2 and Bax and phosphorylation of GSK-3ß (Ser9) in the hippocampus of mice. The administration of TNF-α (0.001fg/site, i.c.v.) increased the immobility time in the TST, which was prevented by the administration of α-tocopherol at the doses of 10, 30 and 100mg/kg (p.o.). Subeffective doses of α-tocopherol (10mg/kg, p.o.) and/or the antidepressants fluoxetine (5mg/kg, p.o.), imipramine (0.1mg/kg, p.o.) and bupropion (1mg/kg, p.o.), the NMDA receptor antagonist MK-801 (0.001mg/kg, p.o.) or the neuronal nitric oxide synthase inhibitor 7-nitroindazole (25mg/kg, i.p.) prevented the depressive-like effect induced by TNF-α. None of the treatments altered the locomotor activity of mice. Treatment with TNF-α and/or α-tocopherol did not alter the levels of Bax and Bcl2 or the phosphorylation of GSK-3ß in the hippocampus of mice. Together, our results show a synergistic antidepressant-like effect of α-tocopherol with antidepressants against the depressive-like behavior induced by an inflammatory insult, suggesting that this vitamin may be useful to optimize conventional pharmacotherapy of depression, including depressive states associated with inflammatory conditions.

Assuntos

Antidepressivos/uso terapêutico , Transtorno Depressivo/induzido quimicamente , Transtorno Depressivo/tratamento farmacológico , Modelos Animais de Doenças , Fator de Necrose Tumoral alfa/toxicidade , alfa-Tocoferol/uso terapêutico , Animais , Transtorno Depressivo/psicologia , Feminino , Camundongos , Resultado do Tratamento

RESUMO

Inhibition of leukocyte adhesion to the vascular endothelium represents a novel and important approach for decreasing sickle cell disease (SCD) vaso-occlusion. Using a humanized SCD-mouse-model of tumor necrosis factor-α-induced acute vaso-occlusion, we herein present data demonstrating that short-term administration of either hydroxyurea or the phosphodiesterase 9 (PDE9) inhibitor, BAY73-6691, significantly altered leukocyte recruitment to the microvasculature. Notably, the administration of both agents led to marked improvements in leukocyte rolling and adhesion and decreased heterotypic red blood cell-leukocyte interactions, coupled with prolonged animal survival. Mechanistically, these rheologic benefits were associated with decreased endothelial adhesion molecule expression, as well as diminished leukocyte Mac-1-integrin activation and cyclic guanosine monophosphate (cGMP)-signaling, leading to reduced leukocyte recruitment. Our findings indicate that hydroxyurea has immediate beneficial effects on the microvasculature in acute sickle-cell crises that are independent of the drug's fetal hemoglobin-elevating properties and probably involve the formation of intravascular nitric oxide. In addition, inhibition of PDE9, an enzyme highly expressed in hematopoietic cells, amplified the cGMP-elevating effects of hydroxyurea and may represent a promising and more tissue-specific adjuvant therapy for this disease.

Assuntos

Anemia Falciforme/tratamento farmacológico , Antidrepanocíticos/uso terapêutico , GMP Cíclico/metabolismo , Hidroxiureia/uso terapêutico , Pirazóis/farmacologia , Pirimidinas/farmacologia , Doenças Vasculares/tratamento farmacológico , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Doença Aguda , Anemia Falciforme/induzido quimicamente , Anemia Falciforme/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Comunicação Celular , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Migração e Rolagem de Leucócitos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Necrose Tumoral alfa/toxicidade , Doenças Vasculares/induzido quimicamente , Doenças Vasculares/metabolismo

RESUMO

Tumor necrosis factor alpha (TNFalpha) is a major mediator of inflammatory responses and also plays a prominent role in bridging the innate and adaptive phases of immunity. In the present work we attempted to study TNFalpha production in endotoxin-stimulated blood of healthy individuals, and the inter-individual variability in TNFalpha production. For this study, we used diluted whole blood stimulated with lipopolysaccharide (LPS). The levels of the pro-inflammatory cytokine TNFalpha were measured by ELISA and by the L929 cytotoxicity bioassay in 16 and 18 healthy donors, respectively. There were highly significant inter-individual variations in the induced TNFalpha production. It is worth noting that there was no difference in sensitivity between ELISA and the cytotoxicity L929 bioassay. We concluded that whole blood culture is a sensitive method to determine the pro-inflammatory cytokine production in response to endotoxin stimuli in a relevant physiologic milieu. Our data indicate that this method provides appropriate information about the state of cellular immunity of the individual.

