Respiratory RNA viruses responsible for the common cold often worsen airway inflammation and bronchial responsiveness, two characteristic features of human asthma. We studied the effects of dsRNA, a nucleotide synthesized during viral replication, on airway inflammation and bronchial hyperresponsiveness in murine models of asthma. Intratracheal instillation of poly I:C, a synthetic dsRNA, increased the airway eosinophilia and enhanced bronchial hyperresponsiveness to methacholine in OVA-sensitized, exposed rats. These changes were associated with induction of cyclooxygenase-2 (COX-2) expression and COX-2-dependent PGD2 synthesis in the lungs, particularly in alveolar macrophages. The direct intratracheal instillation of PGD2 enhanced the eosinophilic inflammation in OVA-exposed animals, whereas pretreatment with a dual antagonist against the PGD2 receptor-(CRTH2) and the thromboxane A2 receptor, but not with a thromboxane A2 receptor-specific antagonist, nearly completely eliminated the dsRNA-induced worsening of airway inflammation and bronchial hyperresponsiveness. CRTH2-deficient mice had the same degree of allergen-induced airway eosinophilia as wild-type mice, but they did not exhibit a dsRNA-induced increase in eosinophil accumulation. Our data demonstrate that COX-2-dependent production of PGD2 followed by eosinophil recruitment into the airways via a CRTH2 receptor are the major pathogenetic factors responsible for the dsRNA-induced enhancement of airway inflammation and responsiveness.

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α–converting enzyme (TACE), critical for the cleavage of membrane-bound pro–TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.

Allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a two-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). Allogeneic CDS can be cryopreserved and transported to other hospitals in a frozen state. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF)-AA, transforming growth factor (TGF)-beta1, keratinocytes growth factor (KGF), interleukin (IL)-6 and IL-8 were contained in the culture medium which was used in preparing CDS over a cultivation period of one week (fresh CDS culture medium sample). After thawing a cryopreserved CDS, the CDS was recultured in a culture medium for one week. VEGF, bFGF, HGF, TGF-beta1 and IL-8 were contained in the culture medium which was used in reculturing CDS for one week (cryopreserved CDS culture medium sample), although some cytokines were detected at a lower level than those before freezing. This finding suggests that the cryopreserved CDS retains its ability to release these cytokines. Clinical research on allogeneic CDS, which was newly developed at the R & D Center for Artificial Skin of Kitasato University, has been carried out in medical centers across Japan with the support of the Millennium Project of the Ministry of Health, Labor and Welfare. It was demonstrated that the allogeneic CDS functions as an excellent cell therapy for intractable skin ulcers as well as burn injuries. The spongy matrix itself, as well as the cytokines released from the allogeneic CDS, seemed to be beneficial for the treatment of intractable skin defect.

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