Vera Luiza Capelozzi

Evidence of type II pneumocyte apoptosis in the pathogenesis of idiopathic pulmonary fibrosis (IFP)/usual interstitial pneumonia (UIP)

Background/Aims—The pathogenesis of idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP), a chronic and incurable human respiratory disease, is not well established. This study was designed to investigate whether the apoptosis of type II pneumocytes could be the precipitating factor in the pathogenesis of IPF. Methods—Nineteen specimens obtained by retrospective review of the medical and pathological records of 55 patients with IPF, four normal subjects, and 10 disease control lungs were analysed. The selected specimens had normal alveoli with intervening patchy scarring of the lung parenchyma, fulfilling the pathological criteria for UIP. To identify individual cells undergoing apoptosis in the normal alveoli, electron microscopy and in situ end labelling of fragmented DNA were performed on paraffin wax embedded sections using digoxigenin-11-dUTP and the enzyme terminal deoxynucleotidyl transferase. Results—Apoptosis was detected in the normal alveoli of 17 of the 19 patients with IPF/UIP and was absent in the controls. Electron microscopy demonstrated apoptotic changes in type II pneumocytes. These results indicate that apoptotic type II pneumocyte death occurs in normal alveoli of IPF/UIP and could be the principal cause of several events that account for the histological, clinical, and functional alterations seen in IPF/UIP. Conclusions—In conclusion, numerous type II pneumocytes from the normal alveoli of most patients with IPF/UIP actively undergo programmed cell death. This finding may shed new light on the pathogenesis of this disease, with implications mainly for the treatment of affected patients.

Recruitment maneuver in pulmonary and extrapulmonary experimental acute lung injury

Objective:The aim of this study is to test the hypothesis that recruitment maneuvers (RMs) might act differently in models of pulmonary (p) and extrapulmonary (exp) acute lung injury (ALI) with similar transpulmonary pressure changes. Design:Prospective, randomized, controlled experimental study. Setting:University research laboratory. Subjects:Wistar rats were randomly divided into four groups. In control groups, sterile saline solution was intratracheally (0.1 mL, Cp) or intraperitoneally (1 mL, Cexp) injected, whereas ALI animals received Escherichia coli lipopolysaccharide intratracheally (100 &mgr;g, ALIp) or intraperitoneally (1 mg, ALIexp). After 24 hrs, animals were mechanically ventilated (tidal volume, 6 mL/kg; positive end-expiratory pressure, 5 cm H2O) and three RMs (pressure inflations to 40 cm H2O for 40 secs, 1 min apart) applied. Measurements and Main Results:Pao2, lung resistive and viscoelastic pressures, static elastance, lung histology (light and electron microscopy), and type III procollagen messenger RNA expression in pulmonary tissue were measured before RMs and at the end of 1 hr of mechanical ventilation. Mechanical variables, gas exchange, and the fraction of area of alveolar collapse were similar in both ALI groups. After RMs, lung resistive and viscoelastic pressures and static elastance decreased more in ALIexp (255%, 180%, and 118%, respectively) than in ALIp (103%, 59%, and 89%, respectively). The amount of atelectasis decreased more in ALIexp than in ALIp (from 58% to 19% and from 59% to 33%, respectively). RMs augmented type III procollagen messenger RNA expression only in the ALIp group (19%), associated with worsening in alveolar epithelium injury but no capillary endothelium lesion, whereas the ALIexp group showed a minor detachment of the alveolar capillary membrane. Conclusions:Given the same transpulmonary pressures, RMs are more effective at opening collapsed alveoli in ALIexp than in ALIp, thus improving lung mechanics and oxygenation with limited damage to alveolar epithelium.

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.

Immunohistochemistry quantification by a digital computer-assisted method compared to semiquantitative analysis.

