RESUMO
Tumor necrosis factor receptor 1 (TNFR1) is overexpressed in several varieties of carcinoma, including breast cancer. WH701 (Ala-Thr-Ala-Gln-Ser-Ala-Tyr-Gly), which was identified by phage display, can specifically bind to TNFR1. In this study, we labeled WH701 with 18F and investigated its tumor diagnostic value. WH701 was synthesized by standard Fmoc-solid phase synthetic protocols and conjugated by NOTA-NHS. NOTA-WH701 was radiolabeled with 18F using NOTA-AlF chelation reaction. The tumor target properties were evaluated in vitro and in vivo using MCF-7 xenografts and inflammation models. [18F]AlF-NOTA-WH701 was labeled in 25 min with a decay-corrected yield of 38.1 ± 4.8% (n = 5) and a specific activity of 10.4-13.0 GBq/µmol. WH701 had relatively high affinity for MCF-7 cells in vitro and [18F]AlF-NOTA-WH701 displayed relatively high tumor uptake in vivo. The tumor to muscle ratio was 4.25 ± 0.56 at 30 min post-injection (p.i.); further, there was a significant difference between the tumor/muscle and inflammation/muscle (3.22 ± 0.56) ratio, which could differentiate the tumor and inflammation. The tumor uptake of [18F]AlF-NOTA-WH701 could be inhibited by 71.1% by unlabeled WH701 at 30 min p.i. We have developed a promising PET tracer [18F]AlF-NOTA-WH701 for the noninvasive detection of breast cancer in vivo.
Assuntos
Neoplasias da Mama/diagnóstico , Inflamação/diagnóstico , Peptídeos/administração & dosagem , Receptores Tipo I de Fatores de Necrose Tumoral/química , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Meios de Contraste/administração & dosagem , Feminino , Fluordesoxiglucose F18/administração & dosagem , Fluordesoxiglucose F18/química , Compostos Heterocíclicos/administração & dosagem , Compostos Heterocíclicos/química , Compostos Heterocíclicos com 1 Anel , Humanos , Inflamação/diagnóstico por imagem , Inflamação/genética , Inflamação/patologia , Células MCF-7 , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Peptídeos/síntese química , Peptídeos/química , Tomografia por Emissão de Pósitrons , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Lymphotoxin alpha (LTα) is expressed in human atherosclerotic lesions and genetic variations in the LTα pathway have been linked to myocardial infarction. Activation of the P2Y2 nucleotide receptor (P2Y2R) regulates the production of LTα. in vitro. We aimed to uncover a potential pathway linking purinergic receptor to LTα-mediated inflammatory processes pivotal to the early stages of atherosclerosis in apolipoprotein E (ApoE(-)(/)(-)) deficient mice. METHODS AND RESULTS: En face immunostaining revealed that P2Y2R and VCAM-1 are preferentially expressed in the atherosclerosis prone site of the mouse aortic sinus. Deletion of the P2Y2R gene suppresses VCAM-1 expression. Compared with ApoE(-)(/)(-) mice, ApoE(-)(/)(-) mice lacking the P2Y2R gene (ApoE(-)(/)(-)/P2Y2R(-)(/)(-)) did not develop fatty streak lesions when fed a standard chow diet for 15weeks. Systemic and CD4(+) T cell production of the pro-inflammatory cytokine lymphotoxin-alpha (LTα) were specifically inhibited in ApoE(-)(/)(-)/P2Y2R(-)(/)(-)mice. Anti-LTα preventive treatment was initiated in ApoE(-)(/)(-)mice with intraperitoneal administration of recombinant human tumor necrosis factor receptor 1 fusion protein (TNFR1-Fc) on 5 consecutive days before the disease onset. Remarkably, none of the TNFR1:Fc-treated ApoE(-)(/)(-)mice exhibited atherosclerotic lesions at any developmental stage. SIGNIFICANCE: ApoE(-)(/)(-) mice deficient in P2Y2R exhibit low endothelial cell VCAM-1 levels, decreased production of LTα and delayed onset of atherosclerosis. These data suggest that targeting this nucleotide receptor could be an effective therapeutic approach in atherosclerosis.
