Partial characterization of normal and Haemophilus influenzae-infected mucosal complementary DNA libraries in chinchilla middle ear mucosa.
OBJECTIVE
We sought to construct and partially characterize complementary DNA (cDNA) libraries prepared from the middle ear mucosa (MEM) of chinchillas to better understand pathogenic aspects of infection and inflammation, particularly with respect to leukotriene biogenesis and response.


METHODS
Chinchilla MEM was harvested from controls and after middle ear inoculation with nontypeable Haemophilus influenzae. RNA was extracted to generate cDNA libraries. Randomly selected clones were subjected to sequence analysis to characterize the libraries and to provide DNA sequence for phylogenetic analyses. Reverse transcription-polymerase chain reaction of the RNA pools was used to generate cDNA sequences corresponding to genes associated with leukotriene biosynthesis and metabolism.


RESULTS
Sequence analysis of 921 randomly selected clones from the uninfected MEM cDNA library produced approximately 250,000 nucleotides of almost entirely novel sequence data. Searches of the GenBank database with the Basic Local Alignment Search Tool provided for identification of 515 unique genes expressed in the MEM and not previously described in chinchillas. In almost all cases, the chinchilla cDNA sequences displayed much greater homology to human or other primate genes than with rodent species. Genes associated with leukotriene metabolism were present in both normal and infected MEM.


CONCLUSIONS
Based on both phylogenetic comparisons and gene expression similarities with humans, chinchilla MEM appears to be an excellent model for the study of middle ear inflammation and infection. The higher degree of sequence similarity between chinchillas and humans compared to chinchillas and rodents was unexpected. The cDNA libraries from normal and infected chinchilla MEM will serve as useful molecular tools in the study of otitis media and should yield important information with respect to middle ear pathogenesis.
Kerschner
Joseph E
JE
Department of Otolaryngology and Communication Sciences, Medical College of Wisconsin, Children's Hospital of Wisconsin, Milwaukee 53226, USA.
Erdos
Geza
G
Hu
Fen Ze
FZ
Burrows
Amy
A
Cioffi
Joseph
J
Khampang
Pawjai
P
Dahlgren
Margaret
M
Hayes
Jay
J
Keefe
Randy
R
Janto
Benjamin
B
Post
J Christopher
JC
Ehrlich
Garth D
GD
D000818:Animals; D002682:Chinchilla; D018076:DNA, Complementary / Q000032:analysis*; D004432:Ear, Middle / Q000737:chemistry*; D015723:Gene Library; D006192:Haemophilus Infections / Q000235:genetics*; D006193:Haemophilus influenzae; D006801:Humans; D009092:Mucous Membrane / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary