Striking deposition of toxic eosinophil major basic protein in mucus: implications for chronic rhinosinusitis.

BACKGROUND: The mechanisms by which eosinophilic inflammation damages the epithelium and contributes to recurrent acute exacerbations in chronic rhinosinusitis (CRS) have not been fully elucidated. OBJECTIVE: We tested the hypotheses that eosinophils deposit toxic major basic protein (MBP) in the mucus and that MBP reaches concentrations able to damage the sinonasal epithelium. METHODS: Tissue specimens with mucus attached to the tissue were carefully collected from 22 patients with CRS and examined by using immunofluorescence staining for MBP. This immunofluorescence was digitally analyzed to determine the area covered by MBP and the intensity of the staining (estimating MBP concentration). Levels of MBP in extracts from nasal mucus were quantitated by means of RIA. RESULTS: Heterogeneous eosinophilia was evident within tissue and mucus specimens. All tissue specimens showed intact eosinophils, but diffuse extracellular MBP deposition, as a marker of eosinophil degranulation, was rare. In contrast, all mucus specimens showed diffuse MBP throughout and abundant diffuse extracellular MBP deposition within clusters of eosinophils. Digitized analyses of MBP immunofluorescence revealed increased area coverage (P < .0001) in mucus compared with that seen in tissue. Estimated concentrations of MBP within the clusters suggested toxic levels. MBP concentrations in mucus extract reached 11.7 microg/mL; MBP was not detectable in healthy control subjects. CONCLUSION: In patients with CRS, eosinophils form clusters in the mucus where they release MBP, which is diffusely deposited on the epithelium, a process not observed in the tissue. Estimated MBP levels far exceed those needed to damage epithelium from the luminal side and could predispose patients with CRS to secondary bacterial infections.

Human aortic valve calcification is associated with an osteoblast phenotype.

BACKGROUND: Calcific aortic stenosis is the third most common cardiovascular disease in the United States. We hypothesized that the mechanism for aortic valve calcification is similar to skeletal bone formation and that this process is mediated by an osteoblast-like phenotype. METHODS AND RESULTS: To test this hypothesis, we examined calcified human aortic valves replaced at surgery (n=22) and normal human valves (n=20) removed at time of cardiac transplantation. Contact microradiography and micro-computerized tomography were used to assess the 2-dimensional and 3-dimensional extent of mineralization. Mineralization borders were identified with von Kossa and Goldner's stains. Electron microscopy and energy-dispersive spectroscopy were performed for identification of bone ultrastructure and CaPO4 composition. To analyze for the osteoblast and bone markers, reverse transcriptase-polymerase chain reaction was performed on calcified versus normal human valves for osteopontin, bone sialoprotein, osteocalcin, alkaline phosphatase, and the osteoblast-specific transcription factor Cbfa1. Microradiography and micro-computerized tomography confirmed the presence of calcification in the valve. Special stains for hydroxyapatite and CaPO4 were positive in calcification margins. Electron microscopy identified mineralization, whereas energy-dispersive spectroscopy confirmed the presence of elemental CaPO4. Reverse transcriptase-polymerase chain reaction revealed increased mRNA levels of osteopontin, bone sialoprotein, osteocalcin, and Cbfa1 in the calcified valves. There was no change in alkaline phosphatase mRNA level but an increase in the protein expression in the diseased valves. CONCLUSIONS: These findings support the concept that aortic valve calcification is not a random degenerative process but an active regulated process associated with an osteoblast-like phenotype.

Centrin-2 is required for centriole duplication in mammalian cells.

BACKGROUND: Centrosomes are the favored microtubule-organizing framework of eukaryotic cells. Centrosomes contain a pair of centrioles that normally duplicate once during the cell cycle to give rise to two mitotic spindle poles, each containing one old and one new centriole. However, aside from their role as an anchor point for pericentriolar material and as basal bodies of flagella and cilia, the functional attributes of centrioles remain enigmatic. RESULTS: Here, using RNA interference, we demonstrate that "knockdown" of centrin-2, a protein of centrioles, results in failure of centriole duplication during the cell cycle in HeLa cells. Following inhibition of centrin-2 synthesis, the preexisting pair of centrioles separate, and functional bipolar spindles form with only one centriole at each spindle pole. Centriole dilution results from the ensuing cell division, and daughter cells are "born" with only a single centriole. Remarkably, these unicentriolar daughter cells may complete a second and even third bipolar mitosis in which spindle microtubules converge onto unusually broad spindle poles and in which cell division results in daughter cells containing either one or no centrioles at all. Cells thus denuded of the mature or both centrioles fail to undergo cytokinesis in subsequent cell cycles, give rise to multinucleate products, and finally die. CONCLUSIONS: These results demonstrate a requirement for centrin in centriole duplication and demonstrate that centrioles play a role in organizing spindle pole morphology and in the completion of cytokinesis.

