combined-vivo-optical-ct-imaging-to-monitor-infection-inflammation

Combined <em>In vivo</em> Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

PerkinElmer

Vaginal infections caused by the opportunistic yeast Candida albicans are a significant problem in women of child-bearing age. Several factors are recognized as playing a crucial role in the pathogenesis of superficial candidiasis; these factors include hyphal formation, phenotypic switching, and the expression of virulence factors, including a 10-member family of secreted aspartic proteinases. In the present investigation, we analyzed the secreted aspartic proteinase gene (SAP) expression profile of C. albicans that is elicited in the course of vaginal infection in mice and how this in vivo expression profile is associated with hyphal formation. We utilized two different genetic reporter systems that allowed us to observe SAP expression on a single-cell basis, a recombination-based in vivo expression technology and green fluorescent protein-expressing Candida reporter strains. Of the six SAP genes that were analyzed (SAP1 to SAP6), only SAP4 and SAP5 were detectably induced during infection in this model. Expression of both of these genes was associated with hyphal growth, although not all hyphal cells detectably expressed SAP4 and SAP5. SAP5 expression was induced soon after infection, whereas SAP4 was expressed at later times and in fewer cells compared with SAP5. These findings point to a link between morphogenetic development and expression of virulence genes during Candida vaginitis in mice, where host signals induce both hyphal formation and expression of SAP4 and SAP5, but temporal gene expression patterns are ultimately controlled by other factors.

Profile of Candida albicans-secreted aspartic proteinase elicited during vaginal infection.

SAP7 of Candida albicans is induced after vaginal infection of mice. Conversely, virulence during vaginal infection was not affected in a Deltasap7/Deltasap7 mutant strain. Only a partial virulence phenotype was detectable after intravenous injection. In conclusion, SAP7 expression does not correlate with C. albicans virulence in mice.

Induction of SAP7 correlates with virulence in an intravenous infection model of candidiasis but not in a vaginal infection model in mice.

Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, alpha-helical peptides are unique. Unlike the cationic cell-penetrating peptides, alpha-helical antennapedia-like, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively.

Noncationic peptides obtained from azurin preferentially enter cancer cells.

Amino acids 50-77 (p28) of azurin, a 128 aa cupredoxin isolated from Pseudomonas aeruginosa, is essentially responsible for azurin's preferential penetration of cancer cells. We now report that p28 also preferentially penetrates human umbilical vein endothelial cells (HUVEC), co-localized with caveolin-1 and VEGFR-2, and inhibits VEGF- and bFGF-induced migration, capillary tube formation and neoangiogenesis in multiple xenograft models. The antiangiogenic effect of p28 in HUVEC is associated with a dose-related non-competitive inhibition of VEGFR-2 kinase activity. However, unlike other antiangiogenic agents that inhibit the VEGFR-2 kinase, p28 decreased the downstream phosphorylation of FAK and Akt that normally precedes cellular repositioning of the cytoskeletal (F-actin), focal adhesion (FAK and paxillin), and cell to cell junction protein PECAM-1, inhibiting HUVEC motility and migration. The decrease in pFAK and pAkt levels suggests that p28 induces a pFAK-mediated loss of HUVEC motility and migration and a parallel Akt-associated reduction in cell matrix attachment and survival. This novel, direct antiangiogenic effect of p28 on endothelial cells may enhance the cell cycle inhibitory and apoptotic properties of this prototype peptide on tumor cell proliferation as it enters a Phase II clinical trial.

Brad N. Taylor

PerkinElmer

Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice