Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background: We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE. Methodology/Principal Findings: Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice. Conclusions: Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.

Incidence and predictors of severe liver fibrosis in HIV-infected patients with chronic hepatitis C in Brazil

The aim of this study was to examine the incidence and factors associated with the severity of liver fibrosis in 234 coinfected patients in Brazil. Patients were cared for in our clinic, from 1996 to 2004. Eligible patients were defined as patients with documented HIV and hepatitis C virus (HCV) infections and had previously undergone a liver biopsy. Patients with persistently normal alanine aminotransferase (ALT) were also included. The variables selected for study were age, gender, risk category, history of high alcohol consumption, CD4(+) T cell count, antiretroviral therapy usage, HCV genotype and duration of HCV infection. Stage of fibrosis was scored as follows: F0, no fibrosis; F1, portal fibrosis with no septa; F2, portal fibrosis with few septa; F3, bridging fibrosis with many septa; and F4, cirrhosis. The liver fibrosis stage was F3 in 39 (16.6%) and F4 in 20(8.5%) patients. Among patients with normal ALT, the liver fibrosis stage was F3-F4 in three patients (5.6%). Predictors of severe liver fibrosis (17344) by multivariate analysis were age (older patients) and genotype 3 (genotype I odds ratio [OR], 0.28; 95% confidence interval [0], 0.12 0.65). In summary, in the present study severe liver fibrosis was found in 25% of our patients and was associated with an age of more than 38 years at the time of liver biopsy as well as, HCV genotype 3. No differences were found with respect to CD4(+) T cell counts although patients with a CD4(+) T cell count greater than 50 were excluded.

Induction of Heme Oxygenase-1 Can Halt and Even Reverse Renal Tubule-Interstitial Fibrosis

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed. Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease. Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed. Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-beta protein production was significantly lower in Hemin-treated animals. Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

PKC beta II inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses

Protein kinase C beta II (PKC beta II) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKC beta II in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKC beta II in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKC beta II on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKC beta II selective inhibitor (beta IIV5-3 conjugated to TAT(47-57) carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT(47-57) carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKC beta II inhibitor. Further, a 90% decrease in active TGF beta 1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKC beta II attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKC beta II in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis

Background & Aims: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that occasionally progresses to cirrhosis but usually has a benign course. The aim of this study was to investigate the role of the hemochromatosis mutation Cys282Tyr in development of the mild hepatic iron overload found in some patients with NASH and its association with hepatic damage in these patients. Methods: Fifty-one patients with NASH were studied. The presence of the Cys282Tyr mutation was tested in all patients, and the data were analyzed with respect to the histological grade of steatosis, inflammation, Perls' staining, hepatic iron concentration (HIC), and serum iron indices. Results: Thirty-one percent of patients with NASH were either homozygous or heterozygous for the Cys282Tyr mutation. This mutation was significantly associated with Perls' stain grade (P < 0.005), HIC (P < 0.005), and transferrin saturation percentage (P < 0.005) but not with serum ferritin levels. Linear regression analysis showed that increased hepatic iron (Perls' stain or HIC) had the greatest association with the severity of fibrosis (P < 0.0001). Conclusions: The Cys282Tyr mutation is responsible for most of the mild iron overload found in NASH and thus has a significant association with hepatic damage in these patients. Heterozygosity for the hemochromatosis gene mutation therefore cannot always be considered benign.

Prognostic Significance of Myocardial Fibrosis Quantification by Histopathology and Magnetic Resonance Imaging in Patients With Severe Aortic Valve Disease

Objectives We sought to determine whether the quantitative assessment of myocardial fibrosis (MF), either by histopathology or by contrast-enhanced magnetic resonance imaging (ce-MRI), could help predict long-term survival after aortic valve replacement. Background Severe aortic valve disease is characterized by progressive accumulation of interstitial MF. Methods Fifty-four patients scheduled to undergo aortic valve replacement were examined by ce-MRI. Delayed-enhanced images were used for the quantitative assessment of MF. In addition, interstitial MF was quantified by histological analysis of myocardial samples obtained during open-heart surgery and stained with picrosirius red. The ce-MRI study was repeated 27 +/- 22 months after surgery to assess left ventricular functional improvement, and all patients were followed for 52 +/- 17 months to evaluate long-term survival. Results There was a good correlation between the amount of MF measured by histopathology and by ce-MRI (r = 0.69, p < 0.001). In addition, the amount of MF demonstrated a significant inverse correlation with the degree of left ventricular functional improvement after surgery (r = -0.42, p = 0.04 for histopathology; r = -0.47, p = 0.02 for ce-MRI). Kaplan-Meier analyses revealed that higher degrees of MF accumulation were associated with worse long-term survival (chi-square = 6.32, p = 0.01 for histopathology; chi-square = 5.85, p = 0.02 for ce-MRI). On multivariate Cox regression analyses, patient age and the amount of MF were found to be independent predictors of all-cause mortality. Conclusions The amount of MF, either by histopathology or by ce-MRI, is associated with the degree of left ventricular functional improvement and all-cause mortality late after aortic valve replacement in patients with severe aortic valve disease. (J Am Coll Cardiol 2010; 56: 278-87) (c) 2010 by the American College of Cardiology Foundation

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

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. Copyright (C) 2008 S. Karger AG, Basel

Abnormal expression of telomerase/apoptosis limits type II alveolar epithelial cell replication in the early remodeling of usual interstitial pneumonia/idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a distinctive, usually fatal, type of chronic fibrosing interstitial pneumonia of unknown cause that increases in prevalence with advanced age, characterized by failure of alveolar re-epithelization and progressive scar formation. Recently, limitation of the replicative capacity of tissues determined by telomerase/apoptosis balance has been implicated in pathogenesis of age-related diseases. In this study, we validated the importance of the expression of type 2 alveolar epithelial cells telomerase protein and studied the relationships between telomerase and apoptosis in early remodeling of usual interstitial pneumonia. We determined type 2 alveolar epithelial cells density, telomerase expression, and apoptosis in surgical lung biopsies from 24 patients with usual interstitial pneumonia, and in normal lung tissues from 18 subjects. We used immunohistochemistry, deoxynucleotidyl transferase method of end labeling, electron microscopy, and histomorphometry to evaluate the amount of type 2 alveolar epithelial cells staining for surfactant-A, telomerase, and in situ detection of apoptotic cells. Unaffected areas of usual interstitial pneumonia and normal lung tissue had similar densities of type 2 alveolar epithelial cells, but a significant minor subpopulation of type 2 alveolar epithelial cells was telomerase positive and a large population was telomerase negative. A significant inverse association was found between low type 2, alveolar. epithelial cell telomerase expression and high apoptosis in unaffected areas of usual interstitial pneumonia. Although type 2 alveolar epithelial cell telomerase expression was higher than apoptosis in NLT group, no significant association was found between them. Electron microscopy confirmed epithelial apoptosis, alveolar collapse, and initial fibroplasia. We conclude that abnormal type 2 alveolar epithelial cells telomerase/apoptosis balance may reduce alveolar epithelial regenerative capacity, thus contributing to the early remodeling response in usual interstitial pneumonia. (C) 2010 Elsevier Inc. All rights reserved.