Atorvastatin inhibits hypercholesterolemia-induced cellular proliferation and bone matrix production in the rabbit aortic valve.

BACKGROUND: Despite the common occurrence of aortic stenosis, the cellular causes of the disorder are unknown, in part because of the absence of experimental models. We hypothesized that atherosclerosis and early bone matrix expression in the aortic valve occurs secondary to experimental hypercholesterolemia and that treatment with atorvastatin modifies this transformation. METHODS AND RESULTS: To test this hypothesis, we developed an experimental hypercholesterolemic rabbit model. New Zealand White rabbits (n=48) were studied: group 1 (n=16), normal diet; group 2 (n=16), 1% (wt/wt) cholesterol diet; and group 3 (n=16), 1% (wt/wt) cholesterol diet plus atorvastatin (3 mg/kg per day). The aortic valves were examined with hematoxylin and eosin stain, Masson trichrome, macrophage (RAM 11), proliferation cell nuclear antigen (PCNA), and osteopontin immunostains. Cholesterol and highly sensitive C-reactive protein (hsCRP) serum levels were obtained by standard assays. Computerized morphometry and digital image analysis were performed for quantifying PCNA (% area). Electron microscopy and immunogold labeling were performed for osteopontin. Semiquantitative RT-PCR was performed for the osteoblast bone markers [alkaline phosphatase, osteopontin, and osteoblast lineage-specific transcription factor (Cbfa-1)]. There was an increase in cholesterol, hsCRP, PCNA, RAM 11, and osteopontin and osteoblast gene markers (alkaline phosphatase, osteopontin, and Cbfa-1) in the cholesterol-fed rabbits compared with control rabbits. All markers except hsCRP were reduced by atorvastatin. CONCLUSIONS: These findings of increased macrophages, PCNA levels, and bone matrix proteins in the aortic valve during experimental hypercholesterolemia provide evidence of a proliferative atherosclerosis-like process in the aortic valve associated with the transformation to an osteoblast-like phenotype that is inhibited by atorvastatin.

Immunomagnetic separation reagents as markers in electron microscopy.

Antibodies coupled to magnetic particles have been employed for immunomagnetic cell isolation, but their consequent use for electron microscopy (EM) has not been evaluated. We used commercial antibodies coupled to iron-dextran to isolate T cells and monocytes/macrophages by immunomagnetic adsorption from normal human peripheral blood mononuclear cells. Subsequently, we studied the association of electron-dense immunomagnetic reagents with cell membranes. CD14-positive monocytes/macrophages isolated from fixed peripheral blood mononuclear cells retained electron-dense beads on the plasma membrane, while live cells internalized them. Flow cytometry and electron microscopy measurements of the percentage of cells that bound a CD4-specific immunomagnetic reagent in pan-T cell isolates (containing numerous T cell subtypes) were indistinguishable. The immunomagnetic reagent associated with cells could be secondarily labeled by secondary antibody coupled to colloidal gold. This study shows that these reagents used for cell isolation or just labeling, remain associated with their targets at the cell membrane. Immunomagnetic reagents allow "capturing" of rare cells from complex mixtures, purifying and concentrating them in a single step for subsequent electron microscopy. The large number of commercially available immunomagnetic reagents specific for different human, mouse and rat antigens provides additional resources for visualization of cellular ultrastructure.

Localization of caveolin 1 in aortic valve endothelial cells using antigen retrieval.

Ultrastructural analysis of aortic valve endothelial cells subjected to growth arrest revealed many vesicles defined as caveolae by the localization of caveolin. Translocation of caveolin after exposure to oxidized LDL suggests that the localization of caveolin may be a valuable tool to study models of early atherogenesis. In this study, several antigen retrieval protocols were tested in osmium-fixed and Spurr-embedded cells to determine the optimal method of antigen retrieval in our model system. SDS produced the most consistent labeling pattern. A quantitative evaluation revealed that SDS significantly increased the labeling density in Spurr-embedded cells. The labeling pattern appeared as clusters of gold particles, 15-40 nm in diameter, that were associated with membranes of a similar size which may represent the neck region of the caveolae.