Increased ER Stress and Unfolded Protein Response Activation in Epithelial and Inflammatory Cells in Hypersensitivity Pneumonitis.

Several types of cytotoxic insults disrupt endoplasmic reticulum (ER) homeostasis, cause ER stress, and activate the unfolded protein response (UPR). The role of ER stress and UPR activation in hypersensitivity pneumonitis (HP) has not been described. HP is an immune-mediated interstitial lung disease that develops following repeated inhalation of various antigens in susceptible and sensitized individuals. The aim of this study was to investigate the lung expression and localization of the key effectors of the UPR, BiP/GRP78, CHOP, and sXBP1 in HP patients compared with control subjects. Furthermore, we developed a mouse model of HP to determine whether ER stress and UPR pathway are induced during this pathogenesis. In human control lungs, we observed weak positive staining for BiP in some epithelial cells and macrophages, while sXBP1 and CHOP were negative. Conversely, strong BiP, sXBP1- and CHOP-positive alveolar and bronchial epithelial, and inflammatory cells were identified in HP lungs. We also found apoptosis and autophagy markers colocalization with UPR proteins in HP lungs. Similar results were obtained in lungs from an HP mouse model. Our findings suggest that the UPR pathway is associated with the pathogenesis of HP.

Changes in the levels of free sialic acid during ex vivo lung perfusion do not correlate with pulmonary function. Experimental model.

BACKGROUND: Ex vivo lung perfusion (EVLP) constitutes a tool with great research potential due to its advantages over in vivo and in vitro models. Despite its important contribution to lung reconditioning, this technique has the disadvantage of incurring high costs and can induce pulmonary endothelial injury through perfusion and ventilation. The pulmonary endothelium is made up of endothelial glycocalyx (EG), a coating of proteoglycans (PG) on the luminal surface. PGs are glycoproteins linked to terminal sialic acids (Sia) that can affect homeostasis with responses leading to edema formation. This study evaluated the effect of two ex vivo perfusion solutions on lung function and endothelial injury. METHODS: We divided ten landrace swine into two groups and subjected them to EVLP for 120 min: Group I (n = 5) was perfused with Steen® solution, and Group II (n = 5) was perfused with low-potassium dextran-albumin solution. Ventilatory mechanics, histology, gravimetry, and sialic acid concentrations were evaluated. RESULTS: Both groups showed changes in pulmonary vascular resistance and ventilatory mechanics (p < 0.05, Student's t-test). In addition, the lung injury severity score was better in Group I than in Group II (p < 0.05, Mann-Whitney U); and both groups exhibited a significant increase in Sia concentrations in the perfusate (p < 0.05 t-Student) and Sia immunohistochemical expression. CONCLUSIONS: Sia, as a product of EG disruption during EVLP, was found in all samples obtained in the system; however, the changes in its concentration showed no apparent correlation with lung function.

Clinical, Histological, and Profibrotic Extracellular Matrix Protein Changes in a Model of Tracheal Stenosis Induced by Cervical Tracheal Autotransplantation.

BACKGROUND: Tracheal stenosis (TS) is a complication of prolonged intubation, tracheotomy, and tracheal surgery that compromises the vascular supply. Animal models are essential for studying its pathophysiology and the effect of interventions. OBJECTIVE: To establish a TS model in rats secondary to tracheal autotransplantation with a graft submerged in bleomycin (Atx-Bleo). Additionally, to evaluate the clinical and histological changes, as well as the expression of newly formed collagen (NFC), isoforms of transforming growth factor beta (TGFß), fibronectin (FN), elastin (ELN), integrin ß1 (ITGß1), and matrix metalloproteinase 1 (MMP1) in TS. METHODS: Twenty Wistar rats were divided into three groups: group I (n = 20) control; group II (n = 10) end-to-end anastomosis of the trachea (tracheoplasty); and group III (n = 10) Atx-Bleo. The animals were evaluated clinically, tomographically, macroscopically, morphometrically, and microscopically. NFC deposition, and the expression of profibrotic and antifibrotic proteins were evaluated in tracheal scars. RESULTS: All animals survived the surgical procedure and the study period. Compared with the other study groups, the Atx-Bleo group developed TS and fibrosis, exhibited higher expression of NFC, TGFß1, TGFß2, FN, ELN, and ITGß1, and mild expression of TGFß3 and MMP1 (p < 0.005; analysis of variance, Dunnett and Tukey tests). CONCLUSION: Atx-Bleo in TS model rats produces tomographic and histological changes, and induces the upregulation of profibrotic proteins (TGFß1, TGFß2, collagen, FN, ELN, ITGß1) and downregulation of antifibrotic proteins (TGFß3, MMP1). Therefore, this model may be used to test new pharmacological treatments for reversing or preventing TS, and conduct basic studies regarding its pathophysiology.

Endothelin-Converting Enzyme 1 and Vascular Endothelial Growth Factor as Potential Biomarkers during Ex Vivo Lung Perfusion with Prolonged Hypothermic Lung-Sparing.

Lung transplantation requires optimization of donor's organ use through ex vivo lung perfusion (EVLP) to avoid primary graft dysfunction. Biomarkers can aid in organ selection by providing early evidence of suboptimal lungs during EVLP and thus avoid high-risk transplantations. However, predictive biomarkers of pulmonary graft function such as endothelin-converting enzyme (ECE-1) and vascular endothelial growth factor (VEGF) have not been described under EVLP with standard prolonged hypothermic preservation, which are relevant in situations where lung procurement is difficult or far from the transplantation site. Therefore, this study is aimed at quantifying ECE-1 and VEGF, as well as determining their association with hemodynamic, gasometric, and mechanical ventilatory parameters in a swine model of EVLP with standard prolonged hypothermic preservation. Using a protocol with either immediate (I-) or delayed (D-) initiation of EVLP, ECE-1 levels over time were found to remain constant in both study groups (p > 0.05 RM-ANOVA), while the VEGF protein was higher after prolonged preservation, but it decreased throughout EVLP (p > 0.05 RM-ANOVA). Likewise, hemodynamic, gasometric, mechanical ventilatory, and histological parameters had a tendency to better results after 12 hours of hypothermic preservation in the delayed infusion group.

Expression of Claudin-4 in Lung Ischemia-Reperfusion Injury in Experimental Lung Transplantation.

OBJECTIVE: To assess the presence of CLDN4 in bronchoalveolar lavage fluid (BALF) and pulmonary tissue as an early indicator of LIRI and its relationship with changes in pulmonary physiology, edema formation and histology in an experimental porcine model of LTx with CIT of 50 min or 6 h. METHODS: In 12 pigs, LIRI was produced by: group I (n = 6) LTx with 50 min of CIT (LTx-50 min-CIT); and group II (n = 6) LTx with 6 h of CIT (LTx-6h-CIT). The lung function, edema formation, macroscopic and microscopic changes were assessed. CLDN4 expression in BALF and pulmonary tissue were determined. RESULTS: Both groups presented similar clinical, edema, and histological damage, as well as similar expression of CLDN4 in BALF and tissue (p > 0.05, RM-ANOVA). CONCLUSION: CLDN4 expressed in BALF and the pulmonary tissue during the first 5 h within 72 h of the PGD window are not associated by the deterioration of lung function, edema and lung histological injury, in LTx with CIT 50 min or 6 h, CLDN4 does not seem to be a valuable indicator of LIRI.

In vivo performance of decellularized tracheal grafts in the reconstruction of long length tracheal defects: Experimental study.

BACKGROUND: The repair of long-segment tracheal lesions remains an important challenge. Nowdays no predictable and dependable substitute has been found. Decellularized tracheal scaffolds have shown to be a promising graft for tracheal transplantation, since it is non-immunogenic. OBJECTIVE: Evaluate in vivo decellularized tracheal allografts performance to replace long tracheal segment. METHODS: Forty-five swines underwent surgery as follows: Fifteen trachea donors and 30 receptors of decellularized trachea allografts. The receptors were randomly divided in five groups (n = 6). In groups I and II, donor trachea segment was decellularized by 15 cycles with sodium deoxycholate and deoxyribonuclease, in group II, the allograft was reinforced with external surgical steel wire. Groups, III, IV, and V decellularization was reduced to seven cycles, supplemented with cryopreservation in group IV and with glutaraldehyde in group V. A 10 rings segment was excised from the receptor swine and the decellularized trachea graft was implanted to re-establish trachea continuity. RESULTS: Both decellularization cycles caused decreased stiffness. All trachea receptors underwent euthanasia before the third post-implant week due to severe dyspnea and trachea graft stenosis, necrosis, edema, inflammation, hemorrhage, and granulation tissue formation in anastomotic sites. Histologically all showed total loss of epithelium, separation of collagen fibers, and alterations in staining. CONCLUSIONS: Both decellularization techniques severely damaged the structure of the trachea and the extracellular matrix of the cartilage, resulting in a no functional graft, in spite of the use of surgical wire, cryopreservation or glutaraldehyde treatment. An important drawback was the formation of fibrotic stenosis in both anastomosis.

Experimental Tracheal Replacement: Angiogenesis and Null Apoptosis Promote Stenosis.

BACKGROUND: Tracheal replacement is a challenge for thoracic surgeons due to stenosis in the trachea-prosthesis anastomosis. We propose that stenosis occurs due to fibrosis as a result of an abnormal healing process, characterized by an increased expression of wound healing growth factors (vascular endothelial growth factor [VEGF], survivin, and CD31), which promote angiogenesis and decrease apoptosis. We analyzed the immunoreactivity of VEGF, survivin, CD31, and caspase-3 in the development of fibrotic stenosis in prosthetic tracheal replacement. METHODS: Fourteen dogs were operated on: group I (n=7) received a 6-ring cervical tracheal segment autograft, while in group II (n=7), a 6-ring segment of the cervical trachea was resected and tracheal continuity was restored with a Dacron prosthesis. The follow-up was 3 months. Immunoreactivity studies for VEGF, survivin, CD31, and caspase-3 were performed. A statistical analysis was done using the Wilcoxon signed rank test. RESULTS: Four animals in group I were euthanized on the 10th postoperative day due to autograft necrosis. Three animals completed the study without anastomotic stenosis. Moderate expression of VEGF (p=0.038), survivin (p=0.038), and CD31 (p=0.038) was found. All group II animals developed stenosis in the trachea-prosthesis anastomotic sites. Microscopy showed abundant collagen and neovascularization vessels. Statistically significant immunoreactive expression of VEGF (p=0.015), survivin (p=0.017), and CD31 (p=0.011) was observed. No expression of caspase-3 was found. CONCLUSION: We found a strong correlation between fibrosis in trachea-prosthesis anastomoses and excessive angiogenesis, moderate to intense VEGF, CD31, and survivin expression, and null apoptotic activity. These factors led to uncontrolled collagen production.

Treatment with Hyaluronic Acid and Collagen-Polyvinylpyrrolidone Improves Extracellular Matrix Assembly for Scarring after Tracheal Resection.

Treatment of tracheal stenosis is occasionally performed in combination with wound healing modulators to manipulate new extracellular matrix (ECM) formation and prevent fibrosis. Hyaluronic acid (HA) and collagen-polyvinylpyrrolidone (collagen-PVP) decrease fibrosis in experimental tracheal healing. However, they have not been used clinically as their effect on ECM components, which modify tracheal scarring, has not been described. Objective. To evaluate the effect of the application of HA, collagen-PVP, a mixture of HA and collagen-PVP (HA+collagen-PVP), and mitomycin C on the expression of decorin, matrix metalloproteinase 1 (MMP1), and MMP9, as well as the type of collagen and deposits formed in the scar after resection and end-to-end anastomosis (REEA) of the cervical trachea using an experimental model. Materials and Methods. Thirty dogs underwent REEA of the cervical trachea and were treated with different wound healing modulators: group I (n = 6), control; group II (n = 6), HA; group III (n = 6), collagen-PVP; group IV (n = 6), HA+collagen-PVP; and group V (n = 6), mitomycin C. The dogs were evaluated clinically and endoscopically for 4 weeks. Subsequently, macroscopic and microscopic changes, expression of ECM proteins, and collagen deposition in tracheal scars were analysed. Results. Groups II, III, and IV showed reduced endoscopic, macroscopic, and microscopic inflammation, improved neovascularization, high decorin expression (p < 0.01, analysis of variance (ANOVA)), and moderate expression of MMP1 (p < 0.003, ANOVA) and type I and III collagen (p < 0.05, Kruskal-Wallis). Groups IV and V developed fewer collagen deposits (p < 0.001, ANOVA). Conclusion. Treatment with HA and collagen-PVP improved post-REEA healing by increasing neovascularization, stimulating the expression of decorin, and regulating the expression of MMP1, as well as type I and III collagen and their deposition.

Decreased expression of transmembrane TNFR2 in lung leukocytes subpopulations of patients with non-fibrotic hypersensitivity pneumonitis compared with the fibrotic disease.

Hypersensitivity pneumonitis (HP) is an interstitial lung disease, characterized by lung inflammation (non-fibrotic HP) that may often progresses to fibrosis (Fibrotic HP). The tumor necrosis factor (TNF) and its receptors (TNFR1 and TNFR2) can be found as soluble (sol) and transmembrane (tm) forms, playing pro-inflammatory functions but also has been related to immune regulatory functions. Bronchioalveolar lavage from fibrotic and non-fibrotic HP patients was obtained, and immune cells were characterized by flow cytometry, whereas soluble proteins were analyzed by ELISA. Compare to fibrotic HP patients, HP patients with non-fibrotic disease have accumulation of pro-inflammatory CD3+ myeloid cells, cell subpopulations that have decreased tmTNFR2 expression, and low frequency of regulatory-T cells. Whereas solTNF, solTNFR2, and IL-8 are increased. These findings suggest that the TNF pathway may explain, at least partially, the differences between both HP clinical forms. The evaluation of the TNF family molecules may help to develop new therapeutic approaches.

An Open-label Study With Pirfenidone on Chronic Hypersensitivity Pneumonitis.

BACKGROUND: Chronic hypersensitivity pneumonitis (cHP) represents a severe lung disease often evolving to fibrosis with the subsequent destruction of the lung parenchyma. There are no approved therapies with confirmed efficacy to deal with this disease. METHODS: We performed an open-label, proof of concept study, to evaluate the efficacy and safety of pirfenidone added to immunosuppressive drugs on the treatment of cHP. We included 22 patients assigned to two groups: Group 1, nine patients that received prednisone plus azathioprine and Group 2, thirteen patients, received prednisone plus azathioprine and pirfenidone (ClinicalTrials.gov identifier NCT02496182). There were no significant imbalances in clinically relevant baseline characteristics between two study groups. RESULTS: After 1 year of treatment, inclusion of pirfenidone was not associated with improved forced vital capacity (primary end-point). A not significant tendency to show higher improvement of diffusion capacity of the lung for carbon monoxide (DLCO) was observed in the group receiving pirfenidone (p=0.06). Likewise, a significant improvement in the total score on the SGRQ was found in the group 2 (p=0.02) without differences in other two questionnaires related to quality of life (ATAQ-IPF and EQ-5D-3L). HRCT showed a decrease of the ground glass attenuation without changes in the fibrotic lesions and without differences between both groups. CONCLUSIONS: These findings suggest that the addition of pirfenidone to the anti-inflammatory treatment in patients with chronic HP may improve the outcome with acceptable safety profile. However, prospective randomized double-blind, placebo-controlled trials in largest cohorts are needed to validate its efficacy.

Transmembrane protease, serine 4 (TMPRSS4) is upregulated in IPF lungs and increases the fibrotic response in bleomycin-induced lung injury.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease characterized by epithelial cell activation, expansion of the fibroblast population and excessive extracellular matrix accumulation. The mechanisms are incompletely understood but evidence indicates that the deregulation of several proteases contributes to its pathogenesis. Transmembrane protease serine 4 (TMPRSS4) is a novel type II transmembrane serine protease that may promote migration and facilitate epithelial to mesenchymal transition (EMT), two critical processes in the pathogenesis of IPF. Thus, we hypothesized that over-expression of TMPRSS4 in the lung could promote the initiation and/or progression of IPF. In this study we first evaluated the expression and localization of TMPRSS4 in IPF lungs by real time PCR, western blot and immunohistochemistry. Then we examined the lung fibrotic response in wild-type and TMPRSS4 deficient mice using the bleomycin-induced lung injury model. We found that this protease is upregulated in IPF lungs, where was primarily expressed by epithelial and mast cells. Paralleling the findings in vivo, TMPRSS4 was expressed by alveolar and bronchial epithelial cells in vitro and unexpectedly, provoked an increase of E-cadherin. No expression was observed in normal human or IPF lung fibroblasts. The lung fibrotic response evaluated at 28 days after bleomycin injury was markedly attenuated in the haplodeficient and deficient TMPRSS4 mice. By morphology, a significant reduction of the fibrotic index was observed in KO and heterozygous mice which was confirmed by measurement of collagen content (hydroxyproline: WT: 164±21.1 µg/lung versus TMPRSS4 haploinsufficient: 110.2±14.3 µg/lung and TMPRSS4 deficient mice: 114.1±24.2 µg/lung (p<0.01). As in IPF, TMPRSS4 was also expressed in epithelial and mast cells. These findings indicate that TMPRSS4 is upregulated in IPF lungs and that may have a profibrotic role.

