Statistics were based on values obtained during the 5 minutes at the end of baseline

, vimentin and MHC confirms the preponderance of NGF in CFs as judged by the preferential merge of 22223206 fluorescently labeled vimentin with NGF. NGF secreted in response to T cruzi infection is bioactive because 1) conditioned medium obtained from T cruziinfected primary CFs at 72 h post-infection, but not medium from uninfected cells strongly promotes neurite outgrowth in PC12 cells in a timeof-infection dependent manner, and 2) an NGF anti-serum blocks the neurite-enhancement effect of active CoM nearly completely. T cruzi Spurs Cardiac Fibroblast-Myocyte Crosstalk Targeting TrkA in Cardiac Fibroblasts for NGF Upregulation To determine whether T cruzi exploits fibroblast TrkA or TrkC, or both, to trigger NGF production, CFs were pre-incubated with antibodies against the neurotrophin receptors prior to T cruzi infection. If T cruzi uses Trks to increase NGF expression, then neutralizing antibodies against relevant Trks should block the T cruzi action. We find 19239230 that antibodies against TrkA significantly block secretion of NGF while antibodies against TrkC are less effective . Further underlying the selectivity of TrkA blockage, antibodies against TrkB and against the panneurotrophin receptor p75NTR, which do not interact with T cruzi, exhibit no inhibitory activity. Therefore, these results suggest that T cruzi boosts NGF production in cardiac fibroblasts predominantly through TrkA recognition. Experiments designed to reduce Trk expression with shRNA validate the conclusion from the antibody blocking experiments. We Saracatinib transfected CFs with lentivirus encoding shRNA constructs for control green fluorescence protein, TrkA, or TrkC and infected the transfected cardiac fibroblasts with T cruzi for 24 h. Compared to GFP-transfected fibroblasts, the cell lines transfected with two shTrkA constructs, but not the cell line transfected with the TrkC mRNA silencer, selectively block NGF secretion in response to T cruzi infection. left panel). NGF mRNA increases in infected hearts maximally 2224 d PI, trailing heart parasite burden, supporting the premise from in vitro experiments that NGF upregulation results from T cruzi recognition of fibroblast-TrkA. Upregulation of NGF transcript is reflected in NGF protein, which increases in infected hearts as quantified with NIH ImageJ software in tissue sections obtained at peak heart parasitism and stained by indirect immunofluorescence. Visualization of the fluorescence also reveals a sharp NGF upturn in T cruzi-infected hearts. Early studies showed increased NGF in T cruzi-infected hearts. To determine whether the observed fluorescence is due to antibody recognition of NGF and not to nonspecific staining, we preincubated the NGF antibody with a low concentration of NGF, which eliminates NGF visualization almost completely, establishing the specificity of NGF detection. Co-staining analysis reveals that NGF merges preponderantly with vimentin , confirming the preferential increase in NGF in the CFs of chagasic hearts. The T cruzi Trk-ligand PDNF Upregulates NGF Preferentially in Cardiac Fibroblasts In Vitro and in the Heart Following Intravenous Administration Given that T cruzi upregulates NGF in cardiac fibroblasts through TrkA, which in turn is recognized by T cruzi via PDNF, then PDNF must be the T cruzi ligand mediating the NGFstimulatory effect. This prediction was tested in CFs and myocytes in culture and in the myocardium. PDNF consists of a N-terminal region, containing the catalytic and Trk-binding sit