Edwin Roger Parra

Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis

BackgroundThe purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance.MethodsFemale New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freunds adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 μg/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05.ResultsIM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 ± 0.118 vs. 0.874 ± 0.282, p < 0.001), bronchioles (0.294 ± 0.139 vs. 0.646 ± 0.172, p < 0.001) and in the septal interstitium (0.027 ± 0.014 vs. 0.067 ± 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 ± 0.07 vs. 1.0 ± 0.528, p = 0.002) and V (1.12 ± 0.42 vs. 4.74 ± 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression (p < 0.0001).ConclusionsCollagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

Inflammatory Cell Phenotyping of the Pulmonary Interstitium in Idiopathic Interstitial Pneumonia

Background: Several studies have implicated the role of inflammation in the pathogenesis of lung damage in idiopathic interstitial pneumonias (IIPs). Investigations of inflammatory cells in IIP have show that eosinophils, neutrophils and T cells may be associated with a poorer prognosis. Objectives: The aim of our study was to map, by quantitative analysis, the number of inflammatory cells in the lung tissue of patients with non-specific interstitial pneumonia/non-specific interstitial pneumonia (NSIP/NSIP), acute interstitial pneumonia/diffuse alveolar damage (AIP/DAD) and idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP) and to correlate them with lung function tests and survival. Methods: After immunohistochemical staining, we quantified the content of inflammatory cells [macrophages, neutrophils (elastase+), plasma cells, and CD3, CD4 and CD8 T lymphocytes (TLs)] in 20 NSIP, 20 DAD and 20 UIP surgical lung biopsies. Results: The total density of inflammatory cells was significantly increased in DAD and NSIP when compared to UIP (p = 0.04). TLs were increased in DAD and NSIP when compared to UlP lungs (p < 0.05). The density of inflammatory cells in UIP showed significant differences in normal, intervening and dense fibrosis areas (p < 0.05). The most numerous cells infiltrating the mural fibrosis and honeycombing areas were plasma cells, neutrophils (elastase+), CD20+, CD3+, CD4+ and CD8+ (p < 0.05). In UIP, CD3+ TLs were directly correlated with forced expiratory volume in 1 s/forced vital capacity ratio × 100 (p = 0.05). CD68+ cells presented a significant positive correlation with the forced expiratory volume in 1 s (p = 0.04); neutrophil (elastase+) cells significantly correlated with residual volume (p = 0.02), residual volume/total lung capacity (p = 0.04) and carbon monoxide transfer factor (p = 0.03). The most important predictor of survival in UIP was CD3+ TLs (p = 0.05). Conclusion: The total density of inflammatory cells and lymphocytes presents a different distribution within the pulmonary parenchyma in AIP/DAD, NSIP/NSIP and IPF/UIP evolutionary adapted responses to injury. There is a localized distribution of inflammation in the normal, intervening and dense fibrosis areas of UIP for CD3+, associated with a lethal deterioration of the pulmonary function and poor survival. Our findings provide further evidence of the importance of inflammation in the pathophysiology of IIPs.

Association of Interferon‐ and Transforming Growth Factor β–Regulated Genes and Macrophage Activation With Systemic Sclerosis–Related Progressive Lung Fibrosis

Systemic sclerosis (SSc)–related interstitial lung disease (ILD) is one of the leading causes of mortality. We undertook this study to analyze the gene expression of lung tissue in a prospective cohort of patients with SSc‐related ILD and to compare it with that in control lungs and with 2 prospective clinical parameters in order to understand the molecular pathways implicated in progressive lung disease.

Centrilobular Fibrosis: An Underrecognized Pattern in Systemic Sclerosis

Background: The impressive association of lung involvement and gastroesophageal reflux in scleroderma raises the possibility of a cause-effect relationship. Objectives: To determine clinical, radiological and histopathological features of systemic sclerosis (SSc) patients according the presence or absence of centrilobular fibrosis (CLF). Methods: Twenty-eight SSc patients with lung involvement were submitted to open lung biopsy and the specimens classified for the presence of CLF (bronchocentric distribution of the lesions and intraluminal matter according to the classification of idiopathic interstitial pneumonia). HRCT, pulmonary function tests and esophageal analysis were also performed. Subsequently, cyclophosphamide was introduced for the nonspecific interstitial pneumonia subgroup and antireflux treatment was intensified for isolated CLF patients. Results: Isolated CLF was found in 21% of the biopsies and also found associated to nonspecific interstitial pneumonia in 84% of these patients. The other 3 cases had usual interstitial pneumonia, pulmonary hypertension and respiratory bronchiolitis-associated interstitial lung disease. The histopathological analysis revealed that all 6 patients with isolated CLF had the bronchocentric distribution and intraluminal basophilic content, with foreign bodies detected in one third of them. The central distribution of lung involvement on HRCT was found in 67% of these patients with a consistent patchy distribution (100%). Ground glass (67%) and consolidation (33%) were the predominant patterns found. The constant clinical finding in all isolated CLF cases was dyspnea, esophageal abnormalities and a moderate lung impairment (FVC: 63.83 ± 16.31%; DLCO: 61.66 ± 18.84%). Lung function parameters in isolated CLF patients remained stable after 1 year of exclusively intensive antireflux treatment (FVC, p = 0.23; DLCO, p = 0.59). Conclusions: The novel description of CLF pattern in SSc lung disease with peculiar histological, tomographic and clinical features will certainly contribute to a more appropriate therapeutic approach.

Interstitial and vascular type V collagen morphologic disorganization in usual interstitial pneumonia

Recent evidence suggests that type V collagen plays a role in organizing collagen fibrils, thus maintaining fibril size and spatial organization uniform. In this study we sought to characterize the importance of type V collagen morphological disorganization and to study the relationship between type V collagen, active remodeling of the pulmonary vascular/parenchyma (fibroblastic foci), and other collagen types in usual interstitial pneumonia (UIP). We examined type V collagen and several other collagens in 24 open lung biopsies with histological pattern of UIP from patients with idiopathic pulmonary fibrosis (IPF). We used immunofluorescence, morphometry, and three-dimensional reconstruction to evaluate the amount of collagen V and its interaction with the active remodeling progression in UIP, as well as types I and III collagen fibers. Active remodeling progression was significantly related to type V collagen density (p<0.05), showing a gradual and direct increase to minimal, moderate, and severe fibrosis degree in UIP and in the three different areas: normal, intervening, and mural-organizing fibrosis in UIP. Parenchymal changes were characterized by morphological disorganization of fibrillar collagen with diverse disarray and thickness when observed by three-dimensional reconstruction. We concluded that in the different temporal stages of UIP, vascular/parenchyma collagen type V is increased, in disarray, and is the most important predictor of survival.

Pathological and ultrastructural analysis of surgical lung biopsies in patients with swine-origin influenza type A/H1N1 and acute respiratory failure

BACKGROUND: Cases of H1N1 and other pulmonary infections evolve to acute respiratory failure and death when co‐infections or lung injury predominate over the immune response, thus requiring early diagnosis to improve treatment. OBJECTIVE: To perform a detailed histopathological analysis of the open lung biopsy specimens from five patients with ARDS with confirmed H1N1. METHODS: Lung specimens underwent microbiologic analysis, and examination by optical and electron microscopy. Immunophenotyping was used to characterize macrophages, natural killer, T and B cells, and expression of cytokines and iNOS. RESULTS: The pathological features observed were necrotizing bronchiolitis, diffuse alveolar damage, alveolar hemorrhage and abnormal immune response. Ultrastructural analysis showed viral‐like particles in all cases. CONCLUSIONS: Viral‐like particles can be successfully demonstrated in lung tissue by ultrastructural examination, without confirmation of the virus by RT‐PCR on nasopharyngeal aspirates. Bronchioles and epithelium, rather than endothelium, are probably the primary target of infection, and diffuse alveolar damage the consequence of the effect of airways obliteration and dysfunction on innate immunity, suggesting that treatment should be focused on epithelial repair.

Abnormal collagen V deposition in dermis correlates with skin thickening and disease activity in systemic sclerosis.

OBJECTIVE The physiological and mechanical properties of the skin, the primary tissue affected by systemic sclerosis, depend on the assembly of collagen types I, III and V, which form heterotypic fibers. Collagen V (COLV) regulates heterotypic fiber diameter, and the maintenance of its properties is important for maintaining normal tissue architecture and function. Based on a COLV-induced experimental SSc model, in which overexpression of abnormal COLV was a prominent feature, we assumed that this abnormality could be present in SSc patients and could be correlated to disease duration, skin thickening and disease activity. METHODS Skin biopsies from 18 patients (6 early-stage and 12 late-stage) and 10 healthy controls were studied. Skin thickening assessment was performed with the Modified Rodnan Skin Score (MRSS), and activity was calculated using the Valentini Disease Activity Index. Morphology, morphometry of COLV deposition in dermis, as well as, quantitative RT-PCR and 3D-reconstruction of the dermal fibroblast culture were performed. RESULTS Structurally abnormal COLV was overexpressed in SSc skin, mainly in the early stages of the disease, when compared to normal controls and late-stage. A positive correlation between COLV expression and MRSS and disease activity was observed. Collagen V alpha-1 and alpha-2 mRNA expression levels were higher in SSc. Tridimensional reconstruction of SSc dermal heterotypic fibers confirmed the presence of atypical COLV. CONCLUSION Increased synthesis of abnormal COLV and its correlation with disease stage, activity and MRSS suggest that this collagen can be a possible trigger involved in the pathogenesis of SSc.

