RESUMO
To identify rate-limiting steps in T cell-independent type 2 antibody production against polysaccharide antigens, we performed a genome-wide screen by immunizing several hundred pedigrees of C57BL/6 mice segregating N-ethyl-N-nitrosurea-induced mis-sense mutations. Two independent mutations, Tilcara and Untied, were isolated that semi-dominantly diminished antibody against polysaccharide but not protein antigens. Both mutations resulted from single-amino-acid substitutions within the kinase domain of protein kinase C-ß (PKCß). In Tilcara, a Ser552>Pro mutation occurred in helix G, in close proximity to a docking site for the inhibitory N-terminal pseudosubstrate domain of the enzyme, resulting in almost complete loss of active, autophosphorylated PKCßI, whereas the amount of alternatively spliced PKCßII protein was not markedly reduced. Circulating B cell subsets were normal and acute responses to B-cell receptor stimulation such as CD25 induction and initiation of DNA synthesis were only measurably diminished in Tilcara homozygotes, whereas the fraction of cells that had divided multiple times was decreased to an intermediate degree in heterozygotes. These results, coupled with evidence of numerous mis-sense PRKCB mutations in the human genome, identify Prkcb as a genetically sensitive step likely to contribute substantially to population variability in anti-polysaccharide antibody levels.
Assuntos
Heterozigoto , Imunoglobulinas/biossíntese , Mutação de Sentido Incorreto , Proteína Quinase C beta/genética , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Sítios de Ligação , Genoma , Imunoglobulinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linhagem , Proteína Quinase C beta/químicaRESUMO
Accurate identification of sparse heterozygous single-nucleotide variants (SNVs) is a critical challenge for identifying the causative mutations in mouse genetic screens, human genetic diseases and cancer. When seeking to identify causal DNA variants that occur at such low rates, they are overwhelmed by false-positive calls that arise from a range of technical and biological sources. We describe a strategy using whole-exome capture, massively parallel DNA sequencing and computational analysis, which identifies with a low false-positive rate the majority of heterozygous and homozygous SNVs arising de novo with a frequency of one nucleotide substitution per megabase in progeny of N-ethyl-N-nitrosourea (ENU)-mutated C57BL/6j mice. We found that by applying a strategy of filtering raw SNV calls against known and platform-specific variants we could call true SNVs with a false-positive rate of 19.4 per cent and an estimated false-negative rate of 21.3 per cent. These error rates are small enough to enable calling a causative mutation from both homozygous and heterozygous candidate mutation lists with little or no further experimental validation. The efficacy of this approach is demonstrated by identifying the causative mutation in the Ptprc gene in a lymphocyte-deficient strain and in 11 other strains with immune disorders or obesity, without the need for meiotic mapping. Exome sequencing of first-generation mutant mice revealed hundreds of unphenotyped protein-changing mutations, 52 per cent of which are predicted to be deleterious, which now become available for breeding and experimental analysis. We show that exome sequencing data alone are sufficient to identify induced mutations. This approach transforms genetic screens in mice, establishes a general strategy for analysing rare DNA variants and opens up a large new source for experimental models of human disease.
Assuntos
Análise Mutacional de DNA , Modelos Animais de Doenças , Exoma , Camundongos Endogâmicos C57BL/genética , Camundongos Mutantes/genética , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA , Animais , Cruzamentos Genéticos , Etilnitrosoureia , Feminino , Genes Recessivos , Heterozigoto , Homozigoto , Endogamia , Antígenos Comuns de Leucócito/genética , Masculino , Camundongos , MutagêneseRESUMO
Protein energy malnutrition (PEM) is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group) were submitted to PEM with a low-protein diet (4 percent) or were fed a control diet (20 percent protein) ad libitum. When the experimental group had lost about 20 percent of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.
