RESUMEN
Infectious pathogens contribute to the development of autoimmune disorders, but the mechanisms connecting these processes are incompletely understood. Here we show that Plasmodium DNA induces autoreactive responses against erythrocytes by activating a population of B cells expressing CD11c and the transcription factor T-bet, which become major producers of autoantibodies that promote malarial anaemia. Additionally, we identify parasite DNA-sensing through Toll-like receptor 9 (TLR9) along with inflammatory cytokine receptor IFN-γ receptor (IFN-γR) as essential signals that synergize to promote the development and appearance of these autoreactive T-bet+ B cells. The lack of any of these signals ameliorates malarial anaemia during infection in a mouse model. We also identify both expansion of T-bet+ B cells and production of anti-erythrocyte antibodies in ex vivo cultures of naive human peripheral blood mononuclear cells (PBMC) exposed to P. falciprum infected erythrocyte lysates. We propose that synergistic TLR9/IFN-γR activation of T-bet+ B cells is a mechanism underlying infection-induced autoimmune-like responses.
Asunto(s)
Anemia Hemolítica Autoinmune/etiología , Anemia Hemolítica Autoinmune/inmunología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/parasitología , ADN Protozoario/inmunología , Malaria Falciparum/complicaciones , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Receptor Toll-Like 9/metabolismo , Anemia Hemolítica Autoinmune/parasitología , Animales , Autoanticuerpos/biosíntesis , Eritrocitos/inmunología , Eritrocitos/parasitología , Femenino , Humanos , Activación de Linfocitos , Malaria Falciparum/parasitología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Plasmodium falciparum/patogenicidad , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Receptor Toll-Like 9/deficiencia , Receptor Toll-Like 9/genética , Receptor de Interferón gammaRESUMEN
Reactive oxygen species (ROS) are important second messengers generated in response to many types of environmental stress. In this setting, changes in intracellular ROS can activate signal transduction pathways that influence how cells react to their environment. In sepsis, a dynamic proinflammatory cellular response to bacterial toxins (e.g. lipopolysaccharide or LPS) leads to widespread organ damage and death. The present study demonstrates for the first time that the activation of Rac1 (a GTP-binding protein), and the subsequent production of ROS, constitutes a major pathway involved in NFkappaB-mediated tumor necrosis factor-alpha (TNFalpha) secretion following LPS challenge in macrophages. Expression of a dominant negative mutant of Rac1 (N17Rac1) reduced Rac1 activation, ROS formation, NFkappaB activation, and TNFalpha secretion following LPS stimulation. In contrast, expression of a dominant active form of Rac1 (V12Rac1) mimicked these effects in the absence of LPS stimulation. IKKalpha and IKKbeta were both required downstream modulators of LPS-activated Rac1, since the expression of either of the IKK dominant mutants (IKKalphaKM or IKKbetaKA) drastically reduced NFkappaB-dependent TNFalpha secretion. Moreover, studies using CD14 blocking antibodies suggest that Rac1 induces TNFalpha secretion through a pathway independent of CD14. However, a maximum therapeutic inhibition of LPS-induced TNFalpha secretion occurred when both CD14 and Rac1 pathways were inhibited. Our results suggest that targeting both Rac1- and CD14-dependent pathways could be a useful therapeutic strategy for attenuating the proinflammatory cytokine response during the course of sepsis.
Asunto(s)
Etidio/análogos & derivados , Lipopolisacáridos/metabolismo , FN-kappa B/metabolismo , Oxidación-Reducción , Proteínas Serina-Treonina Quinasas/metabolismo , Especies Reactivas de Oxígeno , Factor de Necrosis Tumoral alfa/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Adenoviridae/genética , Animales , Western Blotting , Catalasa/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática , Etidio/farmacología , Regulación de la Expresión Génica , Genes Dominantes , Glutatión Transferasa/metabolismo , Humanos , Quinasa I-kappa B , Receptores de Lipopolisacáridos/metabolismo , Luciferasas/metabolismo , Ratones , Modelos Biológicos , Mutación , Unión Proteica , Isoformas de Proteínas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Superóxidos/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
PURPOSE: The reproducibility of functional magnetic resonance imaging (fMRI) has been studied on 1.5 Tesla (T) (high field strength) scanners. We report the reproducibility of visual activation in fMRI at 4 T (very high field strength). METHODS: Five healthy subjects were scanned twice in the same session with a 4 T scanner during binocular flashing visual stimulation. The activated areas during the first and second acquisition were compared. RESULTS: Activation of the visual cortex was observed in all subjects and activation of lateral geniculate nucleus was also detected in four subjects. The ratio of overlapping activated voxels in the first and second acquisition was 0.81 +/- 0.05. CONCLUSIONS: Reproducibility of visual activation using fMRI at 4 T was found to be acceptable, and the results from 4T scanners show a reliability similar to those at 1.5 T.
Asunto(s)
Imagen por Resonancia Magnética , Visión Binocular/fisiología , Corteza Visual/fisiología , Vías Visuales/fisiología , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Mutations in the RPGR gene at the RP3 locus have been found to cause x-linked retinitis pigmentosa in some families. OBJECTIVES: To identify a previously undescribed 2-base pair insertion in codon 99 of the RPGR gene and to describe the phenotype in a well-characterized family with X-linked retinitis pigmentosa. DESIGN: Case reports with clinical features, fluorescein angiography, kinetic perimetry, electrophysiological studies, and molecular genetics. SETTING: University medical centers. PATIENTS: Eight members of the family were screened for the codon 99 insertion in the RPGR gene. RESULTS: Three affected males were found to be hemizygous for the 2-base pair insertion; 2 carriers were heterozygous. This insertion creates a frameshift that would be expected to cause a premature arrest of translation after only 132 amino acids (683 amino acids less than the normal protein). The affected males had typical retinitis pigmentosa with visual field contraction and abnormal findings on electroretinograms with little to no rod activity, profoundly subnormal residual cone responses to single flash and 30-Hz flicker stimuli, and prolonged b-wave implicit times. The electroretinogram of a 49-year-old carrier showed amplitudes that were roughly half of normal. Carrier women did not show a tapetallike fundus reflex but showed asymmetrical patchy pigmentary disturbances consistent with lyonization. CONCLUSION: A frameshifting 2-base pair insertion at codon 99 of the RPGR gene produced typical retinitis pigmentosa and carrier findings (but no tapetallike reflex) in this family.
Asunto(s)
Proteínas Portadoras/genética , Codón/genética , Proteínas del Ojo , Ligamiento Genético/genética , Mutagénesis Insercional , Proteínas/genética , Retinitis Pigmentosa/genética , Cromosoma X/genética , Adolescente , Adulto , Análisis Mutacional de ADN , Cartilla de ADN/química , Elementos Transponibles de ADN/genética , Electrorretinografía , Femenino , Angiografía con Fluoresceína , Mutación del Sistema de Lectura , Humanos , Masculino , Persona de Mediana Edad , Linaje , Polimorfismo Conformacional Retorcido-Simple , Retina/patología , Retina/fisiopatología , Retinitis Pigmentosa/patología , Retinitis Pigmentosa/fisiopatología , Agudeza Visual , Pruebas del Campo Visual , Campos VisualesRESUMEN
PURPOSE: To evaluate the clinical and electrophysiologic findings in a family with two heterozygous sequence changes in the peripherin-retinal degeneration slow (RDS) gene. METHODS: A family study was done of a pedigree obtained by screening for rhodopsin, peripherin/RDS, or rom-1 gene mutations in probands from families with hereditary retinal diseases. The patients consisted of three affected and four unaffected members from a family with cone dystrophy. Ophthalmoscopy, visual field testing, electroretinography, and DNA analysis were performed. RESULTS: Denaturing gradient gel electrophoresis showed the presence of two different sequence changes in the RDS genes of this family. In three members with a retinal disease, the authors observed the substitution of phenylalanine for serine in codon 27 (serine-27-phenylalanine). The clinical and functional findings in these three patients were most consistent with autosomal-dominant cone dystrophy. Three other family members, unaffected with retinal disease, were found to show a substitution of serine for cysteine in codon 72 of the peripherin protein. CONCLUSION: A peripherin/RDS sequence change may produce a cone dystrophy with minimal ophthalmoscopic changes in the macula and limited peripheral degenerative changes. Caution is warranted to avoid ascribing nondisease-causing sequence polymorphisms in candidate genes as responsible for determining the development of a retinal disease phenotype.
Asunto(s)
Proteínas del Ojo/genética , Proteínas de Filamentos Intermediarios/genética , Glicoproteínas de Membrana , Proteínas del Tejido Nervioso , Fenilalanina/genética , Mutación Puntual , Células Fotorreceptoras Retinianas Conos/patología , Degeneración Retiniana/genética , Serina/genética , Adulto , Anciano , ADN/análisis , Electroforesis en Gel de Agar , Electrorretinografía , Fondo de Ojo , Humanos , Masculino , Linaje , Periferinas , Reacción en Cadena de la Polimerasa , Células Fotorreceptoras Retinianas Conos/fisiopatología , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Análisis de Secuencia , Campos VisualesRESUMEN
The Essex faculty of the Royal College of General Practitioners organized a collaborative study for trainees in Essex between October 1986 and July 1988. Of the trainees in post during the study period, 28 (46%) participated. The study was performed not only as an educational exercise for trainees in their practice year but also to assess the feasibility of collaborative study as a research tool in general practice. The authors feel that facilitating collaborative research is a faculty activity worthy of consideration.