Effects of Pirfenidone and Collagen-Polyvinylpyrrolidone on Macroscopic and Microscopic Changes, TGF-ß1 Expression, and Collagen Deposition in an Experimental Model of Tracheal Wound Healing.

Tracheal stenosis (TS) is a fibrosis originated by prolonged inflammation and increased transforming growth factor beta 1 (TGF-ß1) expression and collagen deposition (CD) in the tracheal wound. Several wound-healing modulators (WHMs) have been used to modulate the tracheal healing process and prevent TS, but they have failed, justifying the need to evaluate alternative WHM. The pirfenidone (PFD) and collagen-polyvinylpyrrolidone (Collagen-PVP) decrease inflammation and fibrosis. This study assessed the effect of PFD administration and Collagen-PVP topical application on macroscopic and microscopic changes, TGF-ß1 expression, and CD in an experimental model of tracheal wound healing. Forty Wistar rats underwent cervical tracheoplasty, were divided into 4 groups (n = 10), and were treated with different WHM: group I, saline solution (SS); group II, Collagen-PVP; group III, mitomycin C (MMC); and group IV, 40 mg/kg PFD. Four weeks after surgery, the macroscopic and microscopic changes, in situ TGF-ß1 expression, and CD in posttracheoplasty scars were evaluated. The animals treated with Collagen-PVP and PFD developed less inflammation and fibrosis than animals in the other study groups (p < 0.05, Kruskal-Wallis) and, moreover, showed lower TGF-ß1 expression and CD than animals in group I (p < 0.05, ANOVA and Tukey's test). In conclusion, PFD and Collagen-PVP decrease inflammation, fibrosis, TGFß-1 expression, and CD in the posttracheoplasty rats' scar.

Comparison of lyophilized glutaraldehyde-preserved bovine pericardium with different vascular prostheses for use as vocal cords implants: experimental study.

This study compared the use of lyophilized glutaraldehyde-preserved bovine pericardium (LGPBP), polytetrafluoroethylene (PTFE), polyethylene terephthalate (PET), and Teflon felt (TF) as implants for vocal cords (VC) medialization and aimed to assess the endoscopic, macroscopic, and microscopic VC changes after medialization in a canine model. In 18 mongrel dogs, the right VC were medialized with LGPBP and the left were implanted as follows: Group I (n = 6): LGPBP and PTFE; Group II (n = 6): LGPBP and PET; Group III (n = 6): LGPBP and TF. Surgical handling of the implants was compared. Three months after surgery, macroscopic and microscopic changes of VC and implants were evaluated. LGPBP offered the best surgical handling (p = 0.005, Kruskal-Wallis). TF implants showed extrusion (p = 0.005, Kruskal-Wallis) and severe inflammation. All VC formed fibrous capsules around the implants; the ones developed by LGPBP implants were thinner (p = 0.001, ANOVA, Tukey). VC implanted with synthetic materials showed eosinophilic infiltration (p = 0.01, Kruskal-Wallis). We concluded that the LGPBP could be used as an implant for VC medialization because it is biocompatible, easy to handle and remove during surgical procedures, and nonabsorbable or extrudable and produces an inflammatory reaction similar to PTFE and PET.

Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis.

Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.

Respiratory mechanics and plasma levels of tumor necrosis factor alpha and interleukin 6 are affected by gas humidification during mechanical ventilation in dogs.

The use of dry gases during mechanical ventilation has been associated with the risk of serious airway complications. The goal of the present study was to quantify the plasma levels of TNF-alpha and IL-6 and to determine the radiological, hemodynamic, gasometric, and microscopic changes in lung mechanics in dogs subjected to short-term mechanical ventilation with and without humidification of the inhaled gas. The experiment was conducted for 24 hours in 10 dogs divided into two groups: Group I (n = 5), mechanical ventilation with dry oxygen dispensation, and Group II (n = 5), mechanical ventilation with oxygen dispensation using a moisture chamber. Variance analysis was used. No changes in physiological, hemodynamic, or gasometric, and radiographic constants were observed. Plasma TNF-alpha levels increased in group I, reaching a maximum 24 hours after mechanical ventilation was initiated (ANOVA p = 0.77). This increase was correlated to changes in mechanical ventilation. Plasma IL-6 levels decreased at 12 hours and increased again towards the end of the study (ANOVA p>0.05). Both groups exhibited a decrease in lung compliance and functional residual capacity values, but this was more pronounced in group I. Pplat increased in group I (ANOVA p = 0.02). Inhalation of dry gas caused histological lesions in the entire respiratory tract, including pulmonary parenchyma, to a greater extent than humidified gas. Humidification of inspired gases can attenuate damage associated with mechanical ventilation.

[Glutaraldehyde-preserved and lyophilized bovine pericardium as materials for medialization of the vocal folds in an animal model]. / Pericardio bovino tratado con glutaraldehído y liofilizado como materiales para la medialización de cuerdas vocales en modelo animal.

INTRODUCTION: Glutaraldehyde-preserved bovine pericardium (GBP) and lyophilized GBP (LGBP) have been used successfully in repairing several anatomical defects, but their effectiveness and safety as implants to vocal cords (VC) have not been reported. OBJECTIVE: The aim of this study was to evaluate the use of GBP and LGBP as materials for medialization thyroplasty, as well as to assess the endoscopic, macroscopic and microscopic VC changes after medialization in an experimental canine model. MATERIAL AND METHODS: In 12 healthy mongrel dogs, the right VC were medialized using pericardium and the left with polytetrafluoroethylene (PTFE). Group 1 (n=6): GBP and Group 2 (n=6): LGBP. The surgical manoeuvrability of the implants was compared. The animals were evaluated clinically and endoscopically. Three months after surgery, the larynges were assessed macro- and microscopically. RESULTS: Both GBP and LGBP implants showed better surgical manoeuvrability (Kruskal-Wallis, P=.005). Endoscopic and macroscopic studies showed no evidence of granulomas, absorption or extrusion of the implant. At the end of the study, greater thickness was observed in VC implanted with PTFE. Microscopically, all the VC developed fibrous capsules surrounding the implants and similar chronic inflammation reaction. The VC implanted with PTFE presented eosinophilic infiltration (Kruskal-Wallis, P<.05). CONCLUSION: Both GBP and LGBP can be used as implants for VC medialization because they are biocompatible, have easy surgical manoeuvrability, do not suffer absorption, migration or extrusion and produce inflammation reactions similar to those of PTFE.

Role of Sonic Hedgehog in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of unknown etiology and uncertain pathogenic mechanisms. Recent studies indicate that the pathogenesis of the disease may involve the abnormal expression of certain developmental pathways. Here we evaluated the expression of Sonic Hedgehog (SHH), Patched-1, Smoothened, and transcription factors glioma-associated oncogene homolog (GLI)1 and GLI2 by RT-PCR, as well as their localization in IPF and normal lungs by immunohistochemistry. The effects of SHH on fibroblast proliferation, migration, collagen and fibronectin production, and apoptosis were analyzed by WST-1, Boyden chamber chemotaxis, RT-PCR, Sircol, and annexin V-propidium iodide binding assays, respectively. Our results showed that all the main components of the Sonic signaling pathway were overexpressed in IPF lungs. With the exception of Smoothened, they were also upregulated in IPF fibroblasts. SHH and GLI2 localized to epithelial cells, whereas Patched-1, Smoothened, and GLI1 were observed mainly in fibroblasts and inflammatory cells. No staining was detected in normal lungs. Recombinant SHH increased fibroblast proliferation (P < 0.05), collagen synthesis, (2.5 ± 0.2 vs. 4.5 ± 1.0 µg of collagen/ml; P < 0.05), fibronectin expression (2-3-fold over control), and migration (190.3 ± 12.4% over control, P < 0.05). No effect was observed on α-smooth muscle actin expression. SHH protected lung fibroblasts from TNF-α/IFN-γ/Fas-induced apoptosis (14.5 ± 3.2% vs. 37.3 ± 7.2%, P < 0.0001). This protection was accompanied by modifications in several apoptosis-related proteins, including increased expression of X-linked inhibitor of apoptosis. These findings indicate that the SHH pathway is activated in IPF lungs and that SHH may contribute to IPF pathogenesis by increasing the proliferation, migration, extracellular matrix production, and survival of fibroblasts.

Cancer progression mediated by horizontal gene transfer in an in vivo model.

It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy.

Morphologic diversity of chronic pigeon breeder's disease: clinical features and survival.

Morphology in chronic HP is characterized by bronchiolocentric mononuclear inflammation, poorly formed granulomas and variable degree of fibrosis. However, recent findings suggest that this disease may present different pathologic patterns. In this study we evaluated the clinical behavior and survival of patients with pigeon breeder's disease according to the pathologic pattern. One-hundred ten biopsies were classified as "typical" (n = 58), non-specific interstitial pneumonia (NSIP)-pattern (n = 22), usual interstitial pneumonia (UIP)-like (n = 10), mixed pattern (n = 9), organizing pneumonia (OP)-pattern (n = 3), airway-centered interstitial fibrosis (ACIF)-pattern (n = 3), and non-classified (n = 5). Clinical features and survival were compared between patients with "typical", NSIP, and UIP patterns. There were no statistical differences between the groups in age, gender, time of symptoms, smoking, clubbing, and PaO(2). By the one-way ANOVA test we found differences in the percent of lymphocytes in bronchoalveolar lavage (BAL; p < 0.002) and in the forced vital capacity (p < 0.05) between the 3 groups. After Bonferroni correction the difference in BAL lymphocytes remained significant among the UIP-like and the typical pattern (36.1 ± 22.9 versus 64.6 ± 20.9, p = 0.001). UIP-like patients exhibited the worst survival rate (HR: 4.19; 95% CI: 1.66-14.47; p < 0.004) while NSIP-like pattern showed the best survival (HR: 0.18; 95% CI: 0.04-0.82; p < 0.03). Multivariate Cox regression analysis revealed that patients with a UIP-like pattern retained a significantly worse survival (HR: 3.4 (IC 95%: 1.15-10.29; p < 0.03), and mortality for the NSIP group was best and approached statistical significance (p = 0.07). These findings demonstrate that a variety of histopathologic and imaging patterns are seen in PBD, and the presence of a UIP-like pattern confers the worst prognosis.

Lyophilized glutaraldehyde-preserved bovine pericardium for experimental atrial septal defect closure.

A variety of patch materials has been used to close large atrial septal defects (ASD). Autologous pericardium and glutaraldehyde-preserved bovine pericardium are the most used. Lyophilized bovine pericardium has not been tested inside the cardiovascular system. The aim of this work was to study the behaviour and effectiveness of lyophilized glutaraldehyde-preserved bovine pericardium in ASD closure. Sixteen mongrel dogs were operated on. A 3 cm diameter atrial septal defect was created, and closed with: Group I (n=8): Lyophilized glutaraldehyde preserved bovine pericardium (LGPBP). Group II (n=8): Vascular Dacron patch. The animals were evaluated clinically, by echocardiography, macroscopically, and microscopically. Statistical analysis was done with analysis of variance (ANOVA) and Student's t-test. All the animals survived the surgical procedure and study time (6 months). Clinically all the animals displayed normal physical activity, with normal cardiac sounds. Echocardiography showed that both groups had a normal heart without intracardiac shunts, no thrombus formation, and no vegetations. Macroscopically all the animals showed good integration of the lyophilized bioprosthesis and Dacron patch. All group I animals presented a decrease of the area of the ASD in the left atrium (p<0.001 by ANOVA and Student's t-test). Microscopically, group I presented dense and well-organized collagenous tissue, areas of cartilaginous metaplasia and remnants of the lyophilized bioprosthesis (p<0.001 by ANOVA and Student's t-test). Group II showed encapsulated Dacron patch covered with collagenous tissue and cartilaginous metaplasia. In conclusion, the new lyophilized bioprosthesis is well integrated into the atrial septum, without complications and is effective for ASD closure.