Based on our observations, we propose a model where immune activation as demonstrated by gene expression changes phosphatase inhibitor in the lungs, as early as 3 hours post infection, and associated differential recruitment and activation of inflammation associated innate immune cells, such as PMN and macrophages, significantly influences the overall outcome of Mtb infection in rabbits at later time points. Methods Ethics statement All rabbit procedures were performed in accordance with Animal Welfare Act guidelines and approved by the Institutional Animal Care and Use and Institutional Biosafety Committees of UMDNJ. Mycobacteria for infection Mycobacterium tuberculosis HN878 and CDC1551 were grown in Middlebrook 7H9 in oculum for rabbit infections were prepared, as de scribed.

Aerosol infection of rabbits Female New Zealand White rabbits were exposed to HN878 or CDC1551 aerosols, as described. Uninfected rabbits served as controls. At 3 hrs and 4 weeks post infection, rabbits were sedated with intramuscular Inhibitors,Modulators,Libraries administration of Keta mine plus Xylazine and euthanized by intravenous injec tion of Euthasol. Lung and blood samples were collected for gene expression analysis. Enumeration of lung bacillary load Portions of lung lobes were homogenized in saline, serially diluted and plated on 7H11 agar, as described. Plates were incubated at 37 C for 4 to 5 weeks bacterial CFU were counted and calculated for the entire lung. Detection limit of this assay was 25 CFU. Rabbit lung histology Five micron sections of formalin fixed, paraffin embedded lung tissues from Mtb infected rabbits were stained with hematoxylin and eosin.

Leukocytes were enumer ated microscopically at 40x magnification. Four independ ent counts per animal, Inhibitors,Modulators,Libraries each of 4 random fields, were used for calculations. Measurement of myeloperoxidase activity MPO activity was determined calorimetrically in the lung homogenates, as described. Color development was read at 460 nm at one minute intervals for 10 minutes and MPO activity was expressed as total MPO activityminutegram of protein. Total protein was estimated using BCA Kit. Isolation of total RNA from rabbit lungs Portions of tissue were homogenized in TRIzol, extracted with bromo chloropropane, and su pernatants were processed using NucleoSpin kit as per in structions to prepare total RNA, as described. RNA quantityquality was estimated by NanoDrop.

Microarray analysis of rabbit gene expression Total lung RNA from each uninfected or Mtb infected rabbit was used for cDNA synthesis, as described. For each infected class, Inhibitors,Modulators,Libraries cDNA from Inhibitors,Modulators,Libraries 4 infected animals was hybridized Inhibitors,Modulators,Libraries separately selleck products with a single pool of cDNA from 4 uninfected animals using a two color rabbit microarray fol lowing the manufacturers procedures. The expression data sets for the 43,803 probes were collected from two sets of experiments, HN878 versus uninfected and CDC1551 versus uninfected.