Assuntos
Animais , Masculino , Camundongos , Células da Medula Óssea/fisiologia , Proliferação de Células , Fase de Repouso do Ciclo Celular/fisiologia , Fase G1/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Desnutrição Proteico-Calórica/fisiopatologia , Ensaio de Unidades Formadoras de Colônias , Ciclo Celular/fisiologia , Citometria de Fluxo , Fluoruracila , Desnutrição Proteico-Calórica/sangueRESUMO
Protein energy malnutrition (PEM) is a syndrome that often results in immunodeficiency coupled with pancytopenia. Hemopoietic tissue requires a high nutrient supply and the proliferation, differentiation and maturation of cells occur in a constant and balanced manner, sensitive to the demands of specific cell lineages and dependent on the stem cell population. In the present study, we evaluated the effect of PEM on some aspects of hemopoiesis, analyzing the cell cycle of bone marrow cells and the percentage of progenitor cells in the bone marrow. Two-month-old male Swiss mice (N = 7-9 per group) were submitted to PEM with a low-protein diet (4%) or were fed a control diet (20% protein) ad libitum. When the experimental group had lost about 20% of their original body weight after 14 days, we collected blood and bone marrow cells to determine the percentage of progenitor cells and the number of cells in each phase of the cell cycle. Animals of both groups were stimulated with 5-fluorouracil. Blood analysis, bone marrow cell composition and cell cycle evaluation was performed after 10 days. Malnourished animals presented anemia, reticulocytopenia and leukopenia. Their bone marrow was hypocellular and depleted of progenitor cells. Malnourished animals also presented more cells than normal in phases G0 and G1 of the cell cycle. Thus, we conclude that PEM leads to the depletion of progenitor hemopoietic populations and changes in cellular development. We suggest that these changes are some of the primary causes of pancytopenia in cases of PEM.
Assuntos
Células da Medula Óssea/fisiologia , Proliferação de Células , Fase G1/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Desnutrição Proteico-Calórica/fisiopatologia , Fase de Repouso do Ciclo Celular/fisiologia , Animais , Ciclo Celular/fisiologia , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Fluoruracila , Masculino , Camundongos , Desnutrição Proteico-Calórica/sangueRESUMO
The identification of the TLRs as key sensors of microbial infection has presented a series of new targets for drug development. The TLRs are linked to the most powerful inflammatory pathways in mammals. The question arises from the start: do we wish to stimulate TLR signaling in order to eradicate specific infections and/or neoplastic diseases? Or do we wish to block TLR signaling to treat inflammatory diseases? If we accept that it would be useful to modulate TLR signaling, the next step is to identify the correct molecular target(s) for the task. Perhaps it might even be possible to exercise selectivity, modulating some aspects of TLR signaling and not others. Classical and reverse genetic analyses offer insight into the possibilities that exist, and point to specific checkpoints within signaling pathways at which modulation might normally be imposed.
Assuntos
Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/agonistas , Receptores Toll-Like/antagonistas & inibidores , Infecções Bacterianas/tratamento farmacológico , Receptores Toll-Like/imunologiaRESUMO
With the mouse genome almost entirely sequenced and readily accessible to all who wish to examine it, the challenge across most biological disciplines now lies in the decipherment of gene and protein function rather than in the realm of gene identification per se. In the field of innate immunity, forward genetic methods have repeatedly been applied to identify key sensors, adapters, and effector molecules. However, most spontaneous mutations that affect innate immune function have been mapped and cloned, and the need for new monogenic phenotypes has been felt evermore keenly. N-Ethyl-N-nitrosourea (ENU) mutagenesis is an efficient tool for the creation of aberrant monogenic innate immune response phenotypes. In this review, we will discuss the potential of the forward genetic approach and ENU mutagenesis to identify new genes and new functions of known genes related to innate immunity.
Assuntos
Técnicas Genéticas , Imunidade Inata/genética , Mutagênese , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/fisiologia , Antígenos CD36/genética , Antígenos CD36/fisiologia , Etilnitrosoureia/farmacologia , Marcação de Genes , Genes Recessivos , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Modelos Imunológicos , Mutagênicos/farmacologia , Fator 88 de Diferenciação Mieloide , Fenótipo , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos/genética , Receptores Imunológicos/fisiologia , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/fisiologia , Receptores Toll-LikeRESUMO
Macrophages and dendritic cells are in the front line of host defense. When they sense host invasion, they produce cytokines that alert other innate immune cells and also abet the development of an adaptive immune response. Although lipolysaccharide (LPS), peptidoglycan, unmethylated DNA, and other microbial products were long known to be the primary targets of innate immune recognition, there was puzzlement as to how each molecule triggered a response. It is now known that the Toll-like receptors (TLRs) are the principal signaling molecules through which mammals sense infection. Each TLR recognizes a restricted subset of molecules produced by microbes, and in some circumstances, only a single type of molecule is sensed (e.g., only LPS is sensed by TLR4). TLRs direct the activation of immune cells near to and far from the site of infection, mobilizing the comparatively vast immune resources of the host to confine and defeat an invasive organism before it has become widespread. The biochemical details of TLR signaling have been analyzed through forward and reverse genetic methods, and full elucidation of the molecular interactions that transpire within the first minutes following contact between host and pathogen will soon be at hand.