Assuntos
Apolipoproteínas E/genética , Aterosclerose/patologia , Linfotoxina-alfa/genética , Receptores Purinérgicos P2Y2/genética , Animais , Aterosclerose/genética , Aterosclerose/prevenção & controle , Células Endoteliais/metabolismo , Humanos , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Receptores Tipo I de Fatores de Necrose Tumoral/farmacologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Tumor necrosis factor-α (TNF-α) is one of the key players in stroke progression and can interfere with brain functioning. We previously documented an impairment of experience-dependent plasticity in the cortex neighboring the stroke-induced lesion, which was accompanied with an upregulation of Tnf-α level in the brain of ischemic mice 1 week after the stroke. Because TNF receptor 1 (TnfR1) signaling is believed to be a major mediator of the cytotoxicity of Tnf-α through activation of caspases, we used an anti-inflammatory intervention aimed at Tnf-α R1 pathway, in order to try to attenuate the detrimental effect of post-stroke inflammation, and investigated if this will be effective in protecting plasticity in the infarct proximity. Aged mice (12-14 months) were subjected to the photothrombotic stroke localized near somatosensory cortex, and immediately after ischemia sensory deprivation was introduced to induce plasticity. Soluble TNF-α R1 (sTNF-α R1), which competed for TNF-α with receptors localized in the brain, was delivered chronically directly into the brain tissue for the whole period of deprivation using ALZET Micro-Osmotic pumps. We have shown that such approach undertaken simultaneously with the stroke reduced the level of TNF-α in the peri-ischemic tissue and was successful in preserving the post-stroke deprivation-induced brain plasticity.
Assuntos
Plasticidade Neuronal/genética , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Córtex Somatossensorial/fisiopatologia , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/terapia , Animais , Ligação Competitiva , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Feminino , Bombas de Infusão , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/genética , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/fisiopatologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that has been implicated in the airway pathology of asthma and result in resistance to hormone therapy. Tumor necrosis factor α inhibitors have become a major research focus in the treatment of asthma. METHODS: Recombinant adenovirus (Ad-sTNFR1-IgGFc) expressing a fusion protein (sTNFR1-IgGFc), which was consisted of the soluble extracellular region of TNF receptor 1 and Fc fragment of IgG (sTNFR1-IgGFc), was used to transduce primary human airway smooth muscle cells (HASMCs). Enzyme-linked immunosorbent assay, flow cytometry, and immunocytochemistry confirmed the expression of sTNFR1-IgGFc. MTT was used to test the effect of sTNFR1-IgGFc to antagonism TNF-α-induced proliferates of HASMCs. To investigate the in vivo effectiveness of sTNFR1-IgGFc, mouse model of asthma was established. Ad-sTNFR1-IgGFc was delivered to the lung via nasal spray. Expression of sTNFR1-IgGFc in the tissue was confirmed by in situ hybridization and immunohistochemistry. The 2 major cell types that are involved in the inflamed asthmatic airway, neutrophils and eosinophils, in bronchoalveolar lavage fluid were observed. RESULTS: The sTNFR1-IgGFc isolated from transduced HASMC culture supernatant was able to antagonize HASMC proliferation stimulated by TNF-α. Asthma-induced pathologies and alterations in the cell composition in bronchoalveolar lavage fluid were reduced in mice subjected to Ad-sTNFR1-IgGFc therapy. CONCLUSIONS: The soluble extracellular region of TNF receptor 1 and Fc fragment of IgG was able to functionally antagonize TNF-α in vitro and showed promise as a therapeutic agent for the localized treatment of severe refractory asthma.
Assuntos
Adenoviridae , Asma/terapia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Adenoviridae/genética , Animais , Asma/genética , Asma/patologia , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genéticaRESUMO
PURPOSE: We evaluated an anti-inflammatory effect of TNF receptor 1 (TNFR1) ectodomain shedding in ocular surface. METHODS: Human corneal epithelial cell (HCEC) was first pretreated by TNF-α. Ectodomain shedding was stimulated by uridine triphosphate (UTP) or peptidoglycan (PGN), with or without shedding inhibition using TNF-α processing inhibitor (TAPI). The phosphorylation of the NF-κB inhibitory protein, IκB, was assessed by Western blotting and concentrations of soluble TNFR1 (sTNFR1) in culture medium were analyzed by ELISA. Tear fluid from patients with Sjögren syndrome and graft-versus-host disease (GVHD) was collected and analyzed by ELISA for sTNFR1 concentration. Five dry eye patients underwent topical treatment using diquafosol sodium eye drops, a purinergic P2Y2 receptor agonist, and the tear fluid of the patients was sampled before and 4 weeks after the treatment for sTNFR1 ELISA. RESULTS: Phosphorylation of IκB was diminished by adding UTP or PGN, and this down-regulation of IκB phosphorylation was reversed by adding TAPI. In HCEC medium, sTNFR1 release was increased significantly by adding UTP or PGN, and inhibited significantly by adding TAPI. In the tears of the patients with Sjögren syndrome and GVHD, sTNFR1 expression was upregulated. In the tears of the patients with short breakup time (BUT) dry eye, sTNFR1 concentrations (ng/mL) in the tears were 1.30 ± 0.58 ng/mL for the pretreatment baseline, and 1.64 ± 0.70 after treatment, statistically significantly higher than those for the pretreatment (P < 0.01). CONCLUSIONS: Ectodomain shedding of sTNFR1 blocked TNF-α-induced intracellular signaling in corneal epithelium. The upregulation of sTNFR1 in inflamed ocular surfaces suggests an anti-inflammatory role of sTNFR1 ectodomain shedding at the ocular surface.
Assuntos
Síndromes do Olho Seco/tratamento farmacológico , Epitélio Corneano/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/farmacocinética , Lágrimas/química , Western Blotting , Células Cultivadas , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Ensaio de Imunoadsorção Enzimática , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Humanos , Soluções Oftálmicas , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Transdução de SinaisRESUMO
OBJECTIVE: MicroRNAs (miRs) regulate angiogenesis by posttranscriptional silencing of target genes. The significance of angiostatic miR-200b in switching on skin wound angiogenesis was tested. METHODS AND RESULTS: Wounding caused imminent and transient downregulation of miR-200b in dermal wound-edge endothelial cells. Derailing this injury response by lentiviral delivery of miR-200b in vivo impaired wound angiogenesis. Computational prediction, target reporter luciferase assay, and Western blot analysis provided first evidence that miR-200b targets globin transcription factor binding protein 2 (GATA2) and vascular endothelial growth factor receptor 2 (VEGFR2). Overexpression of GATA2 or VEGFR2 in endothelial cells rescued the angiostatic effect of miR-200b in vitro. Downregulation of miR-200b derepressed GATA2 and VEGFR2 expression to switch on wound angiogenesis, which was disrupted in diabetic wounds. Treatment of endothelial cells with tumor necrosis factor-α, a proinflammatory cytokine abundant in diabetic wounds, induced miR-200b expression, silenced GATA2 and VEGFR2, and suppressed angiogenesis. These outcomes were attenuated using anti-miR-200b strategy. Neutralization of tumor necrosis factor-α in the diabetic wounds improved wound angiogenesis and closure, which was accompanied by downregulation of miR-200b expression and desilencing of GATA2 and VEGFR2. CONCLUSIONS: Injury-induced repression of miR-200b turned on wound angiogenesis. In mice with diabetes mellitus,excessive tumor necrosis factor-α induced miR-200b blunting proangiogenic functions of GATA2 and VEGFR2.
Assuntos
Diabetes Mellitus/terapia , Fator de Transcrição GATA2/metabolismo , Terapia Genética , MicroRNAs/metabolismo , Neovascularização Fisiológica , Pele/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização , Ferimentos Penetrantes/terapia , Animais , Western Blotting , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Modelos Animais de Doenças , Regulação para Baixo , Células Endoteliais/metabolismo , Fator de Transcrição GATA2/genética , Genes Reporter , Vetores Genéticos , Células HEK293 , Humanos , Injeções Intradérmicas , Fluxometria por Laser-Doppler , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Fluxo Sanguíneo Regional , Transdução de Sinais , Pele/irrigação sanguínea , Pele/lesões , Pele/patologia , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Ferimentos Penetrantes/genética , Ferimentos Penetrantes/metabolismo , Ferimentos Penetrantes/patologiaRESUMO
Post-radiation inflammatory reaction leads to an irreversible pulmonary fibrosis which may cause lethal respiratory insufficiency. Pathological inflammatory and fibrotic changes might be attenuated by inhibiting tumour necrosis factor (TNF)-α activity using TNF-α soluble receptors. Thus, an experimental antifibrotic gene therapy with the plasmid vector encoding a mouse soluble receptor I for TNF-α (psTNFR-I) was assessed. Soluble TNFR-I encoding gene was cloned into pcDNA3.1 plasmid. The ability of psTNFR-I expressing vector to transfect cells, and its biological activity in vitro and in vivo were examined by PCR, RT-PCR, MTT assay and ELISA. The C57Bl/6J mice received single intramuscular injection of psTNFR-I, conjugated with polyetylenimine (PEI) 25 kDa, equally divided to both hind legs, 3 days before irradiation (20 Gy, Co60), and either a single injection or ten injections once a week after irradiation. The data proved the effectiveness of psTNFR-I product to neutralise TNF-α activity in vitro. The in vivo plasmid incorporation and maintenance was confirmed. Measurements of plasma soluble TNFR-I levels showed that the in vivo gene transfer was effective. PEI was found to enhance transfection efficiency in vivo. The psTNFR-I/PEI complexes caused no toxicity in the transfected mice. C57Bl/6J mice that received prolonged psTNFR-I/PEI injections developed lethal fibrotic syndrome and died 8 weeks later than the mice treated with a double plasmid injection and the control mice treated with a control plasmid. Sequential administration of soluble TNFR-I by a nonviral, intramuscular gene transduction in the early and late post-radiation inflammatory phase prolonged survival of irradiated mice and attenuated the symptoms of lung fibrosis. The psTNFR-I gene transduction may provide a safe and simple method to partially neutralise TNF-α activity and prevent radiation-induced lung injury.
Assuntos
Pneumonite por Radiação/terapia , Receptores Tipo I de Fatores de Necrose Tumoral , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Colágeno/análise , Modelos Animais de Doenças , Terapia Genética/métodos , Vetores Genéticos/uso terapêutico , Humanos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE: The purpose was to determine whether systemic tumor necrosis factor α (TNF-α) blockade can improve rotator cuff healing in a rat model. METHODS: One hundred twenty Lewis rats underwent unilateral detachment and repair of the supraspinatus. Rats were randomized into 2 groups. The experimental group received injections of pegylated soluble tumor necrosis factor receptor type I (3.0 mg/kg every other day for 3 doses). The control group received saline solution on the same dosing schedule. At 2, 4, and 8 weeks, 20 animals in each group were killed (4 for histologic assessment and 16 for biomechanical testing). Outcomes included qualitative histologic assessment to determine new fibrocartilage formation and collagen fiber organization. Immunohistochemical staining was performed to localize TNF-α, ED1 and ED2 macrophages, and tartrate-resistant acidic phosphatase. Biomechanical testing was performed to determine the ultimate load to failure, stiffness, cross-sectional area, and ultimate stress to failure. RESULTS: Qualitative assessments of histology showed that the experimental group had more cartilage formation at 4 weeks but not at 2 or 8 weeks. There was less TNF-α staining in the experimental group at 4 and 8 weeks, and there were fewer ED1 macrophages at 4 weeks compared with controls. The ultimate load to failure was greater in the experimental group compared with controls at 2 weeks (13.3 ± 2.6 N v 11.2 ± 2.7 N, P = .05) and at 4 weeks (21.7 ± 4.6 N v 18.5 ± 2.1 N, P = .04). The experimental group also had a higher stiffness at 2 weeks (7.2 ± 2.3 N/mm v 5.8 ± 1.4 N/mm, P = .04) and at 4 weeks (10.5 ± 2.7 N/mm v 8.4 ± 1.7 N/mm, P = .01). There were no differences in any biomechanical variable at 8 weeks. CONCLUSIONS: TNF-α blockade can improve the biomechanical strength of tendon-bone healing in a rat rotator cuff model at early time points, which corresponded with modest qualitative improvements in histology. However, these differences were not maintained at 8 weeks. CLINICAL RELEVANCE: TNF-α blockade may influence rotator cuff tendon healing.
Assuntos
Polietilenoglicóis/uso terapêutico , Receptores Tipo I de Fatores de Necrose Tumoral/uso terapêutico , Manguito Rotador/cirurgia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Cicatrização/efeitos dos fármacos , Animais , Fenômenos Biomecânicos , Cicatriz/prevenção & controle , Avaliação Pré-Clínica de Medicamentos , Ectodisplasinas/análise , Fibrocartilagem/fisiologia , Úmero/patologia , Macrófagos/química , Macrófagos/patologia , Polietilenoglicóis/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Endogâmicos Lew , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Regeneração/efeitos dos fármacos , Manguito Rotador/patologia , Método Simples-Cego , Técnicas de Sutura , Tendões/patologia , Fator de Necrose Tumoral alfa/análiseRESUMO
OBJECTIVE: To discover the optimal dose, dose interval, and more advantageous administration route of recombinant human soluble tumour necrosis factor receptor type I (rhsTNF-RI) on orthodontic tooth movement. METHODS: Orthodontic tooth movement (OTM) models were established in Sprague-Dawley rats. The maxillary left first molars were given local injections of phosphate-buffered saline (PBS) and four different concentrations of rhsTNF-RI. On day 14, the amount of tooth movement was registered, and tissue sections were stained with haematoxylin-eosin and tartrate-resistant acid phosphatase. After obtaining the optimal dose, the same models were used to investigate the optimal dose interval and more advantageous route of administration. RESULTS: Compared with the control group, the amount of OTM and the number of TRAP-positive cells in the 0.04 µg/ml group showed no significant changes, whilst were greatly reduced in the other three groups. In the experiment of finding the optimal dose interval, no significant differences were observed in every-two-day and every-three-day injection groups compared to the control. However, every-four-day injection group showed significant difference in the amount of OTM and histological changes. There was no significant difference between local and systemic application of rhsTNF-RI. CONCLUSION: The optimal delivery of rhsTNF-RI was local injection of 0.1 ml at 0.1 µg/ml every three days.
Assuntos
Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Receptores Tipo I de Fatores de Necrose Tumoral/farmacologia , Técnicas de Movimentação Dentária , Análise de Variância , Animais , Relação Dose-Resposta a Droga , Injeções , Injeções Intraperitoneais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
UNLABELLED: Inflammatory cytokines contribute to lumbar radiculopathy. Regulation of cytokines for transient cervical injuries, with or without longer-lasting inflammation, remains to be defined. The C7 root in the rat underwent compression (10gf), chromic gut suture exposure (chr), or their combination (10gf+chr). Ipsilateral C7 spinal cord and dorsal root ganglia (DRG) were harvested at 1 hour after injury for real-time PCR analysis of IL-1beta, IL-6, and TNF-alpha. Cytokine mRNA increased after all 3 injuries. TNF-alpha mRNA in the DRG was significantly increased over sham after 10gf+chr (P = .026). Spinal IL-1beta was significantly increased over sham after 10gf and 10gf+chr (P < .024); IL-6 was significantly increased after 10gf+chr (P < .024). In separate studies, the soluble TNF-alpha receptor was administered at injury and again at 6 hours in all injury paradigms. Allodynia was assessed and tissue samples were harvested for cytokine PCR. Allodynia significantly decreased with receptor administration for 10gf and 10gf+chr (P < .005). Treatment also significantly decreased IL-1beta and TNF-alpha mRNA in the DRG for 10gf+chr (P < .028) at day 1. Results indicate an acute, robust cytokine response in cervical nerve root injury with varying patterns, dependent on injury type, and that early increases in TNF-alpha mRNA in the DRG may drive pain-related signaling for transient cervical injuries. PERSPECTIVE: Inflammatory cytokine mRNA in the DRG and spinal cord are defined after painful cervical nerve root injury. Studies describe a role for TNF-alpha in mediating behavioral sensitivity and inflammatory cytokines in transient painful radiculopathy. Results outline an early response of inflammatory cytokine upregulation in cervical pain.
Assuntos
Citocinas/genética , RNA Mensageiro/genética , Radiculopatia/genética , Animais , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-6/genética , Masculino , Síndromes de Compressão Nervosa/complicações , Síndromes de Compressão Nervosa/fisiopatologia , Dor/etiologia , Dor/fisiopatologia , Medição da Dor/métodos , Estimulação Física/métodos , RNA Mensageiro/metabolismo , Radiculopatia/etiologia , Radiculopatia/fisiopatologia , Ratos , Ratos Sprague-Dawley , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/metabolismo , Raízes Nervosas Espinhais/lesões , Raízes Nervosas Espinhais/fisiopatologia , Fator de Necrose Tumoral alfa/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
TNF-alpha overexpression may contribute to motor neuron death in amyotrophic lateral sclerosis (ALS). We investigated the intracellular pathway associated with TNF-alpha in the wobbler mouse, a murine model of ALS, at the onset of symptoms. TNF-alpha and TNFR1 overexpression and JNK/p38MAPK phosphorylation occurred in neurons and microglia in early symptomatic mice, suggesting that this activation may contribute to motor neuron damage. The involvement of TNF-alpha was further confirmed by the protective effect of treatment with rhTNF-alpha binding protein (rhTBP-1) from 4 to 9 weeks of age. rhTBP-1 reduced the progression of symptoms, motor neuron loss, gliosis and JNK/p38MAPK phosphorylation in wobbler mice, but did not reduce TNF-alpha and TNFR1 levels. rhTBP-1 might possibly bind TNF-alpha and reduce the downstream phosphorylation of two main effectors of the neuroinflammatory response, p38MAPK and JNK.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/prevenção & controle , Neurônios Motores/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Receptores Chamariz do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Contagem de Células/métodos , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Receptores Chamariz do Fator de Necrose Tumoral/administração & dosagem , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
BACKGROUND: Anti-inflammatory gene therapy is promising in inflammatory diseases such as rheumatoid arthritis (RA). We have previously demonstrated that intra-muscular (i.m.) electrotransfer (ET) of plasmids encoding three different human tumor necrosis factor-alpha-soluble receptor I variants (hTNFR-Is) exert protective effects in an experimental RA model. However, such a systemic approach could be responsible for side effects. The present study aimed at performing an intra-articular (i.a.) gene therapy by electrotransfer using the hTNFR-Is plasmids. METHODS AND RESULTS: We evaluated targeting of mice joints by CCD optical imaging after i.a. ET of a luciferase-encoding plasmid and we showed that ET led to strongly increased transgene expression in a plasmid dose-dependent manner. Moreover, articular and seric hTNFR-Is was detectable for 2 weeks. As expected, systemic hTNFR-Is rates were lower after i.a. ET than after i.m. ET. A longer protein secretion could be achieved with several i.a. ETs. Also, we observed that hTNFR-Is expression within arthritic joints was slightly higher than in normal joints. CONCLUSIONS: In collagen-induced arthritis (CIA), a mouse model for RA, we demonstrated that hTNFR-Is/mIgG1-encoding plasmid i.a. ET decreased joint destruction in the ankles. In conclusion, our results suggest that local TNFR-Is gene therapy may play a role in decreasing joint destruction in CIA.
Assuntos
Artrite Experimental/terapia , Sistemas de Liberação de Medicamentos/métodos , Eletroporação , Terapia Genética/métodos , Inflamação/terapia , Plasmídeos/administração & dosagem , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Animais , Tornozelo , Expressão Gênica , Humanos , Articulações , Camundongos , Receptores Tipo I de Fatores de Necrose Tumoral/genéticaRESUMO
The role of TNFalpha/LTalpha during EAN induced by active immunization with peripheral nerve myelin was examined by administering a recombinant soluble chimeric form of human TNF receptor 1 (TNFR1-IgG). TNFalpha and LTalpha do not directly contribute to neurological deficit during EAN since treatment with TNFR1-IgG after onset failed to alter the course of disease. Prophylaxis with a single dose of TNFR1-IgG delayed the onset of EAN and was accompanied initially by inhibition of blood-nerve-barrier permeability and inflammation. Subsequently, the number of infiltrating macrophages and blood-nerve-barrier permeability increased but the disease symptoms remained mild for five days (on average a limp tail) after which severe EAN developed. The antibody titer to peripheral nerve myelin was unaltered by prophylaxis with TNFR1-IgG. The markedly altered tempo of disease onset after TNFR1-IgG prophylaxis indicates that TNFalpha and/or LTalpha have a key role in the development of blood-nerve-barrier permeability and the coupling of macrophage activation and recruitment to peripheral nerve pathology during EAN.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Encefalomielite Autoimune Experimental/complicações , Encefalomielite Autoimune Experimental/patologia , Inflamação/etiologia , Inflamação/prevenção & controle , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Animais , Anticorpos/administração & dosagem , Anticorpos/sangue , Barreira Hematoencefálica/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Macrófagos/efeitos dos fármacos , Masculino , Bainha de Mielina/imunologia , Ratos , Ratos Endogâmicos Lew , Fatores de TempoRESUMO
Adjuvant-induced arthritis is associated with body weight loss and decreased pituitary growth hormone (GH) and hepatic insulin-like growth factor-I (IGF-I) synthesis. Cytokines as tumor necrosis factor (TNF) mediate wasting associated with chronic inflammation. The aim of this study was to analyse whether the inhibition of TNF is able to revert the decrease in the body weight and the GH/IGF-I axis in arthritic rats. Male Wistar rats were injected with Freund's adjuvant, and 15 days later arthritic and control rats were daily injected with polyethylene glycol linked to soluble TNF receptor p55 (PEG-sTNFRI) (1 mg/kg, s.c.) or saline for 8 days. There was a significant decrease in pituitary GH mRNA (P<0.05), hepatic IGF-I mRNA (P<0.01) and serum concentrations of IGF-I (P<0.01) in arthritic rats. The 8-day administration of PEG-sTNFRI resulted in an increase in food intake (P<0.05) and body weight gain (P<0.01) in arthritic but not in control rats. There was an increase in pituitary GH mRNA after PEG-sTNFRI treatment both in control and in arthritic rats. There was a significant increase in IGF-I serum concentrations (P<0.05) and hepatic IGF-I mRNA expression (P<0.05) in control rats treated with PEG-sTNFRI, whereas the effect of this anti-TNF agent in arthritic rats was only statistically significant in hepatic IGF-I mRNA expression (P<0.05). These data suggest that TNF seems to be involved in the decrease in GH and IGF-I synthesis in arthritic rats.
Assuntos
Artrite Experimental/tratamento farmacológico , Hormônio do Crescimento/genética , Fator de Crescimento Insulin-Like I/genética , Receptores Tipo I de Fatores de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Artrite Experimental/genética , Artrite Experimental/metabolismo , Northern Blotting , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/metabolismo , Injeções Subcutâneas , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Polietilenoglicóis/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Receptores Tipo I de Fatores de Necrose Tumoral/química , Solubilidade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: In a phase 2 study, to assess the efficacy and safety of pegsunercept, a soluble tumor necrosis factor receptor type I, for the treatment of rheumatoid arthritis (RA). METHODS: Patients were randomized to receive weekly subcutaneous injections of placebo (n = 61) or active drug [400 microg/kg (n = 67) or 800 microg/kg (n = 66)] for 12 weeks. The primary efficacy endpoint was American College of Rheumatology 20% response (ACR20) at Week 12. Secondary efficacy measures included ACR50 and ACR70 responses, and changes in individual ACR components at Week 12. Safety assessments included summaries of adverse events including infectious episodes. RESULTS: Treatment with pegsunercept resulted in a significantly higher ACR20 response at Week 12 in the 800 microg/kg group (45%) compared with the placebo group (26%; p = 0.020). The treatment effect of pegsunercept (both doses) over the study period showed statistically significant improvement for most ACR components and health related quality of life, with the 800 microg/kg group showing greater clinical improvements in efficacy measures. The overall incidence of adverse events and infectious episodes was similar among the treatment and placebo groups. CONCLUSION: In this 12 week dose-finding study of 194 patients, weekly subcutaneous dosing with pegsunercept showed beneficial effects in improving the signs and symptoms of RA. It appeared to be safe and well tolerated in this small number of patients. Significant clinical improvements were seen in patients in the 800 microg/kg group; however, this dose may be suboptimal, and further evaluation of this product with higher doses or a more frequent dosing regimen is warranted.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Receptores Tipo I de Fatores de Necrose Tumoral/uso terapêutico , Adulto , Idoso , Antirreumáticos/uso terapêutico , Artrite Reumatoide/fisiopatologia , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Feminino , Nível de Saúde , Humanos , Injeções Subcutâneas , Masculino , Metotrexato/uso terapêutico , Pessoa de Meia-Idade , Qualidade de Vida , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Proteínas Recombinantes/uso terapêutico , Resultado do TratamentoRESUMO
OBJECTIVE: This study was undertaken to investigate whether the increased thrombogenic potential of cytotrophoblastic cells of women with preeclampsia can be accounted for by increased rates of apoptosis. STUDY DESIGN: Cytotrophoblasts were isolated from the placenta of (a) nulliparous women without hypertensive disease who were delivered at term and (b) nulliparous women with preeclampsia. The cytotrophoblasts were identified by morphology, and cytokeratin and gonadotropin-releasing hormone positivity. The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) was added to cytotrophoblasts in vitro and incubated for 24 hours. Tissue factor antigen, activity, and amount of apoptosis were evaluated both before and after TNF-alpha stimulation. RESULTS: TNF-alpha simulation significantly increased tissue factor activity both in the cytotrophoblasts of women with (0.08 +/- 0.04 pmol/min/10 6 cells to 0.53 +/- 0.19 pmol/min/10 6 cells) and without (0.07 +/- 0.04 pmol/min/10 6 cells to 0.30 +/- 0.16 pmol/min/10 6 cells) preeclampsia. TNF-alpha stimulation of the cytotrophoblasts also significantly increased tissue factor antigen in the cytotrophoblasts of both groups of women (3.6 +/- 0.9 fmol/10 6 cells to 34.0 +/- 7.5 fmol/10 6 cells, and 7.5 +/- 1.4 fmol/10 6 cells to 25.4 +/- 2.2 fmol/10 6 cells, respectively). For both tissue factor antigen and activity, the magnitude of increase after stimulation was significantly greater in the preeclamptic cytotrophoblasts. In contrast, both normal and preeclamptic cytotrophoblasts showed similar increases in their apoptotic indices (approximately 2-fold) after induction by TNF-alpha. CONCLUSION: The greater response of tissue factor activity and antigen to TNF-alpha by preeclamptic cytotrophoblasts cannot be accounted for by the increase in apoptosis. These data suggest that preeclamptic cytotrophoblasts are inherently more thrombogenic and more sensitive to TNF-alpha stimulation.
Assuntos
Apoptose/efeitos dos fármacos , Mediadores da Inflamação/análise , Receptores Tipo I de Fatores de Necrose Tumoral/administração & dosagem , Trofoblastos/efeitos dos fármacos , Adulto , Apoptose/fisiologia , Células Cultivadas , Citocinas/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Idade Gestacional , Humanos , Placenta/citologia , Pré-Eclâmpsia/fisiopatologia , Gravidez , Valores de Referência , Estudos de Amostragem , Sensibilidade e Especificidade , Trofoblastos/citologiaRESUMO
OBJECTIVE: Inflammation plays an important role in atherosclerosis. One of the most potent pro-inflammatory cytokines is tumor necrosis factor-alpha (TNF-alpha), a cytokine identified to have a pathogenic role in chronic inflammatory diseases such as rheumatoid arthritis (RA). The aim of the study was to evaluate the importance of TNF-alpha in atherogenesis. METHODS AND RESULTS: Mice deficient in both apolipoprotein E (apoE) and TNF-alpha were compared regarding their atherosclerotic burden. Mice were fed a Western-style diet (WD) or normal chow. Mice deficient in both apoE and TNF-alpha exhibited a 50% (P=0.035) reduction of relative lesion size after 10 weeks of WD. Bone marrow transplantation of apoE(o) mice with apoE(o)tnf-alpha(o) bone marrow resulted in a 83% (P=0.021) reduction after 25 weeks on WD. In apoE knockout mice treated with recombinant soluble TNF receptor I releasing pellets, there was a reduction in relative lesion size after 25 weeks of 75% (P=0.018). CONCLUSIONS: These findings demonstrate that TNF-alpha is actively involved in the progression of atherosclerosis. Accordingly, TNF-alpha represents a possible target for prevention of atherosclerosis. This may be of particular importance in rheumatoid arthritis because these patients have an increased risk for cardiovascular disease.