Abnormal deposition of collagen/elastic vascular fibres and prognostic significance in idiopathic interstitial pneumonias

Background: Vascular remodelling has recently been shown to be a promising pathogenetic indicator in idiopathic interstitial pneumonias (IIPs). Aim: To validate the importance of the collagen/elastic system in vascular remodelling and to study the relationships between the collagen/elastic system, survival and the major histological patterns of IIPs. Methods: Collagen/elastic system fibres were studied in 25 patients with acute interstitial pneumonia/diffuse alveolar damage, 22 with non-specific interstitial pneumonia/non-specific interstitial pneumonia and 55 with idiopathic pulmonary fibrosis/usual interstitial pneumonia. The Picrosirius polarisation method and Weigert’s resorcin–fuchsin histochemistry and morphometric analysis were used to evaluate the amount of vascular collagen/elastic system fibres and their association with the histological pattern of IIPs. The association between vascular remodelling and the degree of parenchymal fibrosis in usual interstitial pneumonia (UIP) was also considered. Results: The vascular measurement of collagen/elastic fibres was significantly higher in UIP than in the lungs of controls, and in those with diffuse alveolar damage and those with non-specific interstitial pneumonia. In addition, the increment of collagen/elastic fibres in UIP varied according to the degree and activity of the parenchymal fibrosis. The most important predictors of survival in UIP were vascular remodelling classification and vascular collagen deposition. Conclusion: A progressive vascular fibroelastosis occurs in IIP histological patterns, probably indicating evolutionarily adapted responses to parenchymal injury. The vascular remodelling classification and the increase in vascular collagen were related to survival in IIP and possibly play a role in its pathogenesis. Further studies are needed to determine whether this relationship is causal or consequential.

Effects of bone marrow-derived mononuclear cells on airway and lung parenchyma remodeling in a murine model of chronic allergic inflammation

We hypothesized that bone marrow-derived mononuclear cells (BMDMC) would attenuate the remodeling process in a chronic allergic inflammation model. C57BL/6 mice were assigned to two groups. In OVA, mice were sensitized and repeatedly challenged with ovalbumin. Control mice (C) received saline under the same protocol. C and OVA were further randomized to receive BMDMC (2 × 10⁶) or saline intravenously 24 h before the first challenge. BMDMC therapy reduced eosinophil infiltration, smooth muscle-specific actin expression, subepithelial fibrosis, and myocyte hypertrophy and hyperplasia, thus causing a decrease in airway hyperresponsiveness and lung mechanical parameters. BMDMC from green fluorescent protein (GFP)-transgenic mice transplanted into GFP-negative mice yielded lower engraftment in OVA. BMDMC increased insulin-like growth factor expression, but reduced interleukin-5, transforming growth factor-β, platelet-derived growth factor, and vascular endothelial growth factor mRNA expression. In conclusion, in the present chronic allergic inflammation model, BMDMC therapy was an effective pre-treatment protocol that potentiated airway epithelial cell repair and prevented inflammatory and remodeling processes.

Protective effects of ascorbic acid pretreatment in a rat model of intestinal ischemia-reperfusion injury: A histomorphometric study

BACKGROUND Ascorbic acid has shown promise in attenuation of intestinal ischemia-reperfusion (I/R) injury. The aim of this study was to determine the protective effects of ascorbic acid on intestinal morphology during IR injury in rats. MATERIALS AND METHODS We examined morphological changes in the small intestine of Wistar rats after (i) 40 minutes of ischemia (I), (ii) ischemia followed by 30 min of reperfusion (IR), (iii) ischemia with ascorbic acid (IA), (iv) ischemia followed by reperfusion and ascorbic acid (IRA) and (v) in a sham group (S). We used morphometry to evaluate the amount of villous architecture, crypts, necrosis, hemorrhagic infarcts and inflammatory cells at the mesenteric and antimesenteric borders of the small intestine. RESULTS Ascorbic acid caused a significant reduction of antimesenteric villous hemorrhagic infarction (p<0.05) of the small intestine after ischemia followed by reperfusion as well as villous necrosis reduction at both borders after ischemia (p<0.05). The lesions found in the small intestine were more prominent along the antimesenteric margin. CONCLUSIONS Ascorbic acid pretreatment has a protective effect against the intestinal morphological lesions induced by ischemia-reperfusion injury in rats.