PURPOSE To compare immunostaining quantification obtained by a digital computer-assisted method with the well-established semiquantitative analysis. METHODS Cytoplasmic staining of galectin-3 was obtained by standard immunohistochemical reactions in 25 cases of well-differentiated thyroid carcinoma. The expression index that associates the conventional area fraction of labeled cells with the immunostaining intensity score based on visual qualitative observation was used as the semiquantitative analysis. A digital computer-assisted method is described based on the use of an image processing program (ImageLab). Three parameters were obtained: (1) percentage of labeled cells; (2) digital immunostaining intensity, and (3) digital expression index. The proposed method allows numerical analysis of the immunostaining intensity. RESULTS There was a strong correlation between the immunostaining intensity obtained by the two methods (Pearson correlation coefficient, r = 0.71, P = 0.0001). The same was observed between expression indexes (Pearson correlation coefficient, r = 0.66, P = 0.0001). CONCLUSION Results obtained with our proposed digital computer-assisted method for immunoexpression analysis were concordant with the semiquantitative analysis. In addition, digital values can also resolve disagreement among different observers about the quality of staining intensity because the digital method does not classify the results into groups, but rather provides a numerical value for each individual case; thus, it increases the diagnostic and, more importantly, the prognostic sensitivity of the immunohistochemical analysis.

Respiratory effects of lipopolysaccharide-induced inflammatory lung injury in mice

The pathogenic mechanisms of lipopolysaccharide (LPS)-induced lung injury have not been classified. This study examined the physiological changes after endotoxin inhalation and related those to features of pulmonary inflammation in mice. Pulmonary mechanics, histopathology, and bronchoalveolar lavage fluid (BALF) from BALB/c mice were analysed at different occasions (3, 24, 48 and 72 h) after inhalation of saline or LPS from Escherichia coli (0.3 (L0.3) or 10 mg x mL(-1) (L10)). Mice were sedated, anaesthetized, and ventilated. After chest wall resection static (Est) and dynamic (Edyn) elastances, deltaE (Edyn-Est), resistive (deltaP1) and viscoelastic/inhomogeneous pressures (deltaP2), and deltaP1+deltaP2 (deltaPtot) were obtained by end-inflation occlusion method. Lungs were prepared for histopathology. In parallel groups, tumour necrosis factor (TNF)-alpha, neutrophils, and protein were evaluated in the BALF. L0.3 and L10 showed a time-dependent production of TNF-alpha preceding a massive neutrophil infiltration. In L10 BALF there was an increase in protein level at 24 and 48 h. Est and Edyn increased early in L0.3 (65%, 63%) and L10 (41%, 51%). In L10 deltaE, deltaP2, and deltaPtot showed a gradual rise. At 72 h all groups were similar. L0.3 showed an early increase in cellularity, which returned to normal at 72 h. L10 presented the same pattern with the cell count remaining elevated until 72 h. In conclusion, lipopolysaccharide inhalation led to elastic and viscoelastic pulmonary changes together with tumour necrosis factor-alpha production and neutrophil infiltration in mouse lung.

Centrilobular Fibrosis: A Novel Histological Pattern of Idiopathic Interstitial Pneumonia

The classification of idiopathic interstitial pneumonias (IIP) is still under debate. In this context, we observed in some of our patients with a clinical and radiological diagnosis of IIP a different histological picture with an aggressive centrilobular scarring centered in the bronchiolar epithelia, but involving the surrounding parenchyma, which underwent extensive remodeling. We hypothesized that this pattern is a form of IIP that could be separated out histologically from the previously described patterns, in particular from usual interstitial pneumonia (UIP) and non-specific interstitial pneumonia (NSIP). Forty-nine patients with clinical and radiological diagnosis of IIP and open-lung biopsies were retrospectively selected from 1982 to 1998. The biopsies were reviewed according to the following criteria: derangement of lobular architecture, temporal homogeneity and subpleural or bronchocentric distribution of the lesions, fibroblast foci, bronchial epithelium necrosis and regeneration, exposure of the basal membrane, squamous metaplasia, basophilic intraluminal contents, and foreign bodies within the remodeling airspaces. Three groups were found: UIP (24 patients), NSIP (13), and a third that we named centrilobular fibrosis (CLF) (12). All histological parameters were significantly different among the three groups (p < 0.001). CLF is a specific, homogeneous, and recognizable histological pattern of IIP, and can be isolated from UIP and NSIP.

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.