Assuntos

Fator de Necrose Tumoral alfa/biossíntese , Animais , Bioensaio , Sangue/efeitos dos fármacos , Sangue/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Humanos , Lipopolissacarídeos/farmacologia , Camundongos , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/toxicidade

RESUMO

The presence of antibodies against the major stress protein, Hsp70, in patients with autoimmune diseases led us to hypothesize that Hsp70 may occur extracellularly, and could exert chaperoning and regulatory effects on various cells. We examined the action of pure Hsp/Hsc70 on the main physiological functions of human promonocytic U-937 cells. The protein was isolated from calf muscle and was shown to be a mixture of inducible Hsp70 (60%) and constitutive Hsc70 (40%) isoforms. It was observed that the addition of the protein up-regulated two major monocyte/macrophage differentiation markers, CD11c and CD23, by 20-35%, while it had no effect on CD14. The experiments performed to investigate the influence of Hsp/Hsc70 on the reaction of U-937 cells to differentiation stimuli demonstrated that the addition of the protein prior to PMA was able to inhibit binding of proper transcription factors to double-symmetry and cAMP-response elements of the c-fos early response gene promoter. Administration of exogenous Hsp/Hsc70 prior to treatment with the tumor necrosis factor-alpha significantly lowered the number of apoptotic and necrotic cells. In no case did the control protein, ovalbumin, taken in the same concentration give a comparable effect on U-937 cells. Since the Hsp/Hsc70 effects occurred within the first hour of co-incubation, and therefore they might be explained by its interaction with the cell surface, we assayed binding of the biotinylated protein to U-937 cells by immunoenzyme assay, flow cytometry and indirect immunofluorescence. Using these three techniques we were able to detect Hsp/Hsc70 bound to cells after a 20 min incubation. According to flow cytometry data, at this time 32% of cells were positively stained with streptavidin-FITC. Immunofluorescence studies demonstrated Hsp/Hsc70 bound to the cell surface after a 20 min incubation followed by induction of patch and cap-like structures. One hour later, the majority of the protein had been internalized by U-937 cells.

Assuntos

Proteínas de Transporte/farmacologia , Proteínas de Choque Térmico HSP70/farmacologia , Monócitos/citologia , Animais , Antígenos CD/análise , Apoptose , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , Bovinos , Diferenciação Celular , Divisão Celular , Linhagem Celular , DNA/metabolismo , Endocitose , Citometria de Fluxo , Genes fos/genética , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/isolamento & purificação , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Monócitos/metabolismo , Músculo Esquelético , Regiões Promotoras Genéticas/genética , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/toxicidade

RESUMO

Mycobacteria are ubiquitous in the environment, but they are not part of the normal human microbial flora. It has been suggested that variable contact with mycobacteria can influence susceptibility to mycobacterial pathogens and the efficacy of subsequent Mycobacterium bovis BCG vaccination. To test this, mice were immunized with high or low doses of an environmental saprophyte, M. vaccae, that is intensely immunogenic as an autoclaved preparation. Two months later, they received an intratracheal challenge with M. tuberculosis H37Rv. Recipients of a low Th1-inducing dose (10(7) organisms) were partially protected and maintained a high ratio of interleukin 2 (IL-2)-positive to IL-4-positive cells in the perivascular, peribronchial, and granulomatous areas of the lung, whereas in unimmunized controls the IL-4-positive cells increased markedly between days 21 and 28. In contrast, recipients of the high dose (10(9) organisms), which primes Th2 as well as Th1 cytokine production, died more rapidly than unimmunized controls and showed massive pneumonia from day 7. The ratio of IL-2-positive to IL-4-positive cells in all compartments of the lung rapidly fell to 1 by day 14 for these animals. These events correlated with cytokine mRNA profiles and with increases in the local toxicity of tumor necrosis factor alpha (TNF-alpha), demonstrable only when a major Th2 component was present. These data indicate that cross-reactive epitopes present in an environmental saprophyte can evoke either protective responses or responses that increase susceptibility to M. tuberculosis. The latter are associated with the presence of a Th2 component and increased sensitivity to TNF-alpha.

Assuntos

Microbiologia Ambiental , Mycobacterium/imunologia , Tuberculose/etiologia , Animais , Hipersensibilidade Tardia , Imunização , Imuno-Histoquímica , Interleucina-2/análise , Interleucina-4/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células Th2/fisiologia , Tuberculose/imunologia , Tuberculose/patologia , Fator de Necrose Tumoral alfa/toxicidade

RESUMO

Tumor necrosis factor alpha mediated cell death in L929 cells correlates with a late increase in reduction of the superoxide scavenger 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), suggesting an increase in MTT reduction per viable cell. This effect was studied in two TNF-sensitive and in five different TNF-resistant clones. Within 36 hrs TNF promoted a 7-fold increase in the reduction of MTT in TNF-sensitive cells. Exogenous ceramide induced a similar effect prior to cell death. Four of the five TNF-resistant clones were also resistant to ceramide and displayed no increase in MTT reduction with either TNF or ceramide. The remaining TNF-resistant clone was sensitive to ceramide, displaying an increase in MTT reduction. Our results suggest a late increase in superoxide production prior to cellular destruction during TNF and ceramide mediated cell death and support the notion that ceramide can serve as a second messenger for TNF in cell death.

Assuntos

Ceramidas/farmacologia , Estresse Oxidativo , Fator de Necrose Tumoral alfa/toxicidade , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Corantes , Resistência a Medicamentos , Sequestradores de Radicais Livres , Cinética , Células L , Camundongos , Mutagênese , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Puromicina/farmacologia , Proteínas Recombinantes/biossíntese , Sais de Tetrazólio , Tiazóis , Transfecção

RESUMO

1. The effect of interleukin-10 (IL-10) upon the hyperalgesic activities in rats of bradykinin, tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), interleukin-8 (IL-8), prostaglandin E2 (PGE2) and carrageenin were investigated in a model of mechanical hyperalgesia. 2. Hyperalgesic responses to bradykinin (1 micrograms) were inhibited in a dose-dependent manner by prior treatment with IL-10 (1-100 ng). 3. Hyperalgesic responses to TNF alpha (2.5 pg), IL-1 beta (0.5 pg) and IL-6 (1.0 ng) but not to IL-8 (0.1 ng) and PGE2 (50 ng and 100 ng) were inhibited by prior treatment with IL-10 (10 ng). 4. Hyperalgesic responses to carrageenin (100 micrograms) were inhibited by IL-10 (10 ng) when this cytokine was injected before but not after the carrageenin. 5. A monoclonal antibody to mouse IL-10 potentiated the hyperalgesic responses to carrageenin (10 micrograms) and TNF alpha (0.025 pg) but not that to IL-8 (0.01 ng). 6. In in vitro experiments in human peripheral blood mononuclear cells (MNCs), IL-10 (0.25-4.0 ng ml-1) inhibited in a dose-dependent manner PGE2 production by MNCs stimulated with IL-1 beta (1-64 ng ml-1) or endotoxin (lipopolysaccharide, LPS, 1 iu = 143 pg ml-1) but evoked only small increases in IL-1ra production. 7. These data suggest that IL-10 limits the inflammatory hyperalgesia evoked by carrageenin and bradykinin by two mechanisms: inhibition of cytokine production and inhibition of IL-1 beta evoked PGE2 production. Our data suggest that the latter effect is not mediated via IL-10 induced IL-Ira and may result from suppression by IL-10 of prostaglandin H synthase-2 (COX-2).

Assuntos

Hiperalgesia/tratamento farmacológico , Interleucina-10/uso terapêutico , Animais , Bradicinina/administração & dosagem , Bradicinina/toxicidade , Carragenina/administração & dosagem , Carragenina/toxicidade , Dinoprostona/administração & dosagem , Dinoprostona/metabolismo , Dinoprostona/toxicidade , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Excipientes/administração & dosagem , Excipientes/toxicidade , Humanos , Hiperalgesia/induzido quimicamente , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/administração & dosagem , Interleucina-1/toxicidade , Interleucina-10/administração & dosagem , Interleucina-10/farmacologia , Interleucina-6/administração & dosagem , Interleucina-6/toxicidade , Interleucina-8/administração & dosagem , Interleucina-8/toxicidade , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/toxicidade , Camundongos , Prostaglandina-Endoperóxido Sintases/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/metabolismo , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/toxicidade

RESUMO

Data from several laboratories suggest a role for a variety of cytokines in the process of bone resorption. SK&F 86002 [5-(4-pyridyl)-6(4-fluorophenyl)-2,3-dihydroimidazo(2,1-b) thiazole], a potent cytokine-suppressive anti-inflammatory agent, has been shown to inhibit cyclooxygenase (CO) and 5-lipoxygenase (LO) activity and to inhibit the production of cytokines both in vitro and in vivo. In the present study, SK&F 86002 inhibited fetal rat long bone (FRLB) resorption induced by parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D3, tumor necrosis factor alpha, and Escherichia coli lipopolysaccharide in a dose-dependent (IC50 of 0.5-1 microM) and reversible manner. Under identical conditions, selective CO inhibitors (indomethacin, ibuprofen, naproxen) and 5-LO inhibitors (phenidone, SK&F 107649) were inactive. Analogs of SK&F 86002, which are dual CO/LO inhibitors devoid of cytokine inhibitory activity (SK&F 81114 and SK&F 86055), also failed to significantly inhibit PTH-induced FRLB resorption. Analogs of SK&F 86002, which retain cytokine inhibitory activity (SK&F 104493 and SK&F 105561), inhibit bone resorption. These data indicate that the observed inhibition of bone resorption by compounds of this class correlates with their cytokine suppressive activity.

Assuntos

Anti-Inflamatórios não Esteroides/farmacologia , Reabsorção Óssea/prevenção & controle , Citocinas/antagonistas & inibidores , Imidazóis/farmacologia , Tiazóis/farmacologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/embriologia , Calcitriol/toxicidade , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Humanos , Imidazóis/uso terapêutico , Interleucina-1/metabolismo , Leucotrieno B4/metabolismo , Leucotrieno C4/metabolismo , Lipopolissacarídeos/toxicidade , Inibidores de Lipoxigenase/farmacologia , Inibidores de Lipoxigenase/uso terapêutico , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Hormônio Paratireóideo/toxicidade , Ratos , Tiazóis/uso terapêutico , Fator de Necrose Tumoral alfa/toxicidade

RESUMO

Los modelos experimentales de asbestosis han demostrado que la respuesta inflamatoria inicial está mediada por macrófagos alveolares (MA). Aunque la atracción y acumulación de MA vistos en nuestros modelos está fundamentalmente mediada por el complemento, se ha sugerido la participación de otros factores quimiotácticos no bien caracterizados. En este trabajo, buscamos la presencia de factores quimiotácticos en ratas instiladas con asbesto en forma aguda. Demostramos morfoñlógicamente que el depósito de fibras, la respuesta macrofágica y las lesiones inducidas, son equivalentes a lo reportado en modelos por inhalación. Evaluamos la actividad quimiotáctica en el lavado broncoalveolar (LBA) fraccionado de acuerdo a su peso molecular (PM), y la presencia de albúmina y complemento. Encontramos actividad quimiotáctica en las fracciones del LBA correspondientes a picos de alto y bajo PM. La actividad del primer pico se atribuyó al complemento. La actividad del segundo, aumentó conforme al tiempo de exposición y no parece estar relacionada con complemento. Para identificar otros factores quimiotácticos diferentesa complemento, determinamos la presencia de factor de necrosis tumoral (TNFÿ) y fibronectina (FN) en los LBA no fraccionados. No se detectaron diferencias en la cantidad de TNF presente en los diferentes grupos. Observamos un incremento en la concentración de FN en relación al tiempo de exposición. Aunque la presencia de fracciones de FN pudiera explicar parcialmente el fenómeno quimiotáctico observado con el pico de bajo úPM, no podemos descartar la participación de otros factores no identificados

Assuntos

Animais , Ratos , Asbestose/fisiopatologia , Fatores Quimiotáticos/administração & dosagem , Alvéolos Pulmonares/fisiopatologia , Asbestose/etiologia , Fatores Quimiotáticos/imunologia , Fibronectinas/efeitos adversos , Fibronectinas/toxicidade , Fator de Necrose Tumoral alfa/efeitos adversos , Fator de Necrose Tumoral alfa/toxicidade