Assuntos
Imunidade Inata , Infecções/imunologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Antígenos de Diferenciação/fisiologia , Humanos , Inflamação/imunologia , Lipopolissacarídeos/toxicidade , Fator 88 de Diferenciação Mieloide , Receptores Imunológicos/fisiologia , Sepse/imunologia , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
In humans, ten Toll-like receptor (TLR) paralogues sense molecular components of microbes, initiating the production of cytokine mediators that create the inflammatory response. Using N-ethyl-N-nitrosourea, we induced a germline mutation called Lps2, which abolishes cytokine responses to double-stranded RNA and severely impairs responses to the endotoxin lipopolysaccharide (LPS), indicating that TLR3 and TLR4 might share a specific, proximal transducer. Here we identify the Lps2 mutation: a distal frameshift error in a Toll/interleukin-1 receptor/resistance (TIR) adaptor protein known as Trif or Ticam-1. Trif(Lps2) homozygotes are markedly resistant to the toxic effects of LPS, and are hypersusceptible to mouse cytomegalovirus, failing to produce type I interferons when infected. Compound homozygosity for mutations at Trif and MyD88 (a cytoplasmic TIR-domain-containing adaptor protein) loci ablates all responses to LPS, indicating that only two signalling pathways emanate from the LPS receptor. However, a Trif-independent cell population is detectable when Trif(Lps2) mutant macrophages are stimulated with LPS. This reveals that an alternative MyD88-dependent 'adaptor X' pathway is present in some, but not all, macrophages, and implies afferent immune specialization.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antígenos de Diferenciação/fisiologia , Lipopolissacarídeos/farmacologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Antígenos de Diferenciação/genética , Escherichia coli/fisiologia , Homozigoto , Interferon Tipo I/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Macrófagos Peritoneais/virologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fator 88 de Diferenciação Mieloide , Fenótipo , Mapeamento Físico do Cromossomo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/genética , Análise de Sequência de DNA , Especificidade por Substrato , Receptor 3 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/metabolismo , Vaccinia virus/fisiologiaAssuntos
Apoptose , Autoimunidade , Proteínas de Transporte , Doença de Crohn/etiologia , Proteínas de Drosophila , Peptídeos e Proteínas de Sinalização Intracelular , Animais , Fator Apoptótico 1 Ativador de Proteases , Humanos , Glicoproteínas de Membrana/genética , Proteína Adaptadora de Sinalização NOD2 , Proteínas/genética , Proteínas/fisiologia , Receptores de Superfície Celular/genética , Receptores Toll-LikeRESUMO
OBJECTIVE: To review the role of the Toll-like receptors (TLR) as the principal sensors used by the innate immune system in the context of the pathologic processes underlying sepsis and septic shock. DATA SOURCES: Literature review. DATA SUMMARY: Through the Toll-like receptors, macrophages and other defensive cells "see" endotoxin (TLR4), peptidoglycan (TLR2), and bacterial DNA (TLR9). Representatives of the family predated the divergence of plants and animals and, at that time, had already acquired a defensive function. The strengths and liabilities of the innate immune system, which defends against infection and which also may cause shock and death, are rooted in its ancient origins. In the current era of shock research, the nature of the signals that Toll-like receptors transduce and the effects of genetic variation on microbial sensing are two major challenges.
Assuntos
Evolução Biológica , Proteínas de Drosophila , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Sepse/imunologia , Choque Séptico/imunologia , Animais , Anticorpos/imunologia , Antígenos de Superfície/imunologia , DNA Bacteriano/imunologia , Variação Genética/genética , Variação Genética/imunologia , Humanos , Antígeno 96 de Linfócito , Linfócitos/imunologia , Macrófagos/imunologia , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Peptidoglicano/imunologia , Plantas/imunologia , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Sepse/microbiologia , Choque Séptico/microbiologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptor Toll-Like 9 , Receptores Toll-LikeRESUMO
Monospecific, affinity-purified polyclonal antibodies reacting with the amino-terminal half of the mouse Toll-like receptor 4 (Tlr4) ectodomain failed to block LPS effects and, to the contrary, were capable of inducing TNF synthesis when applied to mouse macrophages and cross-linked with a secondary antibody. This effect was observed with macrophages derived from C3H/HeN and C57BL/10ScSn mice, but not with macrophages derived from C3H/HeJ or C57BL/10ScCr mice, indicating a specific, Tlr4-dependent effect. Neither primary nor secondary antibody caused any response if administered in the absence of the other reagent, nor was any response observed in cells from mice lacking Tlr4, or bearing the Lps(d) mutation of Tlr4. These findings support several conclusions. Tlr4, the essential transducer of LPS responses, may act independently of LPS itself. LPS needs not be internalized, nor must it bind to a secondary target within the cell in order to exert its effect; rather, the receptor alone is required for initiation of a signal. The data are consistent with the hypothesis that a conformational change in Tlr4 is required for activation via this receptor, and reveal that the amino-terminal half of the Tlr4 ectodomain is a target sufficient for antibody-mediated activation.
Assuntos
Anticorpos/farmacologia , Proteínas de Drosophila , Endotoxinas/antagonistas & inibidores , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Animais , Linhagem Celular , Células Cultivadas , Endotoxinas/farmacologia , Macrófagos Peritoneais , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mimetismo Molecular , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Proteínas Recombinantes/imunologia , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/imunologiaRESUMO
In mice (and by inference, in all mammals), a single pathway exists to serve lipopolysaccharide (LPS) signal transduction, and as such, allelic mutations at a single locus entirely abolish responses to LPS in C3H/HeJ and C57BL/10ScCr mice. Positional cloning of this locus, known as Lps, revealed that mutations of the Toll-like receptor 4 gene (Tlr4) are responsible for endotoxin resistance. A quick succession of studies have shown Tlr4 to be the critical transmembrane component of the LPS signal transduction complex. As LPS sensing by Tlr4 depends on physical contact between the two molecules, Tlr4 is a direct interface with the microbial world. Eight other molecules with strong similarity to Tlr4 are presently known in mammals, and taking Tlr4 as a model, all may be guessed to participate in the early detection of invasive pathogens. Acting together, the Toll-like receptors may be assumed to present macrophages with a comprehensive "picture" of the micobial world, and thus comprise the principal sensing molecules utilized by cells of the innate immune system.
Assuntos
Proteínas de Drosophila , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Animais , Evolução Biológica , Clonagem Molecular , Humanos , Imunidade Inata/genética , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mutação , Polimorfismo Genético , Transdução de Sinais , Receptor 4 Toll-Like , Receptores Toll-Like , Proteína ran de Ligação ao GTPRESUMO
The protein product of the Toll-like receptor (TLR) 4 gene has been implicated in the signal transduction events induced by lipopolysaccharide (LPS). In mice, destructive mutations of Tlr4 impede the normal response to LPS and cause a high susceptibility to Gram-negative infection. Expression of TLR4 mRNA in humans is restricted to a small number of cell types, including LPS-responsive myeloid cells, B-cells, and endothelial cells. To investigate the molecular basis for TLR4 expression in cells of myeloid origin, we cloned the human TLR4 gene and analyzed its putative 5'-proximal promoter. In transient transfections a region of only 75 base pairs upstream of the major transcription initiation site was sufficient to induce maximal luciferase activity in THP-1 cells. The sequence of this region is similar in human and mouse TLR4 genes and lacks a TATA box, typical Sp1-sites or CCAAT box sequences. Instead, it contains consensus-binding sites for Ets family transcription factors, octamer-binding factors, and a composite interferon response factor/Ets motif. The activity of the promoter in macrophages was strictly dependent on the integrity of both half sites of the composite interferon response factor/Ets motif, which was constitutively bound by the myeloid and B-cell-specific transcription factor PU.1 and interferon consensus sequence-binding protein. These results indicate that the two tissue-restricted transcription factors PU.1 and interferon consensus sequence-binding protein participate in the basal regulation of human TLR4 in myeloid cells. Cloning of the human TLR4 gene provides a basis for further investigation of the possible impact of genetic variations on the susceptibility to infection and sepsis.
