RESUMEN
BACKGROUND: Kawasaki disease is recognized as the most common cause of acquired heart disease in children in the developed world. Clinical, epidemiologic, and pathologic evidence supports an infectious agent, likely entering through the lung. Pathologic studies proposing an acute coronary arteritis followed by healing fail to account for the complex vasculopathy and clinical course. METHODOLOGY/PRINCIPAL FINDINGS: Specimens from 32 autopsies, 8 cardiac transplants, and an excised coronary aneurysm were studied by light (n=41) and transmission electron microscopy (n=7). Three characteristic vasculopathic processes were identified in coronary (CA) and non-coronary arteries: acute self-limited necrotizing arteritis (NA), subacute/chronic (SA/C) vasculitis, and luminal myofibroblastic proliferation (LMP). NA is a synchronous neutrophilic process of the endothelium, beginning and ending within the first two weeks of fever onset, and progressively destroying the wall into the adventitia causing saccular aneurysms, which can thrombose or rupture. SA/C vasculitis is an asynchronous process that can commence within the first two weeks onward, starting in the adventitia/perivascular tissue and variably inflaming/damaging the wall during progression to the lumen. Besides fusiform and saccular aneurysms that can thrombose, SA/C vasculitis likely causes the transition of medial and adventitial smooth muscle cells (SMC) into classic myofibroblasts, which combined with their matrix products and inflammation create progressive stenosing luminal lesions (SA/C-LMP). Remote LMP apparently results from circulating factors. Veins, pulmonary arteries, and aorta can develop subclinical SA/C vasculitis and SA/C-LMP, but not NA. The earliest death (day 10) had both CA SA/C vasculitis and SA/C-LMP, and an "eosinophilic-type" myocarditis. CONCLUSIONS/SIGNIFICANCE: NA is the only self-limiting process of the three, is responsible for the earliest morbidity/mortality, and is consistent with acute viral infection. SA/C vasculitis can begin as early as NA, but can occur/persist for months to years; LMP causes progressive arterial stenosis and thrombosis and is composed of unique SMC-derived pathologic myofibroblasts.
Asunto(s)
Síndrome Mucocutáneo Linfonodular/etiología , Síndrome Mucocutáneo Linfonodular/patología , Aneurisma/etiología , Aneurisma Roto/etiología , Proliferación Celular , Niño , Preescolar , Femenino , Humanos , Lactante , Linfocitos/patología , Masculino , Síndrome Mucocutáneo Linfonodular/complicaciones , Miocarditis/etiología , Miocarditis/metabolismo , Miocarditis/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/ultraestructura , Miofibroblastos/metabolismo , Miofibroblastos/patología , Miofibroblastos/ultraestructura , Neutrófilos/patología , Trombosis/etiologíaRESUMEN
BACKGROUND: Intracytoplasmic inclusion bodies (ICI) have been identified in ciliated bronchial epithelium of Kawasaki disease (KD) patients using a synthetic antibody derived from acute KD arterial IgA plasma cells; ICI may derive from the KD etiologic agent. METHODS: Acute KD bronchial epithelium was subjected to immunofluorescence for ICI and cytokeratin, high-throughput sequencing, and transmission electron microscopy (TEM). Interferon pathway gene expression profiling was performed on KD lung. RESULTS: An intermediate filament cytokeratin "cage" was not observed around KD ICI, making it unlikely that ICI are overproduced or misfolded human protein aggregates. Many interferon-stimulated genes were detected in the bronchial epithelium, and significant modulation of the interferon response pathway was observed in the lung tissue of KD patients. No known virus was identified by sequencing. Aggregates of virus-like particles (VLP) were detected by TEM in all 3 acute KD patients from whom nonembedded formalin-fixed lung tissue was available. CONCLUSIONS: KD ICI are most likely virus induced; bronchial cells with ICI contain VLP that share morphologic features among several different RNA viral families. Expedited autopsies and tissue fixation from acute KD fatalities are urgently needed to more clearly ascertain the VLP. These findings are compatible with the hypothesis that the infectious etiologic agent of KD may be a "new" RNA virus.
Asunto(s)
Cuerpos de Inclusión Viral/patología , Síndrome Mucocutáneo Linfonodular/virología , Virus/aislamiento & purificación , Virus/patogenicidad , Preescolar , Células Epiteliales/virología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Lactante , Recién Nacido , Masculino , Microscopía Electrónica de Transmisión , Síndrome Mucocutáneo Linfonodular/inmunología , Síndrome Mucocutáneo Linfonodular/patología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Mucosa Respiratoria/virología , Virosomas/inmunología , Virosomas/ultraestructura , Virus/inmunología , Virus/ultraestructuraRESUMEN
Propagation of new human respiratory virus pathogens in established cell lines is hampered by a lack of predictability regarding cell line permissivity and by availability of suitable antibody reagents to detect infection in cell lines that do not exhibit significant cytopathic effect. Recently, molecular methods have been used to amplify and identify novel nucleic acid sequences directly from clinical samples, but these methods may be hampered by the quantity of virus present in respiratory secretions at different time points following the onset of infection. Human airway epithelial (HAE) cultures, which effectively mimic the human bronchial environment, allow for cultivation of a wide variety of human respiratory viral pathogens. The goal of the experiments described here was to determine if propagation and identification of a human respiratory virus may be achieved through inoculation of HAE cultures followed by whole transcriptome amplification (WTA) and sequence analysis. To establish proof-of-principle human coronavirus NL63 (HCoV-NL63) was evaluated, and the first visualization of HCoV-NL63 virus by transmission electron microscopy (TEM) is reported. Initial propagation of human respiratory secretions onto HAE cultures followed by TEM and WTA of culture supernatant may be a useful approach for visualization and detection of new human respiratory pathogens that have eluded identification by traditional approaches.
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Coronavirus/aislamiento & purificación , Células Epiteliales/virología , Infecciones del Sistema Respiratorio/virología , Secuencia de Bases , Células Cultivadas , Coronavirus/genética , Coronavirus/crecimiento & desarrollo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Genoma Viral , Humanos , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , ARN Viral/genética , Cultivo de VirusRESUMEN
BACKGROUND: Kawasaki Disease (KD) is the most common cause of acquired heart disease in children in developed nations. The KD etiologic agent is unknown but likely to be a ubiquitous microbe that usually causes asymptomatic childhood infection, resulting in KD only in genetically susceptible individuals. KD synthetic antibodies made from prevalent IgA gene sequences in KD arterial tissue detect intracytoplasmic inclusion bodies (ICI) resembling viral ICI in acute KD but not control infant ciliated bronchial epithelium. The prevalence of ICI in late-stage KD fatalities and in older individuals with non-KD illness should be low, unless persistent infection is common. METHODS AND PRINCIPAL FINDINGS: Lung tissue from late-stage KD fatalities and non-infant controls was examined by light microscopy for the presence of ICI. Nucleic acid stains and transmission electron microscopy (TEM) were performed on tissues that were strongly positive for ICI. ICI were present in ciliated bronchial epithelium in 6/7 (86%) late-stage KD fatalities and 7/27 (26%) controls ages 9-84 years (p = 0.01). Nucleic acid stains revealed RNA but not DNA within the ICI. ICI were also identified in lung macrophages in some KD cases. TEM of bronchial epithelium and macrophages from KD cases revealed finely granular homogeneous ICI. SIGNIFICANCE: These findings are consistent with a previously unidentified, ubiquitous RNA virus that forms ICI and can result in persistent infection in bronchial epithelium and macrophages as the etiologic agent of KD.
Asunto(s)
Cuerpos de Inclusión/virología , Macrófagos Alveolares/virología , Síndrome Mucocutáneo Linfonodular/virología , Virus ARN , Mucosa Respiratoria/virología , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Niño , Humanos , Recién Nacido , Macrófagos Alveolares/patología , Microscopía , Persona de Mediana Edad , Síndrome Mucocutáneo Linfonodular/etiología , Síndrome Mucocutáneo Linfonodular/patología , ARN Viral/análisis , Mucosa Respiratoria/patologíaRESUMEN
BACKGROUND: In developed nations, Kawasaki disease (KD) is the most common cause of acquired heart disease in children. An infectious etiology is likely but has not yet been identified. We have previously reported that oligoclonal immunoglobulin A plasma cells infiltrate acute KD tissues and that synthetic KD antibodies detect a distinctive spheroidal antigen in acute KD ciliated bronchial epithelium. METHODS: To further characterize the antigen in acute KD bronchi, we examined paraffin-embedded ciliated bronchial epithelium using light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: The spheroids observed by immunohistochemistry (IHC) are visualized as inclusion bodies with hematoxylin-eosin and nucleic acid stains and in methylene blue/azure II/basic fuchsin trichrome-stained plastic sections, suggesting the presence of both protein and nucleic acid. The structures visualized by LM correspond to homogeneous electron-dense perinuclear inclusion bodies (up to 1.4 microns in diameter) in ciliated bronchial epithelium from 4 patients with acute KD examined by TEM. Inclusion bodies were not present in control bronchial epithelium or in nonciliated cells. CONCLUSIONS: The antigen detected in acute KD ciliated bronchial epithelium by IHC with synthetic KD antibodies resides in cytoplasmic inclusion bodies that are consistent with aggregates of viral proteins and associated nucleic acid and may derive from the etiologic agent of KD.