Prognostic Values of Stromal Proportion and PCNA, Ki-67, and p53 Proteins in Patients with Resected Adenocarcinoma of the Lung

Data from 64 patients who underwent surgical resection of lung adenocarcinomas were studied to identify clinicopathologic markers that might provide prognostic information on the clinical behavior of this neoplasia. Patient staging was performed in accordance with the tumor-node-metastasis system as follows: Stage I (n = 29), Stage II (n = 11), Stage IIIA (n = 21), and Stage IIIB (n = 3). Overall follow-up time corresponded to the follow-up time for patients who were alive and to the survival time for patients who had died, all of them expressed in months. Data included age, staging, histologic type, morphometric assessment of histologic features related to tumor (stroma and vascularization), and immunohistochemical detection of proliferation cell markers (Ki-67 protein and proliferating cell nuclear antigen) and p53 protein. The morphometric assessment was made by the point-counting procedure. Data analysis included Life Tables for Survival and Cox Regression models. Overall follow-up analysis showed that significant univariate predictors (P <.05) were T stage; N stage; tumor stromal proportion; and immunohistochemical indexes of proliferating cell nuclear antigen, Ki-67, and p53 proteins. Variables that presented independent predictive value for overall follow-up with the multivariate model (P <.05) were sex, T stage, N stage, tumor stromal proportion, and immunohistochemical detection of p53 protein. We conclude that tumor stromal proportion and immunohistochemical detection of p53 protein, controlled for sex, T stage, and N stage, may be of critical value in the evaluation of recurrence of lung adenocarcinoma, serving as indicators for a more accurate prognosis.

Effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema

We sought to assess whether the effects of mesenchymal stromal cells (MSC) on lung inflammation and remodeling in experimental emphysema would differ according to MSC source and administration route. Emphysema was induced in C57BL/6 mice by intratracheal (IT) administration of porcine pancreatic elastase (0.1 UI) weekly for 1 month. After the last elastase instillation, saline or MSCs (1-105), isolated from either mouse bone marrow (BM), adipose tissue (AD) or lung tissue (L), were administered intravenously (IV) or IT. After 1 week, mice were euthanized. Regardless of administration route, MSCs from each source yielded: 1) decreased mean linear intercept, neutrophil infiltration, and cell apoptosis; 2) increased elastic fiber content; 3) reduced alveolar epithelial and endothelial cell damage; and 4) decreased keratinocyte-derived chemokine (KC, a mouse analog of interleukin-8) and transforming growth factor-β levels in lung tissue. In contrast with IV, IT MSC administration further reduced alveolar hyperinflation (BM-MSC) and collagen fiber content (BM-MSC and L-MSC). Intravenous administration of BM- and AD-MSCs reduced the number of M1 macrophages and pulmonary hypertension on echocardiography, while increasing vascular endothelial growth factor. Only BM-MSCs (IV > IT) increased the number of M2 macrophages. In conclusion, different MSC sources and administration routes variably reduced elastase-induced lung damage, but IV administration of BM-MSCs resulted in better cardiovascular function and change of the macrophage phenotype from M1 to M2.

Causes of pulmonary granulomas: a retrospective study of 500 cases from seven countries

Background The frequencies of various causes of pulmonary granulomas in pathological material are unknown, as is the influence of geographical location on aetiology. The aim of this study was to identify the causes of pulmonary granulomas in pathological specimens, to define their frequencies, and to determine whether these causes vary by geographical location. Methods 500 lung biopsies and resections containing granulomas were reviewed retrospectively by expert pulmonary pathologists from 10 institutions in seven countries. Fifty consecutive cases from each location were assigned a diagnosis based on histological features and available clinical/microbiological data. Results A specific cause was identified in 58% of cases (290/500), most commonly sarcoidosis (136, 27%) and mycobacterial or fungal infections (125, 25%). Mycobacteria were identified in 19% of cases outside the USA versus 8% within the USA. In contrast, fungi accounted for 19% cases in the USA versus 4% in other locations. Fungi were mostly detected by histology, whereas most mycobacteria were identified in cultures. In 42% of cases (210/500) an aetiology could not be determined. Conclusions Across several geographical settings, sarcoidosis and infections are the most common causes of pulmonary granulomas diagnosed in pathological specimens. Fungi are more commonly identified than mycobacteria in the USA, whereas the reverse is true in other countries. A definite aetiology cannot be demonstrated in more than a third of all cases of pulmonary granulomas, even after histological examination. These findings highlight the need to submit material for histology as well as cultures in all cases in which granulomatous disease enters the differential diagnosis.