Assuntos
Medula Óssea/metabolismo , Proteínas de Drosophila , Regulação da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptores de Superfície Celular/genética , Proteínas Repressoras/fisiologia , Transativadores/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA , Humanos , Fatores Reguladores de Interferon , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Homologia de Sequência do Ácido Nucleico , Receptor 4 Toll-Like , Receptores Toll-Like , Transativadores/metabolismoRESUMO
Some mammalian species show an ability to discriminate between different lipopolysaccharide (LPS) partial structures (for example, lipid A and its congener LA-14-PP, which lacks secondary acyl chains), whereas others do not. Using a novel genetic complementation system involving the transduction of immortalized macrophages from genetically unresponsive C3H/HeJ mice, we now have shown that the species-dependent discrimination between intact LPS and tetra-acyl LPS partial structures is fully attributable to the species origin of Toll-like receptor 4 (Tlr4), an essential membrane-spanning component of the mammalian LPS sensor. Because Tlr4 interprets the chemical structure of an LPS molecule, we conclude that LPS must achieve close physical proximity with Tlr4 in the course of signal transduction.
Assuntos
Proteínas de Drosophila , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Biomarcadores , Células Cultivadas , Teste de Complementação Genética , Humanos , Lipídeo A/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Receptores de Superfície Celular/genética , Receptor 4 Toll-Like , Receptores Toll-LikeRESUMO
The vast and growing array of cytokines is the subject of intense research for their potential to ameliorate a range of diseases that extends from autoimmune disorders to cancer and beyond. Among the cytokines, tumor necrosis factor-alpha (TNF-alpha) has proven to be a key ligand in triggering many intracellular processes, both physiological and pathological. Understanding its role in rheumatoid arthritis and Crohn's disease has produced effective new therapeutic agents, and there is reason to expect greater success as research proceeds. New synthetic macromolecules are effectively interfering with TNF and other ligands at and before the cellular membrane interface. This article reviews current knowledge of the molecular mechanics of TNF and the therapeutic inhibition of TNF action. Unraveling these processes has led to many insights into cytokine physiology and pathology.
Assuntos
Artrite Reumatoide/metabolismo , Doença de Crohn/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/patologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/patologia , Humanos , Receptores do Fator de Necrose Tumoral/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
BACKGROUND & AIMS: Transfer of T helper cells from DBA/2 mice to irradiated allogeneic B6D2F1 mice leads to development of colonic graft-versus-host disease with pathological features of inflammatory bowel disease. To examine the role of tumor necrosis factor (TNF) in graft-versus-host disease enteropathy, an adenoviral vector encoding a TNF inhibitor protein was administered. METHODS: Irradiated B6D2F1 mice were infused with DBA/2 bone marrow and spleen cells. Mice then received either a control beta-galactosidase-encoding adenovirus or an adenovirus encoding a TNF inhibitor, composed of the extracellular domain of the human 55-kilodalton TNF receptor linked to the murine immunoglobulin G1 heavy chain. Mucosal permeability to sucralose and colonic histology were assessed 14 and 25 days after transplantation. RESULTS: Less diarrhea was observed in DBA/2 --> B6D2F1 mice expressing the TNF inhibitor, and colonic sections from these mice had significantly less inflammation and epithelial cell abnormalities. In TNF inhibitor recipients, mucosal permeability to sucralose was similar to that in nonirradiated control mice and significantly less than in recipients of the control adenovirus. CONCLUSIONS: TNF inhibition decreases the severity of enteropathy in the DBA/2 --> B6D2F1 murine model of colonic graft-versus-host disease.
