RESUMO
Magnetized laser-produced plasmas are central to many novel laboratory astrophysics and inertial confinement fusion studies, as well as in industrial applications. Here we provide the first complete description of the three-dimensional dynamics of a laser-driven plasma plume expanding in a 20 T transverse magnetic field. The plasma is collimated by the magnetic field into a slender, rapidly elongating slab, whose plasma-vacuum interface is unstable to the growth of the "classical," fluidlike magnetized Rayleigh-Taylor instability.
RESUMO
The mathematical model of group of neurons and astrocytes in the ischemic stroke is discussed. The model includes the description of synaptic signal transmission between the neurons, calcium signaling in astrocytes, ionic currents between the cells and extracellular space and the diffusion through extracellular space. The new modeling approach, based on the creation of appropriate graph, is developed to describe the structure of the system. Using the model, we have analyze the influence of inhibitory synapses on system functioning. We also have shown how the use of medications supporting the activity of inhibitory synapses affects the system behavior in ischemic stroke.
Assuntos
Astrócitos/fisiologia , Modelos Neurológicos , Neurônios/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Astrócitos/efeitos dos fármacos , Sinalização do Cálcio , Humanos , Potenciais da Membrana , Neurônios/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Transmissão Sináptica/efeitos dos fármacosRESUMO
Catechol-O-methyltransferase (COMT) is a key regulator of pain perception, cognitive function, and affective mood. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous position, code for differences in COMT enzymatic activity and are associated with pain sensitivity. Haplotypes divergent in synonymous changes exhibited the largest difference in COMT enzymatic activity, due to a reduced amount of translated protein. The major COMT haplotypes varied with respect to messenger RNA local stem-loop structures, such that the most stable structure was associated with the lowest protein levels and enzymatic activity. Site-directed mutagenesis that eliminated the stable structure restored the amount of translated protein. These data highlight the functional significance of synonymous variations and suggest the importance of haplotypes over single-nucleotide polymorphisms for analysis of genetic variations.
Assuntos
Catecol O-Metiltransferase/biossíntese , Catecol O-Metiltransferase/genética , Haplótipos , Conformação de Ácido Nucleico , RNA Mensageiro/química , Alelos , Substituição de Aminoácidos , Animais , Pareamento de Bases , Sequência de Bases , Catecol O-Metiltransferase/metabolismo , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Células PC12 , Dor/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , TransfecçãoRESUMO
The treatment of chronic inflammatory diseases is complicated by their unpredictable, relapsing clinical course. Here, we describe a new strategy in which an inflammation-regulated therapeutic transgene is introduced into the joints to prevent recurrence of arthritis. To this end, we designed a recombinant adenoviral vector containing a two-component, inflammation-inducible promoter controlling the expression of human IL-10 (hIL-10) cDNA. When tested in vitro, this system had a low-level basal activity and was activated four to five orders of magnitude by various inflammatory stimuli, including TNF-alpha, IL-1 beta, IL-6, and LPS. When introduced in joints of rats with recurrent streptococcal cell wall-induced arthritis, the IL-10 transgene was induced in parallel with disease recurrence and effectively prevented the influx of inflammatory cells and the associated swelling of the joints. Levels of inflammation-inducible hIL-10 protein within the joints correlated closely with the severity of recurrence. An endogenously regulated therapeutic transgene can thus establish negative feedback and restore homeostasis in vivo while minimizing host exposure to the recombinant drug.
Assuntos
Artrite Experimental/terapia , Terapia Genética , Homeostase , Interleucina-10/genética , Transgenes , Adenoviridae/genética , Animais , Artrite Experimental/genética , Células Cultivadas , Citocinas/farmacologia , Modelos Animais de Doenças , Feminino , Fibroblastos , Vetores Genéticos , Humanos , Interleucina-10/metabolismo , Regiões Promotoras Genéticas , Ratos , Ratos Endogâmicos LewRESUMO
The transcription factor NF-kappa B has been well recognized as a pivotal regulator of inflammation in rheumatoid arthritis (RA), but recent developments revealed a broad involvement of NF-kappa B in other aspects of RA pathology, including development of T helper 1 responses, activation, abnormal apoptosis and proliferation of RA fibroblast-like synovial cells, and differentiation and activation of bone resorbing activity of osteoclasts. In agreement with this, studies in animal models of RA have demonstrated the high therapeutic efficacy of specific inhibitors of NF-kappa B pathway, indicating the feasibility of anti-NF-kappa B therapy for human disease.
Assuntos
Artrite Reumatoide/metabolismo , NF-kappa B/metabolismo , Sinovite/metabolismo , Animais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Humanos , Hiperplasia , Articulações/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Sinovite/genética , Sinovite/patologiaRESUMO
The family of nuclear factor kappaB (NF-kappaB) transcription factors is a topic of intense interest in the biomedical community stemming from the role NF-kappaB plays in almost every aspect of cell regulation: stress responses, immune cell activation, apoptosis, proliferation, differentiation and oncogenic transformation. The objective of this article is to provide an overview of recent developments in the field with an emphasis on the role of NF-kappaB in chronic inflammation, and to discuss the feasibility of therapeutic approaches based on the specific suppression of the NF-kappaB pathway.
Assuntos
Inflamação/tratamento farmacológico , NF-kappa B/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Doença Crônica , Modelos Animais de Doenças , Deleção de Genes , Humanos , Proteínas I-kappa B/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Th1/imunologia , Ativação Transcricional/efeitos dos fármacosRESUMO
The mechanisms of cell proliferation and transformation are intrinsically linked to the process of apoptosis: the default of proliferating cells is to die unless specific survival signals are provided. Platelet-derived growth factor (PDGF) is a principal survival factor that inhibits apoptosis and promotes proliferation, but the mechanisms mediating its anti-apoptotic properties are not completely understood. Here we show that the transcription factor NF-kappaB is important in PDGF signalling. NF-kappaB transmits two signals: one is required for the induction of proto-oncogene c-myc and proliferation, and the second, an anti-apoptotic signal, counterbalances c-Myc cytotoxicity. We have traced a putative pathway whereby PDGF activates NF-kappaB through Ras and phospatidylinositol-3-kinase (PI(3)K) to the PKB/Akt protein kinase and the IkappaB kinase (IKK); NF-kappaB thus appears to be a target of the anti-apoptotic Ras/PI(3)K/Akt pathway. We show that, upon PDGF stimulation, Akt transiently associates in vivo with IKK and induces IKK activation. These findings establish a role for NF-kappaB in growth factor signalling and define an anti-apoptotic Ras/PI(3)K/Akt/IKK/NF-kappaB pathway, thus linking anti-apoptotic signalling with transcription machinery.
Assuntos
Apoptose , NF-kappa B/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Células 3T3 , Animais , Catálise , Divisão Celular/fisiologia , Células Cultivadas , DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Quinase I-kappa B , Camundongos , Mitógenos/metabolismo , Mitógenos/fisiologia , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas ras/metabolismoRESUMO
The transcription factor NF-kappaB is a pivotal regulator of inflammatory responses. While the activation of NF-kappaB in the arthritic joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role of NF-kappaB in animal models of RA. We demonstrate that in vitro, NF-kappaB controlled expression of numerous inflammatory molecules in synoviocytes and protected cells against tumor necrosis factor alpha (TNFalpha) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-kappaB was found to be activated in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis. In vivo suppression of NF-kappaB by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IkappaBalpha profoundly enhanced apoptosis in the synovium of rats with SCW- and pristane-induced arthritis. This indicated that the activation of NF-kappaB protected the cells in the synovium against apoptosis and thus provided the potential link between inflammation and hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly, the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic effects of local suppression of NF-kappaB. These results establish a mechanism regulating apoptosis in the arthritic joint and indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-kappaB.
Assuntos
Apoptose/genética , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Articulações/imunologia , Articulações/patologia , NF-kappa B/genética , Animais , Apoptose/imunologia , Artrite Reumatoide/genética , Modelos Animais de Doenças , Humanos , Hiperplasia/genética , Inflamação/genética , NF-kappa B/imunologia , RatosRESUMO
One of the most challenging issues of anti-inflammatory gene therapy is the complexity of inflammatory pathways. Transcription factor NF-kappa B plays a pivotal role in activation of multiple inflammatory molecules, and therefore represents the logical target for intervention. We evaluated the feasibility of suppressing the inflammatory responses in different cell lines through specific inhibition of NF-kappa B by gene transfer of I kappa B alpha, the naturally occurring intracellular inhibitor of NF-kappa B. The I kappa B alpha overexpressing cells were established using retroviral gene transfer or stable transfection with the wild-type (wt) I kappa B alpha cDNA. In all cell types, overexpression of wt I kappa B alpha resulted in a profound (> 100-fold) increase of the I kappa B alpha message and a moderate (two- to three-fold) increase of the I kappa B alpha protein. The effects of the I kappa B alpha overexpression on the NF-kappa B activation and the inflammatory responses varied significantly in different cell lines. In conditionally immortalized human endometrial stromal cells, overexpression of I kappa B alpha prevented both interleukin-1 (IL-1)-inducible degradation of endogenous I kappa B alpha protein and activation of NF-kappa B. Accordingly, induction of cytokines interleukin-8 (IL-8) and Gro gamma was markedly suppressed. In monocytic THP-1 cells, both lipopolysaccharide (LPS)-inducible degradation of I kappa B alpha and NF-kappa B activation were only partially inhibited by overexpression of exogenous I kappa B alpha cDNA. None the less, the LPS-induced transcription of IL-1 beta and secretion of cytokines interleukin-6 (IL-6) and IL-8 were virtually abolished. In epithelial HT-29 cells, no inflammatory responses were inhibited. These results demonstrate the range of responses in various cell lines to gene transfer of I kappa B alpha and indicate the feasibility of suppression of inflammatory responses in appropriate target cells and their progeny by suppression of NF-kappa B.
Assuntos
DNA Complementar , Proteínas de Ligação a DNA/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Proteínas I-kappa B , NF-kappa B/antagonistas & inibidores , Linhagem Celular , Endométrio/imunologia , Feminino , Expressão Gênica , Vetores Genéticos , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Inibidor de NF-kappaB alfa , RetroviridaeRESUMO
Restoration of the impaired balance between pro- and antiinflammatory cytokines should provide effective treatment of rheumatoid arthritis. Gene therapy has been proposed as an approach for delivery of therapeutic proteins to arthritic joints. Here, we examined the efficacy of antiinflammatory gene therapy in bacterial cell wall-induced arthritis in rats. Human secreted interleukin 1 receptor antagonist (sIL-1ra) was expressed in joints of rats with recurrent bacterial cell wall-induced arthritis by using ex vivo gene transfer. To achieve this, primary synoviocytes were transduced in culture with a retroviral vector carrying the sIL-1ra cDNA. Transduced cells were engrafted in ankle joints of animals prior to reactivation of arthritis. Animals in control groups were engrafted with synoviocytes transduced with lacZ and neo marker genes. Cells continued to express transferred genes for at least 9 days after engraftment. We found that gene transfer of sIL-1ra significantly suppressed the severity of recurrence of arthritis, as assessed by measuring joint swelling and by the gross-observation score, and attenuated but did not abolish erosion of cartilage and bone. The effect of intraarticularly expressed sIL-1ra was essentially local, as there was no significant difference in severity of recurrence between unengrafted contralateral joints in control and experimental groups. We estimate that locally expressed sIL-1ra was about four orders of magnitude more therapeutically efficient than systemically administered recombinant sIL-1ra protein. These findings provide experimental evidence for the feasibility of antiinflammatory gene therapy for arthritis.
Assuntos
Artrite Experimental/terapia , Sialoglicoproteínas/administração & dosagem , Animais , Artrite Experimental/patologia , Sequência de Bases , DNA Complementar/genética , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Ratos , Ratos Endogâmicos Lew , Sialoglicoproteínas/genética , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Membrana Sinovial/patologiaRESUMO
The epithelium-associated tissue distribution of the intracellular IL-1R antagonist (icIL-1Ra) suggests that it functions as a novel regulatory molecule for IL-1 in somatic tissues. We examined the role of the icIL-1Ra in IL-1 beta-induced responses in human ovarian cancer cells because ovarian surface epithelium expresses transcripts for the icIL-1Ra, and the majority of ovarian cancers arise from these cells. Several human ovarian and cervical cancer cell lines spontaneously express the icIL-1Ra. icIL-1Ra-expressing cells did not have altered growth characteristics or altered short term responses to IL-1 compared with icIL-1Ra-nonexpressing cells. While a 90-min exposure to IL-1 beta resulted in increased steady state cytokine mRNA levels in all cells, icIL-1Ra-positive cells were incapable of maintaining IL-1-beta-induced expression of GRO mRNA. This did not result from decreased transcriptional activity of the GRO gene, but reflected differences in mRNA stability and/or degradation. To determine whether the icIL-1Ra altered mRNA stability, we used a retroviral expression vector to express the icIL-1Ra in an icIL-1Ra-negative cell line. The resulting cells displayed a profile of IL-1 beta-induced genes analogous to that found in cells spontaneously expressing icIL-1Ra. These data show for the first time an intrinsic biologic activity for the icIL-1Ra. The capacity to selectively alter IL-1-induced gene expression suggests that this version of the IL-1Ra is a unique intracellular inhibitor that attenuates IL-1 responses at a point downstream of the initial IL-1/IL-1 receptor interaction.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Sialoglicoproteínas/farmacologia , Transdução de Sinais/genética , Feminino , Genes Precoces/efeitos dos fármacos , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/virologia , RNA Mensageiro/efeitos dos fármacos , Retroviridae/genética , Sialoglicoproteínas/genética , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologiaRESUMO
Gene therapy may provide an effective alternative to conventional approaches for treating rheumatoid arthritis. Direct in vivo gene delivery to synovium has a distinct advantage with respect to clinical use. To date, retroviral vectors are the best studied constructs for gene delivery, and almost all approved gene therapy trials in humans rely on retroviral vectors. However, the applicability of retroviral transduction is limited by requirement for cell division, and attempts to transduce normal synovium in situ using retroviral vectors are reported to fail. The present study was undertaken in order to investigate susceptibility of inflamed synovium to retroviral infection in vivo. Using an experimental model of bacterial cell wall (BCW)-induced arthritis in rats, we attempted two approaches for delivery of retroviral vectors to synovium. In the first approach, recombinant retroviral vectors carrying reporter genes lacZ and neo were directly injected into inflamed rat ankle joints. Alternatively, inflamed joints were inoculated with gamma-irradiated murine retroviral vector-producing packaging cells. We found that about 1% of cells in explants from joints inoculated with packaging cells were lacZ-neo-positive. The lacZ+ neo+ cells in joint explants proliferated in culture and were of rat origin as determined using species-specific polymerase chain reaction (PCR) analysis. There was no evidence of transduction in explants from joints directly injected with retroviral vectors or from contralateral, control joints. These findings show that arthritic joints have a population of cells susceptible to retroviral infection in situ and demonstrate the possibility of using retroviral vectors for direct gene delivery to inflamed synovium.