Immunohistochemical localization of fibronectin as a tool for the age determination of human skin wounds

We analyzed the distribution of fibronectin in routinely embedded tissue specimens from 53 skin wounds and 6 postmortem wounds. In postmortem wounds a faint but focal positive staining was exclusively found at the margin of the specimens which dit not extend into the adjacent stroma. Vital wounds were classified into 3 groups. The first comprising lesions with wound ages ranging from a few seconds to 30 min, the second comprising those with wound ages upt to 3 weeks, and the third group with lesions more than 3 weeks old. Ten out of 17 lesions with a wound age up to 30 min showed a clear positive reaction within the wound area. Three specimens in this group were completely negative, while in 4 additional cases the result was not significantly different from postmortem lesions. These 7 cases were characterized by acute death with extremely short survival times (only seconds). In wounds up to 3 weeks old fibronectin formed a distinct network containing an increasing number of inflammatory cells corresponding to the wound age. In 2 cases with a survival time of 17 days and in all wounds older than 3 weeks fibronectin was restricted to the surface of fibroblasts and to parallel arranged fibers in the granulation tissue without any network structures. We present evidence that fibronectin is a useful marker for vital wounds with a survival time of more than a few minutes. Fibronectin appears before neutrophilic granulocytes migrate into the wound area. Since a faint positive fibronectin staining is seen in postmortem lesions and bleedings, we propose that only those wounds which show strong positive fibronectin staining also extending into the adjacent stroma should be regarded as vital

Functional and phenotypical comparison of myofibroblasts derived from biopsies and bronchoalveolar lavage in mild asthma and scleroderma

BACKGROUND: Activated fibroblasts, which have previously been obtained from bronchoalveolar lavage fluid (BALF), are proposed to be important cells in the fibrotic processes of asthma and scleroderma (SSc). We have studied the motility for BALF derived fibroblasts in patients with SSc that may explain the presence of these cells in the airway lumen. Furthermore, we have compared phenotypic alterations in activated fibroblasts from BALF and bronchial biopsies from patients with mild asthma and SSc that may account for the distinct fibrotic responses. METHODS: Fibroblasts were cultured from BALF and bronchial biopsies from patients with mild asthma and SSc. The motility was studied using a cell migration assay. Western Blotting was used to study the expression of alpha-smooth muscle actin (α-SMA), ED-A fibronectin, and serine arginine splicing factor 20 (SRp20). The protein expression pattern was analyzed to reveal potential biomarkers using two-dimensional electrophoresis (2-DE) and sequencing dual matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF). The Mann-Whitney method was used to calculate statistical significance. RESULTS: Increased migration and levels of ED-A fibronectin were observed in BALF fibroblasts from both groups of patients, supported by increased expression of RhoA, Rac1, and the splicing factor SRp20. However, these observations were exclusively accompanied by increased expression of α-SMA in patients with mild asthma. Compared to BALF fibroblasts in mild asthma, fibroblasts in SSc displayed a differential protein expression pattern of cytoskeletal- and scavenger proteins. These identified proteins facilitate cell migration, oxidative stress, and the excessive deposition of extracellular matrix observed in patients with SSc. CONCLUSION: This study demonstrates a possible origin for fibroblasts in the airway lumen in patients with SSc and important differences between fibroblast phenotypes in mild asthma and SSc. The findings may explain the distinct fibrotic processes and highlight the motile BALF fibroblast as a potential target cell in these disorders

Prolonged ozone exposure in an allergic airway disease model: Adaptation of airway responsiveness and airway remodeling

BACKGROUND: Short-term exposure to high concentrations of ozone has been shown to increase airway hyper-responsiveness (AHR). Because the changes in AHR and airway inflammation and structure after chronic ozone exposure need to be determined, the goal of this study was to investigate these effects in a murine model of allergic airway disease. METHODS: We exposed BALB/c mice to 2 ppm ozone for 4, 8, and 12 weeks. We measured the enhanced pause (Penh) to methacholine and performed cell differentials in bronchoalveolar lavage fluid. We quantified the levels of IL-4 and IFN-γ in the supernatants of the bronchoalveolar lavage fluids using enzyme immunoassays, and examined the airway architecture under light and electron microscopy. RESULTS: The groups exposed to ozone for 4, 8, and 12 weeks demonstrated decreased Penh at methacholine concentrations of 12.5, 25, and 50 mg/ml, with a dose-response curve to the right of that for the filtered-air group. Neutrophils and eosinophils increased in the group exposed to ozone for 4 weeks compared to those in the filtered-air group. The ratio of IL-4 to INF-γ increased significantly after exposure to ozone for 8 and 12 weeks compared to the ratio for the filtered-air group. The numbers of goblet cells, myofibroblasts, and smooth muscle cells showed time-dependent increases in lung tissue sections from the groups exposed to ozone for 4, 8, and 12 weeks. CONCLUSION: These findings demonstrate that the increase in AHR associated with the allergic airway does not persist during chronic ozone exposure, indicating that airway remodeling and adaptation following repeated exposure to air pollutants can provide protection against AHR

Bovine pericardium based non-cross linked collagen matrix for successful root coverage, a clinical study in human

Introduction: The aim of this study was to clinically assess the capacity of a novel bovine pericardium based, non-cross linked collagen matrix in root coverage. Methods: 62 gingival recessions of Miller class I or II were treated. The matrix was adapted underneath a coronal repositioned split thickness flap. Clinical values were assessed at baseline and after six months. Results: The mean recession in each patient was 2.2 mm at baseline. 6 Months after surgery 86.7% of the exposed root surfaces were covered. On average 0,3 mm of recession remained. The clinical attachment level changed from 3.5 ± 1.3 mm to 1,8 ( ± 0,7) mm during the observational time period. No statistically significant difference was found in the difference of probing depth. An increase in the width of gingiva was significant. With a baseline value of 1.5 ± 0.9 mm an improvement of 2.4 ± 0.8 mm after six month could be observed. 40 out of 62 recessions were considered a thin biotype at baseline. After 6 months all 62 sites were assessed thick. Conclusions: The results demonstrate the capacity of the bovine pericardium based non-cross linked collagen matrix for successful root coverage. This material was able to enhance gingival thickness and the width of keratinized gingiva. The percentage of root coverage achieved thereby is comparable to existing techniques. This method might contribute to an increase of patient's comfort and an enhanced aesthetical outcome

Altered monocyte and fibrocyte phenotype and function in scleroderma interstitial lung disease: reversal by caveolin-1 scaffolding domain peptide

Interstitial lung disease (ILD) is a major cause of morbidity and mortality in scleroderma (systemic sclerosis, or SSc). Fibrocytes are a monocyte-derived cell population implicated in the pathogenesis of fibrosing disorders. Given the recently recognized importance of caveolin-1 in regulating function and signaling in SSc monocytes, in the present study we examined the role of caveolin-1 in the migration and/or trafficking and phenotype of monocytes and fibrocytes in fibrotic lung disease in human patients and an animal model. These studies fill a gap in our understanding of how monocytes and fibrocytes contribute to SSc-ILD pathology. We found that C-X-C chemokine receptor type 4-positive (CXCR4+)/collagen I-positive (ColI+), CD34+/ColI+ and CD45+/ColI+ cells are present in SSc-ILD lungs, but not in control lungs, with CXCR4+ cells being most prevalent. Expression of CXCR4 and its ligand, stromal cell-derived factor 1 (CXCL12), are also highly upregulated in SSc-ILD lung tissue. SSc monocytes, which lack caveolin-1 and therefore overexpress CXCR4, exhibit almost sevenfold increased migration toward CXCL12 compared to control monocytes. Restoration of caveolin-1 function by administering the caveolin scaffolding domain (CSD) peptide reverses this hypermigration. Similarly, transforming growth factor β-treated normal monocytes lose caveolin-1, overexpress CXCR4 and exhibit 15-fold increased monocyte migration that is CSD peptide-sensitive. SSc monocytes exhibit a different phenotype than normal monocytes, expressing high levels of ColI, CD14 and CD34. Because ColI+/CD14+ cells are prevalent in SSc blood, we looked for such cells in lung tissue and confirmed their presence in SSc-ILD lungs but not in normal lungs. Finally, in the bleomycin model of lung fibrosis, we show that CSD peptide diminishes fibrocyte accumulation in the lungs. Our results suggest that low caveolin-1 in SSc monocytes contributes to ILD via effects on cell migration and phenotype and that the hyperaccumulation of fibrocytes in SSc-ILD may result from the altered phenotype and migratory activity of their monocyte precursors

