A technique for in vitro culture of canine valvular interstitial cells.

OBJECTIVE: To develop a method for in vitro culture of canine valvular interstitial cells (VICs). ANIMALS, MATERIALS AND METHODS: Canine VICs were isolated from the distal third of the anterior mitral valve leaflet using an explant technique and maintained in cell culture. Molecular phenotyping of the cultured cells was performed using reverse transcription polymerase chain reaction and immunocytochemistry. RESULTS: Cells resembling fibroblasts migrated from canine mitral valve explants and were maintained in culture for up to eight passages. Establishment of the valve explant required collagen but once established, subsequent passages grew on non-coated plastic plates. At confluence the cultured cells exhibited the characteristic whorled pattern of fibroblasts in culture. The isolated valve cells expressed vimentin but not platelet endothelial cell adhesion molecule or von Willebrand's factor, consistent with the molecular phenotype of VICs. CONCLUSIONS: VICs can be readily isolated from canine mitral valve leaflets and successfully maintained in culture using standard culture techniques. The described techniques permit the study of bioactive VICs in a controlled environment and may be a useful in vitro model for investigation of cellular and molecular alterations associated with canine chronic degenerative valve disease.

The antimicrobial peptide PR-39 has a protective effect against HeLa cell apoptosis.

PR-39 is a cathelicidin with antimicrobial properties, which acts as a suppressor of inflammation and exerts a number of additional activities. We investigated for the first time the effect of PR-39 on human cells, by addressing the possible interference with HeLa cell metabolism. We observed that the continuous administration of PR-39 to HeLa cell cultures was not cytotoxic and did not interfere with DNA synthesis. When used in combination with a panel of drugs inducing apoptosis through different mechanisms of action, i.e. etoposide, bleomycin, tert-butylhydroperoxide and 2-deoxy-d-ribose, PR-39 attenuated the apoptotic response of HeLa cells.

Heparan sulfate proteoglycan-dependent neutrophil chemotaxis toward PR-39 cathelicidin.

Cathelicidins are mammalian proteins containing a C-terminal cationic antimicrobial domain. Porcine PR-39 cathelicidin affects leukocyte biology. Mechanisms of action may involve alteration of heparan sulfate proteoglycan-dependent functions in inflammatory cells. It was tested whether PR-39 affects human neutrophil migration and if such effects involve heparan sulphate proteoglycans. Neutrophils were from forearm venous blood of healthy donors. Migration was tested in modified Boyden chamber assays. Involvement of heparan sulfate proteoglycans was tested by their chemical modification and by the use of specific antibodies. PR-39 induced migration in neutrophils in a concentration dependent manner. Modification of heparan sulfate proteoglycans with sodium chlorate inhibited migration whereas chemotaxis toward the chemoattractant formyl-Met-Leu-Phe was not affected. Removal of heparan sulfates or chondroitin sulfates from the surface of neutrophils by heparinase or chondroitinase inhibited migration toward PR-39. In conclusion, antimicrobial PR-39 stimulates human neutrophil chemotaxis in a heparan sulfate proteoglycan-dependent manner. Involvement of syndecans is likely as both heparinase and chondroitinase were abrogating. Data suggest active participation of heparan sulfate proteoglycans of neutrophils in cathelicidin peptide-mediated regulation of the antimicrobial host defense.

Gene silencing and overexpression of porcine peptidoglycan recognition protein long isoforms: involvement in beta-defensin-1 expression.

Peptidoglycan recognition proteins (PGRPs) are a group of newly identified proteins with emerging functions in mammalian innate immunity. Here we report the identification and characterization of two long isoforms of porcine PGRP. Their complete cDNA sequences encode predicted peptides of 252 and 598 residues and are named pPGRP-L1 and pPGRP-L2, respectively. These porcine isoforms share identical PGRP domains at their C terminus, which are highly conserved with human and mouse orthologs. pPGRP-L1 is expressed constitutively in several tissues, including bone marrow, intestine, liver, spleen, kidney, and skin. pPGRP-L2 is highly expressed in the duodenum and liver, and expression in intestinal tissues is increased by Salmonella infection. In intestinal cells, expression of both pPGRP-L1 and pPGRP-L2 is increased by bacterial infection. Recombinant pPGRP-L1 and pPGRP-L2 have N-acetylmuramoyl-L-alanine amidase activity. Loss-of-function and gain-of-function experiments indicate that these two pPGRPs are involved in expression of the antimicrobial peptide beta-defensin-1. Silencing of pPGRP-L2 in intestinal cells challenged with Listeria monocytogenes results in downregulation of beta-defensin-1. Conversely, overexpression of pPGRP-L1 or pPGRP-L2 dramatically upregulates expression of beta-defensin-1. Collectively, these findings suggest that porcine PGRPs are involved in antimicrobial peptide expression.

Central angiotensin II-enhanced splenic cytokine gene expression is mediated by the sympathetic nervous system.

