Project description:Streptococcus pneumoniae (Spn) is the predominant causative organism of acute otitis media (AOM) in children. A human cDNA microarray comprising 30,968 human genome probes was used to evaluate the transcriptional changes that occur in peripheral blood mononuclear cells (PBMC) at the onset of clinical AOM caused by Spn infection in children after comparison of microarray results with the pre-infection healthy stage of the same children.
Project description:Nontypeable Haemophilus influenzae (NTHi) is a common causative organism of acute otitis media (AOM) in children. A human cDNA microarray comprising 30,968 human genome probes was used to evaluate the transcriptional changes that occur in peripheral blood mononuclear cells (PBMC) at the onset of clinical AOM caused by NTHi infection in children after comparison of microarray results with the pre-infection healthy stage of the same children.
Project description:Objective: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media.
Project description:Objective: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media.
Project description:Streptococcus pneumoniae (Spn) is the predominant causative organism of acute otitis media (AOM) in children. A human cDNA microarray comprising 30,968 human genome probes was used to evaluate the transcriptional changes that occur in peripheral blood mononuclear cells (PBMC) at the onset of clinical AOM caused by Spn infection in children after comparison of microarray results with the pre-infection healthy stage of the same children. Four to ten milliliters of heparinized peripheral venous blood was collected from children at 6 to 30 months of age when they were in acute otitis media (AOM) stage and pre-infection healthy stage. The diagnosis of AOM was based on symptoms and signs as well as S. pneumoniae culture positive in the middle ear fluid. Patients with polymicrobial infections, history of immunodeficiency, history of chronic or recurrent AOM, chronic disease, or receiving steroids or other immunomodulatory agents were excluded. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll gradient and total RNA was extracted from PBMCs using the QIAamp RNA blood Mini Kit (Qiagen, Maryland, USA) according to manufacturer’s instructions. Double-stranded cDNA generated from total RNA was labeled with Cyanine-5 and subsequently hybridized to Human OneArray glass slides according to the manufacturer's standard protocols (PhalanxBio Inc, CA, USA). Microarrays were scanned at 5 μm resolution using an Agilent scanner. Raw intensity signals for each microarray were captured using a Molecular Dynamics Axon 4100A scanner, measured using GenePixPro™ Software. The data from all microarrays in each experimental set was then analyzed using Omicsoft Array Studio software; control and missing features were removed, and the remaining signals were quantile normalized.
Project description:Nontypeable Haemophilus influenzae (NTHi) is a common causative organism of acute otitis media (AOM) in children. A human cDNA microarray comprising 30,968 human genome probes was used to evaluate the transcriptional changes that occur in peripheral blood mononuclear cells (PBMC) at the onset of clinical AOM caused by NTHi infection in children after comparison of microarray results with the pre-infection healthy stage of the same children. Four to ten milliliters of heparinized peripheral venous blood was collected from children at 6 to 30 months of age when they were in acute otitis media (AOM) stage and pre-infection healthy stage. The diagnosis of AOM was based on symptoms and signs as well as Nontypeable Haemophilus influenzae culture positive in the middle ear fluid. Patients with polymicrobial infections, history of immunodeficiency, history of chronic or recurrent AOM, chronic disease, or receiving steroids or other immunomodulatory agents were excluded. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll gradient and total RNA was extracted from PBMCs using the QIAamp RNA blood Mini Kit (Qiagen, Maryland, USA) according to manufacturerM-bM-^@M-^Ys instructions. Double-stranded cDNA generated from total RNA was labeled with Cyanine-5 and subsequently hybridized to Human OneArray glass slides according to the manufacturer's standard protocols (PhalanxBio Inc, CA, USA). Microarrays were scanned at 5 M-NM-<m resolution using an Agilent scanner. Raw intensity signals for each microarray were captured using a Molecular Dynamics Axon 4100A scanner, measured using GenePixProM-bM-^DM-" Software. The data from all microarrays in each experimental set was then analyzed using Omicsoft Array Studio software; control and missing features were removed, and the remaining signals were quantile normalized.
Project description:Recurrent otitis (ROM) is a pathological conditions in which a children have at least three episodes of acute otitis media over a course of 6 months. In this study we focused in proteins dysregulated between ROM and adenotonsillar hypertrophy (ATH). Tissue sample of seven ROM and seven ATH were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) for protein identification and western-blotting for data validation. We identified 23 significantly (p<0.05) protein spots. The expression of two proteins were validated by western-blotting. The 2-DE coupled with MS approach identified several proteins involved in cellular and metabolic processes. Further study are needed to better understanding the role of this proteins in ROM.
Project description:Objective: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media. There are 8 control samples and 9 samples trans-tympanically injected with H flu 10e9 for 6 hours. Each sample is from a single animal.
Project description:Objective: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. Methods: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. Results: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. Conclusion: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media. There are 6 control samples and 8 samples trans-tympanically injected with H flu 10e9 for 6 hours. Each sample is a pool of 4 animals