A semi-automated microscopic image analysis method for scoring Ki-67 nuclear immunostaining.

Nuclear proliferation marker MIB-1 (Ki-67) immunohistochemistry (IHC) is used to examine tumor cell proliferation. However, the diagnostic or prognostic value of the Ki-67 nuclear staining intensity and location, defined as nuclear gradient (NG), has not been assessed. This study examined the potential association between Ki-67 NG and cell cycle phases and its effect on the prognosis of pulmonary typical carcinoid (PTC) tumors. We propose a method for classifying the NG of Ki-67 during the cell cycle and compare the results between PTC, pulmonary adenocarcinoma (PAD), and breast ductal carcinoma (BDC). A literature review and objective analysis of IHC-stained paraffin sections were used to determine the Ki-67 labeling index and composed a stratification of the NG into NG1, NG2, and NG3/4 categories. A semi-automated image analysis protocol was established to determine the Ki-67 NG in PTC, PAD, and BDC. High intraobserver consistency and moderate interobserver agreement were achieved in the determination of Ki-67 NG in tumor specimens. NG1 and NG2 were lower in PTC than in PAD and BDC. Cox multivariate analysis of PTC after adjusting for age and number of metastatic lymph nodes showed that Ki-67 NG1 and NG2 significantly predicted clinical outcomes. The semi-automated method for quantification of Ki-67 nuclear immunostaining proposed in this study could become a valuable diagnostic and prognostic tool in PTC.

A semi-automated microscopic image analysis method for scoring Ki-67 nuclear immunostaining

Nuclear proliferation marker MIB-1 (Ki-67) immunohistochemistry (IHC) is used to examine tumor cell proliferation. However, the diagnostic or prognostic value of the Ki-67 nuclear staining intensity and location, defined as nuclear gradient (NG), has not been assessed. This study examined the potential association between Ki-67 NG and cell cycle phases and its effect on the prognosis of pulmonary typical carcinoid (PTC) tumors. We propose a method for classifying the NG of Ki-67 during the cell cycle and compare the results between PTC, pulmonary adenocarcinoma (PAD), and breast ductal carcinoma (BDC). A literature review and objective analysis of IHC-stained paraffin sections were used to determine the Ki-67 labeling index and composed a stratification of the NG into NG1, NG2, and NG3/4 categories. A semi-automated image analysis protocol was established to determine the Ki-67 NG in PTC, PAD, and BDC. High intraobserver consistency and moderate interobserver agreement were achieved in the determination of Ki-67 NG in tumor specimens. NG1 and NG2 were lower in PTC than in PAD and BDC. Cox multivariate analysis of PTC after adjusting for age and number of metastatic lymph nodes showed that Ki-67 NG1 and NG2 significantly predicted clinical outcomes. The semi-automated method for quantification of Ki-67 nuclear immunostaining proposed in this study could become a valuable diagnostic and prognostic tool in PTC.

Extracellular matrix components and regulators in the airway smooth muscle in asthma.

There is an intimate relationship between the extracellular matrix (ECM) and smooth muscle cells within the airways. Few studies have comprehensively assessed the composition of different ECM components and its regulators within the airway smooth muscle (ASM) in asthma. With the aid of image analysis, the fractional areas of total collagen and elastic fibres were quantified within the ASM of 35 subjects with fatal asthma (FA) and compared with 10 nonfatal asthma (NFA) patients and 22 nonasthmatic control cases. Expression of collagen I and III, fibronectin, versican, matrix metalloproteinase (MMP)-1, -2, -9 and -12 and tissue inhibitor of metalloproteinase-1 and -2 was quantified within the ASM in 22 FA and 10 control cases. In the large airways of FA cases, the fractional area of elastic fibres within the ASM was increased compared with NFA and controls. Similarly, fibronectin, MMP-9 and MMP-12 were increased within the ASM in large airways of FA cases compared with controls. Elastic fibres were increased in small airways in FA only in comparison with NFA cases. There is altered extracellular matrix composition and a degradative environment within the airway smooth muscle in fatal asthma patients, which may have important consequences for the mechanical and synthetic functions of airway smooth muscle.

Intraluminal plugs in idiopathic and secondary organizing pneumonia: repair or remodelling?

AIMS: Organizing pneumonia (OP) is an inflammatory lung disease characterized histologically by intraluminal plugs involving alveolar ducts and alveoli. The aim was to examine extracellular matrix repair and remodelling in 12 cases of idiopathic OP and compare these with 11 cases of secondary OP. METHODS AND RESULTS: Collagen/elastic fibre density, myofibroblast proliferation, microvascular density and endothelial activity in the intraluminal plugs were evaluated by histochemistry, immunohistochemistry and morphometry. The density of the collagen system fibres was greater in plugs of idiopathic OP when compared with secondary OP (P < 0.001). Quantification of myofibroblastic cells confirmed differences observed in patterns of immunoexpression; when compared with secondary OP, idiopathic OP contained fewer myofibroblastic cells in intraluminal plugs (P = 0.01). Microvascular density (CD34) and endothelial activity (vascular cell adhesion molecule-1 and E-selectin) were significantly greater in secondary OP (P < 0.05). CONCLUSIONS: Idiopathic and secondary OP show significant variation in morphological features that may represent different responses to injury. Increased collagen synthesis, low myofibroblast proliferation, poor microvascularization and minimal endothelial activity found in idiopathic OP may be more of a remodelling process than secondary OP repair.

Inflammatory cell mapping of the respiratory tract in fatal asthma.

BACKGROUND: The site and distribution of inflammation in the airways of asthmatic patients has been largely investigated. Inflammatory cells are distributed in both large and small airways in asthma. It has been demonstrated that distal lung inflammation in asthma may significantly contribute to the pathophysiology of the disease. The upper airways have also been implicated in the overall asthmatic inflammation. Although it is now accepted that lung inflammation is not restricted to the intrapulmonary airways in asthma, little is known about cell distribution in the other lung compartments and their relation to the intrapulmonary airways. OBJECTIVE: We aimed to map the inflammatory process in fatal asthma (FA), from the upper airways to the lung parenchyma. METHODS: Eosinophil, neutrophil, mast cell and lymphocyte content were determined in nasal mucosa, the trachea, intrapulmonary airways and parenchyma (peribronchiolar and distal) of 20 patients with FA and 10 controls. RESULTS: Eosinophil content was higher in all studied areas in FA compared with controls (P<0.02). Mast cell content was higher in the outer area of larger airways, small membranous bronchioles and in peribronchiolar parenchyma of FA compared with controls (P<0.04). CD3+, CD4+and CD20+cells showed increased content in FA intrapulmonary airways compared with controls (P<0.05). There was a positive correlation between CD4+cell content in nasal mucosa and larger airways in asthmatics. Increased neutrophil content was observed only in peribronchiolar parenchyma of FA (P=0.028). CONCLUSION: Eosinophils present a widespread distribution within the respiratory tract in FA, from the nasal mucosa to the distal lung. The outer wall of small membranous bronchioles is the main site of inflammatory changes in FA. There is a localized distribution of alveolar inflammation at the peribronchiolar region for mast cells and neutrophils. Our findings provide further evidence of the importance of the lung periphery in the pathophysiology of FA.

Acute lung injury in two experimental models of acute pancreatitis: infusion of saline or sodium taurocholate into the pancreatic duct.

OBJECTIVE: To compare acute pulmonary changes secondary to sodium taurocholate hemorrhagic pancreatitis with those changes secondary to a less severe pancreatitis induced by saline infusion into the biliopancreatic duct. DESIGN: Prospective, randomized controlled trial. SETTING: University pulmonary laboratory. SUBJECTS: A total of 110 male Wistar rats. INTERVENTIONS: Pancreatitis was induced by either 0.5 mL of a 4% solution of sodium taurocholate (TAU group) or 0.5 mL of normal saline (SAL group) injection into the biliopancreatic duct. Data were compared with data from control (sham-operated) animals (SHAM group). MEASUREMENTS AND MAIN RESULTS: The severity of pancreatic and pulmonary injuries was evaluated 1, 3, and 8 days after the induction of acute pancreatitis by morphometric and pulmonary mechanical studies. Biliopancreatic duct pressure was measured during infusion of solutions in SAL and TAU groups. SAL and TAU groups developed an intense necrohemorrhagic pancreatitis on day 1 without differences in biliopancreatic duct pressures (134.0+/-45.1 cm H2O vs. 123.3+/-23.4 cm H2O). Acute pancreatic lesions were still intense on day 3 in the TAU group only. Pulmonary resistance in SAL and TAU groups was significantly greater than in the SHAM group on day 3 only. On day 1, there was an increase in intraalveolar edema in both groups (p < .02). There was an increase in polymorphonuclear cells in alveolar septa on day 1 only in the TAU group (p < .001). In contrast, both experimental groups presented greater values of PMN cells on day 8 compared with the SHAM group (p < .001). Both groups with pancreatitis showed an increase in alveolar distention and collapse on day 1 that persisted only in the TAU group on days 3 and 8. No deaths were observed in the control (SHAM) group. In contrast, the SAL group had lower mortality than the TAU group in the first two days (17% and 52%, respectively, p = .03). CONCLUSION: High-pressure infusion of normal saline into the biliopancreatic duct of rats results in significant pancreatic and lung alterations. These changes are worse in the presence of sodium taurocholate.

Neurokinin depletion attenuates pulmonary changes induced by antigen challenge in sensitized guinea pigs.

The role of neurokinins in the acute pulmonary response to antigen was studied in guinea pigs that received ovalbumin (50 mg/kg ip) on days 1 and 3 and capsaicin (50 mg/kg sc) on day 21 (OAC); ovalbumin on days 1 and 3 (OA1); capsaicin on day 1 and OA on days 8 and 10 (COA); and ovalbumin on days 8 and 10 (OA2). On day 28, guinea pigs were submitted to ovalbumin aerosol challenge. Maximal values of pulmonary dynamic elastance (Edyn) and pulmonary resistance (RL) were significantly lower in OAC and COA groups compared with OA1 and OA2 groups (P < 0.001). There was no difference between maximal Edyn and RL values obtained in OAC and COA groups. Morphometric analysis of lungs showed significantly (P < 0.05) lower values of contraction index of airways, peribronchial edema, and alveoli over inflation in guinea pigs that received capsaicin compared with intact guinea pigs. Capsaicin treatment did not influence the formation of specific IgG1 anaphylactic antibodies. We conclude that neurokinin depletion results in a decrease in the pulmonary mechanical and inflammatory responses to antigen challenge in sensitized guinea pigs. These effects are observed when capsaicin is given either before or after sensitization.