Studies on the osmophilic fungus Wallemia sebi as an allergen evaluated by skin prick test and radioallergosorbent test

Recently, Wallemia sebi, a species of osmophilic fungi, has been abundantly detected in house dust using low water activity media. In this study, allergenic activity of W. sebi was assessed by skin prick test and radioallergosorbent test (RAST) in 74 asthmatic patients ranging from 6 to 32 years of age. Aspergillus fumigatus and house dust were used for comparison. In skin prick test, W. sebi extract, A. fumigatus extract and house dust extract elicited positive reactions in 4 (5.4%), 4 (5.4%) and 51 (68.9%) patients, respectively. RAST showed positive results in 14 subjects (18.9%) for W. sebi extract, in 8 (10.8%) for A. fumigatus extract and in 59 (79.7%) for house dust extract. These results indicated that some asthmatic individuals showed immediate-type hypersensitivity to W. sebi, and which means this fungal species may be of importance to atopic diseases as a causative agent.     

Comparative chromosome mapping of sex-linked genes and identification of sex chromosomal rearrangements in the Japanese wrinkled frog (Rana rugosa, Ranidae) with ZW and XY sex chromosome systems

There are regional variations of sex chromosome morphologies in the Japanese wrinkled frog, Rana rugosa (2n = 26): heterogametic ZZ/ZW-type and XX/XY-type sex chromosomes, and two different types of homomorphic sex chromosomes. To search for homology between the ZW and XY sex chromosomes and the chromosome rearrangements that have occurred during sex chromosomal differentiation in R. rugosa, we performed chromosome mapping of sexual differentiation genes for R. rugosa by FISH. Three genes, AR, SF-1/Ad4BP and Sox3, were localized to both the ZW and XY chromosomes, and their locations were all different between the Z and W and between the X and Y. AR and SF-1/Ad4BP were located on the short arms of the W and X and the long arms of Z and Y, and Sox3 was mapped to the different locations on the long arms between the Z and W and between the X and Y, probably as a result of multiple rearrangements that occurred during the process of sex chromosome differentiation. However, the chromosomal locations of three genes were almost consistent between the Z and Y and between the W and X, indicating that the Z and Y chromosomes and the W and X chromosomes were respectively derived from the same origins. Dmrt1, which is located on avian sex chromosomes, was localized to autosomes in R. rugosa with both the ZW and XY sex chromosomes, suggesting that Dmrt1 might not be related to sex determination in this species.     

Corticosteroid enhances TNF‐α‐mediated leukocyte adhesion to pulmonary microvascular endothelial cells

Background: Some severe asthma patients are characterized by elevated levels of tumor necrosis factor alpha (TNF‐α) and neutrophilic inflammation in the airways. Although such phenotypic changes in asthma might contribute to corticosteroid refractoriness, the role of TNF‐α in the process remains unclear. TNF‐α exerts its biological effects mainly by acting on the vascular endothelium, and thereby upregulates leukocyte recruitment into inflamed tissues. The aim of this study was to investigate the effects of dexamethasone (DEX) on the TNF‐α‐mediated responses of human microvascular endothelial cells from lung blood vessels (HMVEC‐LBl) in vitro. Methods: HMVEC‐LBl were cultured with TNF‐α in the presence and absence of DEX. The effects of DEX on various TNF‐α‐mediated responses, such as the expressions of chemokines and cellular adhesion molecules, leukocyte adhesion were determined. Results: TNF‐α significantly induced growth‐related oncogene alpha (GRO‐α), interleukin 8 (IL‐8), regulated on activation, normal T‐cell expressed and secreted (RANTES) and interferon‐inducible protein 10 (IP‐10) productions and cell surface expressions of intracellular adhesion molecule 1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1) on HMVEC‐LBl. TNF‐α‐induced GRO‐α and IL‐8 were slightly attenuated by DEX treatment (reaches to 89% and 79%, respectively), whereas expressions of IP‐10, ICAM‐1 and VCAM‐1 were significantly enhanced by the same treatment (up to 172%, 152% and 139%, respectively). Correspondingly, in vitro adhesion of eosinophils and neutrophils to TNF‐α‐treated HMVEC‐LBl were significantly enhanced by DEX. Conclusions: Some proinflammatory effects of DEX, a corticosteroid, were found in TNF‐α‐mediated in vitro reactions of pulmonary microvascular endothelial cells, i.e. chemokine productions and leukocyte adhesion. These in vitro results may explain, at least in part, the corticosteroid refractoriness accompanied by a marked increase in TNF‐α production that is seen in severe asthmatic patients.     

Interferon-{beta} inhibits bleomycin-induced lung fibrosis by decreasing transforming growth factor-{beta} and thrombospondin

Pulmonary fibrosis is the result of abnormal processes of repair that occur after lung injury. Transforming growth factor (TGF)-beta is a key molecule in the progression of pulmonary fibrosis. Although clinical use of interferon (IFN)-beta did not improve survival in patients with idiopathic pulmonary fibrosis, because some preclinical studies have suggested that IFN-beta is a potent inhibitor of fibrogenesis, beneficial effects of IFN-beta have been expected. We therefore attempted to determine effects of IFN-beta and investigated the mechanism of action of IFN-beta in bleomycin-induced pulmonary fibrosis. Bleomycin at Day 0 and IFN-beta for 4 wk were administered intravenously to ICR mice. At 28 d after bleomycin injection, histologic and chemical analysis was performed for evaluation of effects of IFN-beta. Tissue distribution and amounts of TGF-beta1 and thrombospondin (TSP)-1/2 were analyzed. IFN-beta attenuated prolylhydroxylase activity, resulting in inhibition of pulmonary fibrosis. Bleomycin-induced increase in TGF-beta1 in epithelial cells and extracellular matrix was attenuated by IFN-beta. TSP-1/2 was limited in platelets of control mice, but was present in foamy cells in fibrotic regions induced by bleomycin. These findings suggest that the antifibrotic effect of IFN-beta is inhibition of TGF-beta and its activation via decrease in TSP-1/2 in lung tissue and change in location of TSP-1/2 from platelets to foamy cells.     

Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse

The sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins that are encoded by a single gene in tandem within a large precursor protein (prosaposin) and are required for in vivo degradation of some sphingolipids with relatively short carbohydrate chains. Human patients with prosaposin or specific saposin B or C deficiency are known, and prosaposin- and saposin A-deficient mouse lines have been generated. Experimental evidence suggests that saposin D may be a lysosomal acid ceramidase activator. However, no specific saposin D deficiency state is known in any mammalian species. We have generated a specific saposin D(-/-) mouse by introducing a mutation (C509S) into the saposin D domain of the mouse prosaposin gene. Saposin D(-/-) mice developed progressive polyuria at around 2 months and ataxia at around 4 months. Pathologically, the kidney of saposin D(-/-) mice showed renal tubular degeneration and eventual hydronephrosis. In the nervous system, progressive and selective loss of the cerebellar Purkinje cells in a striped pattern was conspicuous, and almost all Purkinje cells disappeared by 12 months. Biochemically, ceramides, particularly those containing hydroxy fatty acids accumulated in the kidney and the brain, most prominently in the cerebellum. These results not only indicate the role of saposin D in in vivo ceramide metabolism, but also suggest possible cytotoxicity of ceramide underlying the cerebellar Purkinje cell and renal tubular cell degeneration.     

Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung

Combined small cell and non-small cell carcinoma is relatively rare in the lung. Examination of the clonal relationship of different components in this type of tumor may give a clue to the rarity. We retrieved 6 such tumors; all 6 had small cell carcinoma and adenocarcinoma components, and 3 had an additional squamous cell carcinoma component. We examined the point mutations in the p53 gene and allelic loss (ie, the loss of heterozygosity [LOH] pattern) of chromosome 3p in each component. p53 mutations were detected in the small cell carcinoma component of 5 tumors and in the non-small cell carcinoma components of 2 tumors. In 1 case, the squamous cell carcinoma component had a p53 mutation locus identical to that in the small cell carcinoma component, but in the other case, the adenocarcinoma component had a different mutation than that in the small cell carcinoma component. Chromosome 3p LOH loci in the squamous cell carcinoma component were present in the small cell carcinoma component in all 3 cases, but some LOH loci were not identical in the small cell carcinoma and adenocarcinoma components in 3 cases. These results suggest that the small cell and squamous cell carcinoma components of combined small cell lung carcinomas have an intimate clonal relationship. On the other hand, the adenocarcinoma component often may be derived from a separate clone or, more likely, undergo a progressive process separate from the squamous cell-small cell carcinoma beginning in a very early stage, that is, before the appearance of p53 and chromosome 3p abnormalities. This tumorigenesis process may explain the relative rarity of combined small cell and non-small cell carcinoma, which occurs primarily in the peripheral lung, an infrequent site of squamous cell carcinoma.     

Confocal imaging of biofilm formation process using fluoroprobed Escherichia coli and fluoro-stained exopolysaccharide

We developed a novel method of evaluating biofilm architecture on a synthetic material using green fluorescent protein-expressing Escherichia coli and red fluorescence staining of exopolysaccharides. Confocal laser scanning microscopy observation revealed the time course of the change in the in situ three-dimensional structural features of biofilm on a polyurethane film without structural destruction: initially adhered cells are grown to form cellular aggregates and secrete exopolysaccharides. These cells were spottily distributed on the surface at an early incubation time but fused to form a vertically grown biofilm with incubation time. Fluorescence intensity, which is a measure of the number of cells, determined using a fluorometer and biofilm thickness determined from confocal laser scanning microscopy vertical images were found to be effective for quantification of time-dependent growth of biofilms. The curli (surface-located fibers specifically binding to fibronectin and laminin)-producing Escherichia coli strain, YMel, significantly proliferated on fibronectin-coated polyurethane, whereas the curli-deficient isogenic mutant, YMel-1, did not. The understanding of biofilm architecture in molecular and morphological events and new fluorescence microscopic techniques may help in the logical surface design of biomaterials with a high antibacterial potential.     

Inhibitory effect of aerosol WP871 on SRS-A mediated bronchoconstriction in the guinea pig in vivo

Slow-reacting substance of anaphylaxis (SRS-A) is an important factor mediating bronchoconstriction in asthma. We developed a guinea pig model for SRS-A mediated bronchoconstriction induced by antigen inhalation. Using this model, we investigated the effect of inhaled WP871, a new anti-allergic drug, on bronchoconstriction. Aerosol WP871 (0.01 and 0.033%) to some extent inhibited the antigen-induced bronchoconstriction in a dose-dependent fashion, but high-dose WP871 (0.1%) inhalation itself produced a non-specific bronchoconstriction. However, aerosol WP871 (0.033%) showed no inhibitory effect on bronchoconstriction caused by direct inhalation of leukotriene C4, a component of SRS-A. These findings indicate that aerosol WP871 does not antagonize SRS-A, but inhibits synthesis and/or release of SRS-A and has some non-specific bronchoconstrictive effect in high concentration.