RESUMEN
Infection with pathogenic influenza viruses is associated with intense inflammatory disease. Here, we investigated the innate immune response in mice infected with H5N1 A/Vietnam/1203/04 and with reassortant human H1N1 A/Texas/36/91 viruses containing the virulence genes hemagglutinin (HA), neuraminidase (NA) and NS1 of the 1918 pandemic virus. Inclusion of the 1918 HA and NA glycoproteins rendered a seasonal H1N1 virus capable of inducing an exacerbated host innate immune response similar to that observed for highly pathogenic A/Vietnam/1203/04 virus. Infection with 1918 HA/NA:Tx/91 and A/Vietnam/1203/04 were associated with severe lung pathology, increased cytokine and chemokine production, and significant immune cell changes, including the presence of CD11b(+)Gr-1(+) cells in the blood, lung and bone marrow. Significant differential gene expression in the lung included pathways for cell death, apoptosis, production and response to reactive oxygen radicals, as well as arginine and proline metabolism and chemokines associated with monocyte and neutrophil/granulocyte accumulation and/or activation. Arginase was produced in the lung of animals infected with A/Vietnam/1204. These results demonstrate that the innate immune cell response results in the accumulation of CD11b(+)Gr-1(+) cells and products that have previously been shown to contribute to T cell suppression.
Asunto(s)
Médula Ósea/inmunología , Antígeno CD11b/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Pulmón/inmunología , Infecciones por Orthomyxoviridae/inmunología , Linfocitos T/inmunología , Animales , Quimiocinas/inmunología , Femenino , Perfilación de la Expresión Génica , Granulocitos/inmunología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Leucocitos/inmunología , Pulmón/citología , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/virología , Virulencia/inmunologíaRESUMEN
The mechanisms responsible for the virulence of the highly pathogenic avian influenza (HPAI) and of the 1918 pandemic influenza virus in humans remain poorly understood. To identify crucial components of the early host response during these infections by using both conventional and functional genomics tools, we studied 34 cynomolgus macaques (Macaca fascicularis) to compare a 2004 human H5N1 Vietnam isolate with 2 reassortant viruses possessing the 1918 hemagglutinin (HA) and neuraminidase (NA) surface proteins, known conveyors of virulence. One of the reassortants also contained the 1918 nonstructural (NS1) protein, an inhibitor of the host interferon response. Among these viruses, HPAI H5N1 was the most virulent. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.
Asunto(s)
Inmunidad Innata/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Animales , Movimiento Celular/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Perfilación de la Expresión Génica , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Enfermedades Pulmonares/patología , Enfermedades Pulmonares/virología , Ganglios Linfáticos/inmunología , Macaca , Masculino , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Tasa de Supervivencia , Linfocitos T/citología , Linfocitos T/inmunología , Factores de Tiempo , Tropismo , Replicación ViralRESUMEN
Sphingosine-1-phosphate is a bioactive lipid that is mitogenic for human glioma cell lines by signaling through its G protein-coupled receptors. We investigated the role of sphingosine-1-phosphate receptors and the enzymes that form sphingosine-1-phosphate, sphingosine kinase (SphK)-1, and -2 in human astrocytomas. Astrocytomas of various histologic grades expressed three types of sphingosine-1-phosphate receptors, S1P1, S1P2, and S1P3; however, no significant correlation with histologic grade or patient survival was detected. Expression of SphK1, but not SphK2, in human astrocytoma grade 4 (glioblastoma multiforme) tissue correlated with short patient survival. Patients whose tumors had low SphK1 expression survived a median 357 days, whereas those with high levels of SphK1 survived a median 102 days. Decreasing SphK1 expression using RNA interference or pharmacologic inhibition of SphK significantly decreased the rate of proliferation of U-1242 MG and U-87 MG glioblastoma cell lines. Surprisingly, RNA interference to knockdown SphK2 expression inhibited glioblastoma cell proliferation more potently than did SphK1 knockdown. SphK knockdown also prevented cells from exiting G1 phase of the cell cycle and marginally increased apoptosis. Thus, SphK isoforms may be major contributors to growth of glioblastoma cells in vitro and to aggressive behavior of glioblastoma multiforme.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Glioblastoma/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adulto , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Northern Blotting/métodos , Western Blotting/métodos , Recuento de Células/métodos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Aumento de la Célula/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/mortalidad , Glioblastoma/patología , Glioblastoma/fisiopatología , Humanos , Persona de Mediana Edad , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de TiempoRESUMEN
The purpose of this article is to discuss small-group apprenticeships (SGAs) as a method to instruct cell culture techniques to high school participants. The study aimed to teach cell culture practices and to introduce advanced imaging techniques to solve various biomedical engineering problems. Participants designed and completed experiments using both flow cytometry and laser scanning cytometry during the 1-month summer apprenticeship. In addition to effectively and efficiently teaching cell biology laboratory techniques, this course design provided an opportunity for research training, career exploration, and mentoring. Students participated in active research projects, working with a skilled interdisciplinary team of researchers in a large research institution with access to state-of-the-art instrumentation. The instructors, composed of graduate students, laboratory managers, and principal investigators, worked well together to present a real and worthwhile research experience. The students enjoyed learning cell culture techniques while contributing to active research projects. The institution's researchers were equally enthusiastic to instruct and serve as mentors. In this article, we clarify and illuminate the value of small-group laboratory apprenticeships to the institution and the students by presenting the results and experiences of seven middle and high school participants and their instructors.