We tested the hypothesis that central angiotensin II (ANG II) administration would activate splenic sympathetic nerve discharge (SND), which in turn would alter splenic cytokine gene expression. Experiments were completed in sinoaortic nerve-lesioned, urethane-chloralose-anesthetized, splenic nerve-intact (splenic-intact) and splenic nerve-lesioned (splenic-denervated) Sprague-Dawley rats. Splenic cytokine gene expression was determined using gene-array and real-time RT-PCR analyses. Splenic SND was significantly increased after intracerebroventricular administration of ANG II (150 ng/kg, 10 microl), but not artificial cerebrospinal fluid (aCSF). Splenic mRNA expression of IL-1beta, IL-6, IL-2, and IL-16 genes was increased in ANG II-treated splenic-intact rats compared with aCSF-treated splenic-intact rats. Splenic IL-1beta, IL-2, and IL-6 gene expression responses to ANG II were significantly reduced in splenic-denervated compared with splenic-intact rats. Splenic gene expression responses did not differ significantly in ANG II-treated splenic-denervated and aCSF-treated splenic-intact rats. Splenic blood flow responses to intracerebroventricular ANG II administration did not differ between splenic-intact and splenic-denervated rats. These results provide experimental support for the hypothesis that ANG II modulates the immune system through activation of splenic SND, suggesting a novel relation between ANG II, efferent sympathetic nerve outflow, and splenic cytokine gene expression.

Characterization of bovine cDNA encoding triggering receptor expressed on myeloid cells 1 (TREM-1).

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a type I transmembrane receptor of the immunoglobulin superfamily expressed predominantly on neutrophils and monocytes. TREM-1 induces the expression of inflammatory cytokines and adhesion molecules, and augments osteoclast, microglia, oligodendrocyte, and dendritic cell differentiation. Here, we report the cloning of TREM-1 from bovine bone marrow cells. Full-length bovine TREM-1 cDNA is 1202 base pairs in length and encodes a predicted 232 amino acid peptide. Comparative analyses showed that bovine TREM-1 has 48 to 61% amino acid identity with other TREM-1 proteins, sharing the greatest identity with porcine TREM-1. Cysteine residues characteristic of the immunoglobulin superfamily were conserved in bovine TREM-1 and RT-PCR analysis revealed diverse mRNA tissue expression for bovine TREM-1. Molecular cloning of bovine TREM-1 extends the repertoire of bovine pattern recognition receptors and provides information important for investigating their role in bovine innate immunity.

PR-39, a porcine antimicrobial peptide, inhibits apoptosis: involvement of caspase-3.

The porcine antimicrobial peptide, PR-39, has several activities beyond its function of killing bacteria. Here we report that PR-39 alters macrophage viability by inhibiting apoptosis, which was induced by nutrient depletion, LPS stimulation or camptothecin treatment. This antiapoptotic effect was pronounced resulting in significant reductions in annexin-V binding to externalized phosphatidylserine and was associated with a decrease in caspase-3 activity. These findings suggest that PR-39, a porcine neutrophil-derived antimicrobial peptide, might function in the inflammatory milieu not only to kill bacteria, but also to aid in modulating the viability of inflammatory cells by regulating apoptosis.

Characterization of an upstream open reading frame in the 5' untranslated region of PR-39, a cathelicidin antimicrobial peptide.

Cathelicidins are antimicrobial peptides present in humans, and laboratory and domestic animals. These peptides are essential components of innate and acquired immune responses; however, little is known about cathelicidin gene regulation. To investigate the regulatory mechanisms of cathelicidin gene expression, we characterized the promoter of the PR-39 gene, a multifunctional cathelicidin. Deletion analysis identified a negative regulatory element in the 5'-flanking region of the gene located in the sequence from nt (nucleotide(s)) -69 to -63. Site-directed mutagenesis indicated that ATG and its vicinity nucleotides are critical for the repressive activity of this region. A primer extension assay identified a transcription start site upstream from the negative repressor ATG and 76 nt upstream from the major open reading frame (ORF). RT-PCR and 3'-RACE further demonstrated that cDNA of PR-39 and the cathelicidin porcine myeloid antimicrobal peptide (PMAP)-23, which share prepro sequence identity, start from nt -79. Sequencing of this region showed that the 5' untranslated region (UTR) of the gene contains an upstream translation start site and an upstream ORF that functions as a repressor of the PR-39 gene. These findings indicate extensive regulation of the PR-39 gene and suggest a mechanism for the tissue-specific and age-dependent repression of this cathelicidin gene.

Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity.

Antimicrobial peptides are important host-defense molecules of innate immunity. Cathelicidins are a diverse family of potent, rapidly acting and broadly effective antimicrobial peptides, which are produced by a variety of cells. This review examines the classification, antimicrobial spectrum, mechanism of action, and regulation of cathelicidins.