Assuntos
Transplante de Medula Óssea , Colo/transplante , Doença Enxerto-Hospedeiro/terapia , Transfusão de Linfócitos , Receptores do Fator de Necrose Tumoral/biossíntese , Linfócitos T Auxiliares-Indutores/transplante , Transplante Homólogo/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Colo/fisiologia , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Terapia de Imunossupressão/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/biossíntese , Baço/imunologia , Sacarose/análogos & derivados , Sacarose/metabolismo , Transfecção , Irradiação Corporal Total , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
In mouse macrophages (RAW 264.7 cells), toll-like receptor 4 (Tlr4) is a limiting factor in lipopolysaccharide (LPS) signal transduction. The expression of only 1-2 x 10(4) copies of recombinant Tlr4 per cell enhances sensitivity to LPS, shifting the EC50 by 30-fold to the left. Expression of the Tlr4(Lps-d) isoform of Tlr4 (found in C3H/HeJ mice) shifts the EC50 2600-fold to the right, essentially abolishing LPS responses. A truncated form of Tlr4, lacking a cytoplasmic domain, exerts only a weak inhibitory effect on signal transduction. Similarly, the normal or Tlr4(Lps-d) forms of protein lacking an ectodomain [corrected], cause modest inhibition of LPS signaling. Manipulations of Tlr4 structure and expression cause changes in LPS sensitivity that range over 3 to 4 orders of magnitude. These findings support the view that Tlr4 is an integral component of a solitary pathway for LPS signal transduction in macrophages and permit inferences related to the mechanism of signaling and its blockade.
Assuntos
Proteínas de Drosophila , Lipopolissacarídeos/metabolismo , Macrófagos/química , Glicoproteínas de Membrana/farmacologia , Glicoproteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Células L , Macrófagos/fisiologia , Glicoproteínas de Membrana/genética , Camundongos , Mutação , Estrutura Quaternária de Proteína , Receptores de Superfície Celular/genética , Receptor 4 Toll-Like , Receptores Toll-Like , Transfecção , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/efeitos dos fármacosRESUMO
Sustained antibody-mediated inhibition of tumor necrosis factor (TNF) activity offers protection against Type I (insulin-dependent) diabetes mellitus in non-obese diabetic (NOD) mice. The mechanism of this effect, however, has remained obscure: TNFalpha might be required for the development of specific immune responses to islet antigens or it could directly participate in destruction of beta cells. In this study, autoimmune destruction of beta cells was initiated in NOD-severe combined immunodeficient (scid) mice by transfer of NOD splenic T-cells to induce diabetes. The blockade of TNFalpha activity was achieved during a narrow window of time after transfer. Transient inhibition of TNFalpha greatly reduced the number of islet lymphocytes and the incidence of diabetes in recipients of prediabetic NOD spleen cells. Protection extended beyond the interval of effective TNF blockade. Furthermore, the protective effect was only observed if cells were obtained from 6-week-old donors. The suppression of autoimmunity was reversible in the context of adoptive transfer as indicated by the transfer of splenocytes from the primary recipient to a second NOD-scid host led to a diabetic outcome. The blockade of TNFalpha was accompanied by a considerable increase in spleen size and doubling of the total splenocyte count, suggesting that TNFalpha might normally eliminate a transplanted T-cell subset within the recipients. Further analysis showed an increase in the absolute count of CD4 + T cells and pronounced distortion of the CD45RBhigh to CD45RBlow ratio, with a relative augmentation in the CD45RBlow count in the spleen. TNFalpha appears to regulate the number and subtype distribution of a transplanted T cell population.
Assuntos
Transferência Adotiva , Diabetes Mellitus Tipo 1/imunologia , Antígenos Comuns de Leucócito/análise , Linfócitos/patologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Relação CD4-CD8 , Diabetes Mellitus Tipo 1/patologia , Feminino , Hiperplasia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Baço/citologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , beta-Galactosidase/genéticaRESUMO
Mutations of the gene Lps selectively impede lipopolysaccharide (LPS) signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant to endotoxin yet highly susceptible to Gram-negative infection. The codominant Lpsd allele of C3H/HeJ mice was shown to correspond to a missense mutation in the third exon of the Toll-like receptor-4 gene (Tlr4), predicted to replace proline with histidine at position 712 of the polypeptide chain. C57BL/10ScCr mice are homozygous for a null mutation of Tlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces the LPS signal across the plasma membrane. Destructive mutations of Tlr4 predispose to the development of Gram-negative sepsis, leaving most aspects of immune function intact.