RESUMEN
Asthma is a major cause of morbidity worldwide with prevalence and severity still increasing at an alarming pace. Hallmarks of this disease include early-phase bronchoconstriction with subsequent eosinophil infiltration, symptoms that may be mimicked in vivo by the complement-derived C3a anaphylatoxin, following its interaction with the single-copy C3aR. We analyzed the pathophysiological role of the C3a anaphylatoxin in a model of experimental OVA-induced allergic asthma, using an inbred guinea pig strain phenotypically unresponsive to C3a. Molecular analysis of this defect revealed a point mutation within the coding region of the C3aR that creates a stop codon, thereby effectively inactivating gene function. When challenged by OVA inhalation, sensitized animals of this strain exhibited a bronchoconstriction decreased by approximately 30% in comparison to the corresponding wild-type strain. These data suggest an important role of C3a in the pathogenesis of asthma and define a novel target for drug intervention strategies.
Asunto(s)
Asma/inmunología , Broncoconstricción/inmunología , Complemento C3a/fisiología , Proteínas de la Membrana , Receptores de Complemento/deficiencia , Administración por Inhalación , Resistencia de las Vías Respiratorias/genética , Resistencia de las Vías Respiratorias/inmunología , Animales , Asma/etiología , Asma/patología , Línea Celular , Movimiento Celular/inmunología , Complemento C3a/metabolismo , Eosinófilos/patología , Regulación de la Expresión Génica/inmunología , Marcadores Genéticos/inmunología , Cobayas , Humanos , Inyecciones Intraperitoneales , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Mutación Puntual/inmunología , Receptores de Complemento/antagonistas & inhibidores , Receptores de Complemento/genética , Especificidad de la Especie , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Live Ixodes ricinus ticks attached to humans residing in Germany were examined for borreliae by dark-field microscopy and PCR. Borrelia species were identified by 16S rRNA sequence analysis, which showed the presence of several species, some not yet defined, and a high prevalence of multiply infected ticks.
Asunto(s)
Técnicas de Tipificación Bacteriana , Grupo Borrelia Burgdorferi/clasificación , Grupo Borrelia Burgdorferi/genética , Ixodes/microbiología , Enfermedad de Lyme/microbiología , Reacción en Cadena de la Polimerasa/métodos , Piel/microbiología , Piel/parasitología , Animales , Vectores Arácnidos/microbiología , Secuencia de Bases , Grupo Borrelia Burgdorferi/aislamiento & purificación , Cartilla de ADN/genética , Estudios de Evaluación como Asunto , Alemania/epidemiología , Humanos , Enfermedad de Lyme/epidemiología , Enfermedad de Lyme/transmisión , Epidemiología Molecular , Datos de Secuencia Molecular , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
The interaction of human anaphylatoxin C4a with the guinea pig (gp) and human (hu) C3a receptors (C3aR) was analyzed using human rC4a, which exhibited C4a-specific activity on guinea pig platelets. A gpC3aR of 475 residues with a large second extracellular loop and a peptide sequence approximately 60% identical to the huC3aR was isolated from a genomic DNA library and found to be expressed in guinea pig heart, lung, and spleen. HEK-293 cells cotransfected with this clone, and a cDNA encoding G alpha-16 specifically bound (Kd = 1.6+/-0.7 nM) and responded functionally to C3a with an intracellular calcium mobilization (ED50 = 0.18+/-0.02 nM). Human rC4a weakly bound to both the hu- and gpC3aR (IC50 > 1 microM). However, only HEK-293 cells expressing the gpC3aR responded functionally to rC4a (ED50 = 8.7+/-0.52 nM), while cells expressing the huC3aR did not (c < or = 1 microM). Thus, through an interaction with the C3aR, huC4a may elicit anaphylatoxic effects in guinea pigs but not in man.
Asunto(s)
Complemento C4a/agonistas , Proteínas de la Membrana , Receptores de Complemento/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Complemento C4a/genética , Cobayas , Humanos , Riñón , Datos de Secuencia Molecular , Activación Plaquetaria/efectos de los fármacos , Activación Plaquetaria/inmunología , Receptores de Complemento/química , Receptores de Complemento/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologíaRESUMEN
The human C3a receptor (C3aR) mediates the activation of cells by the potent proinflammatory chemoattractant C3a, an anaphylatoxin, generated in the early phase of an inflammatory reaction by proteolytic cleavage of the complement component C3. To understand the molecular mechanisms that regulate C3aR gene expression, we initiated studies to determine its genomic and mRNA organization. We now report the following novel findings: (1) The C3aR is a single-copy gene as shown by Southern hybridization of human genomic DNA. (2) Using PCR amplification of DNA from monochromosomal somatic cell hybrid and radiation hybrid panels, the C3aR locus was mapped to chromosome 12p13. (3) Genomic DNA clones encompassing the C3aR locus were isolated from a human genomic DNA library and characterized by restriction mapping, Southern blotting, PCR analysis and DNA sequencing. Comparison of the genomic with the known cDNA sequences revealed a single 6-kb intron sequence located 1 1 bp upstream of the ATG initiation codon. The open reading frame and the complete 3' untranslated region are encoded on a single exon.
Asunto(s)
Complemento C3a/metabolismo , Proteínas de la Membrana , Receptores de Complemento/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 12/genética , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Exones , Regulación de la Expresión Génica , Biblioteca de Genes , Humanos , Intrones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN Mensajero/genéticaRESUMEN
Recent cloning of the human C3a receptor (C3aR) revealed that this receptor belongs to the large family of rhodopsin-type receptors. A unique feature of the C3aR is the large second extracellular loop comprising about 175 amino acid residues. We constructed combinatorial phage Ab libraries expressing single chain Fv Abs from BALB/c mice immunized with the affinity-purified second extracellular loop of the C3aR, fused to glutathione-S-transferase. A panel of anti-C3aR single chain Fv fragments (scFvs) was selected after four rounds of panning using the second extracellular loop of the C3aR, fused to the maltose binding protein as Ag. Sequencing of the clones obtained revealed three different groups of scFvs, the epitopes of which were mapped to two distinct regions within the loop, i.e., positions 185 to 193 and 218 to 226, representing the immunodominant domains of the loop. By flow cyotmetric analyses, the scFvs bound to RBL-2H3 cells transfected with the C3aR, but not to cells transfected with the C5aR or to nontransfected RBL-2H3 cells. In addition, the scFvs bound to the human mast cell line HMC-1. Immunofluorescence studies showed C3aR expression on polymorphonuclear granulocytes and monocytes, but not on lymphocytes. In addition, no C3aR expression was observed on human erythrocytes or platelets. Surprisingly, none of the scFvs alone or in combination inhibited C3a-induced Ca2+ mobilization from RBL-2H3 cells transfected with the C3aR. In addition, C3a did not displace binding of the scFvs to the receptor, strongly suggesting that the N-terminal part of the second extracellular loop is not involved in ligand binding.