Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling time series of the human polymorphonuclear leukocyte response to exposure to Anaplasma phagocytophilum or Staphylococcus aureus


ABSTRACT: Polymorphonuclear leukocytes (PMNs) were obtained from healthy individuals in accordance with protocols approved by the Institutional Review Board for Human Subjects at the University of Minnesota and the National Institute of Allergy and Infectious Diseases. PMNs (107) were combined on ice with live S. aureus (108) or with live or heat-killed A. phagocytophilum (bacteria isolated from 5x106 infected HL60 cells for a ratio of 1 infected HL60 cell: 2 PMNs, ~ 5-20 A. phagocytophilum: PMN) in wells of a 12-well tissue culture plate (pre-coated with 20% autologous normal human serum). Unstimulated control assays received either buffer (for S. aureus comparisons) or clarified HL60 lysate (for A. phagocytophilum comparisons). Plates were centrifuged at 350 x g for 8 min at 4oC to synchronize phagocytosis and incubated at 37 deg. C in a CO2 incubator for the indicated times. At the indicated times, tissue culture medium was aspirated from the plate and PMNs were lysed directly with RLT buffer (Qiagen, Valencia, CA). Purification of PMN RNA and subsequent preparation of labeled cRNA target was performed as described in Methods. Labeling of samples, hybridization of cRNA with HU133A oligonucleotide arrays (Affymetrix, Santa Clara, CA), and scanning were performed according to standard Affymetrix protocols ( http://www.affymetrix.com/pdf/expression_manual.pdf ). Experiments were performed in triplicate, using PMNs from three healthy individuals for each treatment.

ORGANISM(S): Homo sapiens

SUBMITTER: Frank DeLeo 

PROVIDER: E-GEOD-2405 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Insights into pathogen immune evasion mechanisms: Anaplasma phagocytophilum fails to induce an apoptosis differentiation program in human neutrophils.

Borjesson Dori L DL   Kobayashi Scott D SD   Whitney Adeline R AR   Voyich Jovanka M JM   Argue Cynthia M CM   Deleo Frank R FR  

Journal of immunology (Baltimore, Md. : 1950) 20050501 10


Polymorphonuclear leukocytes (PMNs or neutrophils) are essential to human innate host defense. However, some bacterial pathogens circumvent destruction by PMNs and thereby cause disease. Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis, survives within PMNs in part by altering normal host cell processes, such as production of reactive oxygen species (ROS) and apoptosis. To investigate the molecular basis of A. phagocytophilum survival within neutrophils, we used Affymetrix  ...[more]

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