Immunohistochemical analysis of ocular platelet basic protein expression during infection with Pseudomonas aeruginosa.

Platelet basic protein (PBP) and several of its derivatives are known to express a wide range of biological characteristics. It is the precursor of connective tissue activating peptide (CTAP-III), beta thromboglobulin (beta-TG) and neutrophil activating peptide (NAP-2), which is the proteolytic derived end product. The temporal ocular expression of the chemokine PBP before and during corneal infection over several days by Pseudomonas aeruginosa was examined by immunohistochemistry. Prior to corneal infection, immunohistochemical staining demonstrated the constitutive expression of PBP in the cornea, lens and retina. PBP expression was present in the corneal epithelium, stromal fibroblasts and endothelium. There was a temporal increase in PBP expression in the cornea after infection. The entire cornea exhibited extensive cellular infiltration by positive PBP staining infiltrating cells within 6 days post-infection. The cornea, lens and retina underwent extensive degradation within 5-6 days post-infection with some apparent selective increase in PBP staining in the lens and retina.

Increased invasion through basement membrane by CXCL7-transfected breast cells.

BACKGROUND: CXC chemokines may modify breast cancer cells and surrounding extracellular matrix to facilitate metastasis. CXCL7 is heparin binding, has heparanase activity, and is a ligand to CXCR2, a G-protein-linked receptor. METHODS: Isogenic cell lines, malignant MCF10CA1a.cl1 cells, and premalignant MCF10AT cells were used. CXCR2 and CXCL7 expression levels were quantified by reverse transcriptionase-polymerase chain reaction and Western blot. MCF10AT cells were stably transfected with CXCL7, and matrigel invasion assays were performed. Antibody to CXCL7 was used to inhibit invasion. CXCL7 secretion by transfectants and heparanase activity were quantified by enzyme-linked immunosorbent assay. RESULTS: CXCL7 and CXCR2 expression were significantly higher in malignant MCF10CA1a.cl1 cells than in premalignant MCF10AT cells. Secreted CXCL7, secreted heparanase activity, and invasiveness were all increased in CXCL7-transfected MCF10AT cells. CXCL7 antibody inhibited invasion of CXCL7-transfected MCF10AT cells. CONCLUSIONS: Malignant MCF10CA1a.cl1 cells express more CXCL7 and CXCR2 than premalignant MCF10AT cells. CXCL7-transfected MCF10AT cells are as invasive as malignant breast cancer cells.

Expression patterns of lymphangiogenic and angiogenic factors in a model of breast ductal carcinoma in situ.

BACKGROUND: Foci of invasion are found in greater than 20% of excised specimens of breast ductal carcinoma in situ (DCIS). Since lymphangiogenesis markers are associated with the potential for increased lymph node metastasis, the purpose of the current study was to determine expression of lymphangiogenesis molecular markers in a model of aggressive DCIS. METHODS: From the MCF10A xenograft model, comedo type MCF10DCIS.com cells, premalignant MCF10AT, and invasive MCF10CA1a.cl1 cells were tested. Invasion was tested by Matrigel invasion assays (Becton-Dickinson, Bedford, MA). Gene expression was determined by reverse transcriptase-polymerase chain reaction and protein expression by immunoblot, normalized to beta-actin. RESULTS: MCF10DCIS.com cells were 4-fold more invasive than MCF10AT cells (P < .01), and expressed several-fold more mRNA and protein than MCF10AT and MCF10CA1a.cl1 cells for vascular endothelial growth factor C, vascular endothelial growth factor D, and lymphatic vessel endothelial hyaluronan receptor 1 (P < .01). CONCLUSIONS: A subset of comedo-type DCIS cells are invasive, and expression of lymphangiogenesis markers is greater at the mRNA and protein levels than by invasive cancer cells (P < .01). These additional molecular markers may characterize aggressive DCIS more precisely.

Murine ocular heparanase expression before and during infection with Pseudomonas aeruginosa.

PURPOSE: To demonstrate the constitutive expression and regulation of heparanase (heparan sulfate endoglycosidase) in the normal mouse eye and in mice intracorneally infected with Pseudomonas aeruginosa. METHODS: Naïve (unimmunized) and immunized C57BL/6J mice were infected with P. aeruginosa, and corneal heparanase gene and protein expression were detected by semiquantitative RT-PCR and immunoblot analysis. Immunohistochemistry was also applied to characterize corneal heparanase in naïve mice. RESULTS: Heparanase mRNA and protein expression were detected in uninfected corneas of C57BL/6J mice. Immunohistochemical studies indicated heparanase protein expression was primarily in the corneal epithelium before corneal infection and was also in the corneal stroma after infection. Immunohistochemical studies of uninfected and infected whole eyes of naïve mice indicated heparanase protein expression in most layers of the retina, but the expression did not appear to be upregulated during corneal infection. Staining was most intense in the inner photoreceptor layer of the retina. CONCLUSIONS: Heparanase was constitutively expressed in both the corneal epithelium and several retinal layers before intracorneal infection with P. aeruginosa. Temporal upregulation of corneal heparanase protein expression was detected in naïve mice during infection, most likely due to heparanase positive infiltrating cells, but the protein was not upregulated in corneas from immunized mice because they had a lower inflammatory response, associated with the restoration of corneal clarity. There did not appear to be temporal upregulation of heparanase expression in the retina of infected mice, as determined by immunohistochemistry.