Utility of In Vivo Transcription Profiling for Identifying Pseudomonas aeruginosa Genes Needed for Gastrointestinal Colonization and Dissemination

Microarray analysis of Pseudomonas aeruginosa mRNA transcripts expressed in vivo during animal infection has not been previously used to investigate potential virulence factors needed in this setting. We compared mRNA expression in bacterial cells recovered from the gastrointestinal (GI) tracts of P. aeruginosa-colonized mice to that of P. aeruginosa in the drinking water used to colonize the mice. Genes associated with biofilm formation and type III secretion (T3SS) had markedly increased expression in the GI tract. A non-redundant transposon library in P. aeruginosa strain PA14 was used to test mutants in genes identified as having increased transcription during in vivo colonization. All of the Tn-library mutants in biofilm-associated genes had an attenuated ability to form biofilms in vitro, but there were no significant differences in GI colonization and dissemination between these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI colonization among these mutant strains and their WT counterparts, whereas rates of survival following dissemination were significantly decreased in mice infected by the T3SS mutant strains. However, there was a variable, strain-dependent effect on overall survival between parental and T3SS mutants. Thus, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia

Intracellular Trafficking Considerations in the Development of Natural Ligand-Drug Molecular Conjugates for Cancer

Overexpressed receptors, characteristic of many cancers, have been targeted by various researchers to achieve a more specific treatment for cancer. A common approach is to use the natural ligand for the overexpressed receptor as a cancer-targeting agent which can deliver a chemically or genetically conjugated toxic molecule. However, it has been found that the therapeutic efficacy of such ligand-drug molecular conjugates can be limited, since they naturally follow the intracellular trafficking pathways of the endogenous ligands. Therefore, a thorough understanding of the intracellular trafficking properties of these ligands can lead to novel design criteria for engineering ligands to be more effective drug carriers. This review presents a few commonly used ligand/receptor systems where intracellular trafficking considerations can potentially improve the therapeutic efficacy of the ligand-drug molecular conjugates

Innocent Cardiac Murmur in Puppies: Prevalence, Correlation with Hematocrit, and Auscultation Characteristics

BACKGROUND: The aims of this study were to establish the prevalence of innocent cardiac murmurs in clinically healthy puppies, to investigate a possible correlation between the presence of an innocent murmur and hematocrit, and to describe the auscultation characteristics of innocent murmurs. HYPOTHESIS: Lower hematocrit contributes to the genesis of innocent murmurs. ANIMALS: Five hundred and eighty-four client-owned clinically healthy puppies, between 20 and 108 days old. METHODS: Two cross-sectional surveys with a 1-year (n = 389 pups) pilot and a half-year (n = 195 pups) principal study periods. Cardiac auscultation was performed by a single, board-certified cardiologist. Hematocrit was measured with an automatized hematology analyzer. Echocardiography was performed only on puppies with a cardiac murmur in the principal study. RESULTS: In the pilot study, 15% of the dogs had a murmur. Innocent murmur was diagnosed in 28% of the 195 dogs in the principal study. Innocent murmurs were systolic, mostly with a musical character and with a maximal intensity of 2 of 6, and mostly with the point of maximal intensity in the left cardiac base. The hematocrit was significantly lower in the group with a murmur compared to the group without (P = .023). CONCLUSIONS AND CLINICAL IMPORTANCE: Innocent murmur was a common finding in puppies at the age when the first veterinary controls usually take place. Physiologic anemia contributes to the genesis of innocent murmurs in puppies. Rising hematocrit in growing puppies can explain the spontaneous disappearance of innocent murmurs with aging. Hematocrit did not differentiate innocent murmurs from abnormal murmurs

Thymocyte depletion affects neurotrophin receptor expression in thymic stromal cells

Thymocytes and thymic stromal cells cross-talk in a bidirectional manner within the thymus, thus contributing to the generation of mature T-cells. The thymic stromal cells in the rat express the high- (TrkA, TrkB) and low-affinity (p75NTR) receptors for neurotrophins. In this study we analysed the regulation of TrkA, TrkB and p75NTR expression in the rat thymus by thymocytes. We induced thymocyte apoptosis by administration of corticoids in rats, and then analysed the expression and distribution of these receptors 1, 4 and 10 days later. Thymocyte death was assessed by the activation of caspase-3 in cells undergoing apoptosis. We observed massive thymocyte apoptosis 1 day after injection and, to a lesser extent, after 4 days, which was parallel with a reduction in the density of thymic epithelial cells normally expressing TrkA and p75NTR. Furthermore, TrkA expression was found in cortical thymic epithelial cells, which normally lack this receptor. The expression of TrkB was restricted to a subset of macrophage-dendritic cells, and remained unchanged with treatment. The normal pattern of neurotrophin receptor expression was almost completely restored by day 10. The results demonstrate that the expression of neurotrophin receptors by thymic epithelial cells, but not by macrophage-dendritic cells, is regulated by thymocytes