We developed a method for obtaining viable buccal cells from mouthwash samples for use as a source of mRNA and protein. Immunofluorescent analysis showed that most cells were derived from nonkeratinized parabasal epithelia, with a minor proportion of proliferative cells. Gene expression was detected in buccal cells using reverse transcription PCR, Western blot analysis, and immunofluorescence. Using a keratinocyte-specific medium, buccal cells could be cultured on Matrigel-coated permeable filters for up to 2 weeks while maintaining the expression of some epithelial-specific markers, including cytokeratin 13, cytokeratin 10, transferrin receptor, and beta-integrin. The basal marker cytokeratin 14 and Ki67, an indicator of cellular proliferation, were detected in a few cells. We show that buccal cells can be obtained from a noninvasive procedure for use as a source of material for biochemical analyses. A population of the buccal cells can be maintained in culture for up to 2 weeks using keratinocyte-specific medium in combination with extracellular matrix.

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We describe an experimental model in Wistar rats of non-resolving bronchopneumonia evoked by Streptococcus pneumoniae type 25. In contrast to a model of resolving streptococcal pneumonia that we have previously described, morphological studies reveal that in this model, there is significant early damage to type 1 pneumocytes which progresses to necrosis, leaving isolated areas of denuded alveolar basement membrane. Furthermore, there is accompanying degeneration and necrosis of a proportion of the type 2 pneumocytes, and alveolar epithelial repair by proliferation and differentiation of these cells appears to be retarded. Isolated, hypertrophic, and hyperplastic foci of type 2 pneumocytes persist as the acute inflammatory response subsides, and organization progresses with proliferation and emigration of fibroblasts into the lumina of alveoli and terminal bronchioles. The resultant lesion is morphologically indistinguishable from bronchiolitis obliterans organizing pneumonia. We hypothesize that the abnormal outcome in this model of pneumonia is a consequence of the failure of proliferating type 2 pneumocytes to transform into type 1 pneumocytes and thus maintain the integrity of the alveolar epithelial surface.

1 Tumour necrosis factor-a (TNF-a) is a cytokine with diverse properties consistent with a possible role in in¯ammatory disease. We investigated whether TNF-a is induced during the progression of lung in¯ammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-a expression in¯uences recruitment of speci®c in¯ammatory cell types. 2 A single intravenous injection of Sephadex particles into rats led to extensive granulomatous in¯ammation in lung alveolar and bronchial tissue that peaked in intensity after 24 ± 72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3 Messenger RNA encoding TNF-a was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-a protein was localized to mononuclear cells at all times points preand post-Sephadex administration. 4 Treatment of rats with dexamethasone signi®cantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and signi®cantly reduced TNF-a mRNA expression by BAL cells. 5 Treatment of rats with cyclosporin A was without e ect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-a, but did reduce signi®cantly recruitment of neutrophils and eosinophils to BAL cell populations. 6 These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-a expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.

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