N-α-PGP and PGP, potential biomarkers and therapeutic targets for COPD

© 2009 O'Reilly et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Nrf2 protects against pulmonary fibrosis by regulating the lung oxidant level and Th1/Th2 balance

<p>Abstract</p> <p>Background</p> <p>Pulmonary fibrosis is a progressive and lethal disorder. Although the precise mechanisms of pulmonary fibrosis are not fully understood, oxidant/antioxidant and Th1/Th2 balances may play an important role in many of the processes of inflammation and fibrosis. The transcription factor Nrf2 acts as a critical regulator for various inflammatory and immune responses by controlling oxidative stress. We therefore investigated the protective role of Nrf2 against the development of pulmonary fibrosis.</p> <p>Methods</p> <p>To generate pulmonary fibrosis, both wild-type C57BL/6 mice and Nrf2-deficient mice of the same background were administered bleomycin intratracheally.</p> <p>Results</p> <p>The survival of Nrf2-deficient mice after bleomycin administration was significantly lower than that of wild-type mice. The degree of bleomycin-induced initial pulmonary inflammation and pulmonary fibrosis was much more severe in Nrf2-deficient mice than in wild-type mice. The expression of antioxidant enzymes and phase II detoxifying enzymes was significantly reduced in the lungs of Nrf2-deficient mice, concomitant with an elevation of lung 8-isoprostane level, compared with wild-type mice. The expression of Th2 cytokines, such as interleukin-4 and interleukin-13, was significantly elevated in the lungs of Nrf2-deficient mice with an increase in the number of Th2 cells that express GATA-binding protein 3.</p> <p>Conclusions</p> <p>The results indicated that Nrf2 protects against the development of pulmonary fibrosis by regulating the cellular redox level and lung Th1/Th2 balance. Thus, Nrf2 might be an important genetic factor in the determination of susceptibility to pulmonary fibrosis.</p

Requirement of Podocalyxin in TGF-Beta Induced Epithelial Mesenchymal Transition

Epithelial mesenchymal transition (EMT) is characterized by the development of mesenchymal properties such as a fibroblast-like morphology with altered cytoskeletal organization and enhanced migratory potential. We report that the expression of podocalyxin (PODXL), a member of the CD34 family, is markedly increased during TGF-β induced EMT. PODXL is enriched on the leading edges of migrating A549 cells. Silencing of podocalyxin expression reduced cell ruffle formation, spreading, migration and affected the expression patterns of several proteins that normally change during EMT (e.g., vimentin, E-cadherin). Cytoskeletion assembly in EMT was also found to be dependent on the production of podocalyin. Compositional analysis of podocalyxin containing immunoprecipitates revealed that collagen type 1 was consistently associated with these isolates. Collagen type 1 was also found to co-localize with podocalyxin on the leading edges of migrating cells. The interactions with collagen may be a critical aspect of podocalyxin function. Podocalyxin is an important regulator of the EMT like process as it regulates the loss of epithelial features and the acquisition of a motile phenotype

Latency Associated Peptide Has In Vitro and In Vivo Immune Effects Independent of TGF-β1

Latency Associated Peptide (LAP) binds TGF-β1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-β1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-β1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-β1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation