<i>BRCA1</i> promoter methylation and immunohistochemical analysis of BRCA1 in early-stage breast tumors.

<p>(A) DNA methylation status of the <i>BRCA1</i> promoter determined by methylation-specific PCR. M-labeled lanes represent PCR products amplified with methylation-specific primers (75 bp). U-labeled lanes indicate the presence of unmethylated genes (82 bp). Patients 1, 2, 3, 4, 6, and 7 show the presence of a PCR product in both reactions, indicating methylation of the <i>BRCA1</i> promoter region. Patients 5 shows unmethylated gene. Molecular weight marker used is a 100-bp ladder. (B) Representative corresponding images of immunohistochemical staining of BRCA1. Scale bars, 100 µm.</p

Multivariate analysis of clinical factors and <i>BRCA1</i> promoter methylation in patients with early-stage breast cancer for overall survival and disease-free survival.

<p>Multivariate analysis of clinical factors and <i>BRCA1</i> promoter methylation in patients with early-stage breast cancer for overall survival and disease-free survival.</p

Mycobacterial infection induces higher interleukin-1β and dysregulated lung inflammation in mice with defective leukocyte NADPH oxidase

<div><p>Granulomatous inflammation causes severe tissue damage in mycobacterial infection while redox status was reported to be crucial in the granulomatous inflammation. Here, we used a NADPH oxidase 2 (NOX2)-deficient mice (<i>Ncf1</i>^<i>-/-</i>) to investigate the role of leukocyte-produced reactive oxygen species (ROS) in mycobacterium-induced granulomatous inflammation. We found poorly controlled mycobacterial proliferation, significant body weight loss, and a high mortality rate after <i>M</i>. <i>marinum</i> infection in <i>Ncf1</i>^<i>-/-</i> mice. Moreover, we noticed loose and neutrophilic granulomas and higher levels of interleukin (IL)-1β and neutrophil chemokines in <i>Ncf1</i>^<i>-/-</i> mice when compared with those in wild type mice. The lack of ROS led to reduced production of IL-1β in macrophages, whereas neutrophil elastase (NE), an abundant product of neutrophils, may potentially exert increased inflammasome-independent protease activity and lead to higher IL-1β production. Moreover, we showed that the abundant NE and IL-1β were present in the caseous granulomatous inflammation of human TB infection. Importantly, blocking of IL-1β with either a specific antibody or a recombinant IL-1 receptor ameliorated the pulmonary inflammation. These findings revealed a novel role of ROS in the early pathogenesis of neutrophilic granulomatous inflammation and suggested a potential role of IL-1 blocking in the treatment of mycobacterial infection in the lung.</p></div

Increased severity and mortality of <i>M</i>. <i>marinum</i> pulmonary infection in <i>Ncf1</i>^<i>-/-</i> mice.

<p><i>Ncf1</i>^-/- (loss of function mutation in p47phox) and WT controls were intra-tracheal injected with <i>M</i>. <i>marinum</i> (3 x 10⁷ CFU). Survival (A) and changes in body weight (B) were monitored over the 28 days period following <i>M</i>. <i>marinum</i> infection in WT (n = 20) and <i>Ncf1</i>^-/- mice (n = 23). The number of viable mycobacteria (C) was determined at 7 days and 14 days after <i>M</i>. <i>marinum</i> infection. Data are shown as a mean log of CFU per paired-lung (5 mice per group). The high correlation between changes in body weight and a number of viable mycobacteria in lungs was demonstrated in (D). Data represented mean ± sd. The experiments were analyzed with Log-rank test (A), Kruskal-Wallis test (C), and Spearman’s rank correlation analysis (D) *p < 0.05; **p < 0.005. These experiments were repeated twice with similar results.</p

The loose and neutrophilic granulomas in <i>Ncf1</i>^-/- mice in comparison with the compact granulomas in WT mice.

<p>The representative immunohistochemical stain of macrophages (F4/80) (A) showed compact aggregation of macrophages in WT mice, whereas macrophages were scattered within the granuloma of <i>Ncf1</i>^-/- mice. The immunofluorescent stain (B) (F4/80: red; Neutrophil elastase (NE): green) illustrated much more NE-positive neutrophils interspersed among macrophages in <i>Ncf1</i>^-/- mice compared with sparse neutrophils in WT mice. (C) Neutrophil and macrophage counts of <i>M</i>. <i>marinum</i>-infected WT and <i>Ncf1</i>^-/-mice at day 7 and day 14 were analyzed by flow cytometry, while CD11b⁺Ly6G⁺ represented neutrophils and CD11b⁺F4/80⁺ represented macrophages. Data represent mean ± sd of 4 mice from two independent experiments. The experiments were analyzed with Kruskal-Wallis test. *p < 0.05; **p < 0.005. These experiments were repeated twice with similar results.</p

High levels of IL-1β and neutrophilic chemokines in <i>M</i>. <i>marinum</i>-infected <i>Ncf1</i>^-/- mice.

<p>Cytokine and chemokine responses to <i>M</i>. <i>marinum</i> infection (3 x 10⁶ CFU). Cytokines of innate immunity including IL-1β (A), TNF-α (B) and IL-6 (C); cytokines of adaptive immunity including IFN-γ (D), IL-10 (E) and IL-17 A(F), and neutrophilc chemokines including CXCL5 (G), CCL5 (H) and CXCL1 (I) were assessed in lung homogenates obtained 7 days and 14 days after <i>M</i>. <i>marinum</i> infection (<i>3</i> x 10⁶ CFU). Data represent mean ± sd (n = 4–7 mice each group) The experiments were analyzed with Kruskal-Wallis test. *p < 0.05; **p < 0.005 and repeated with similar results.</p

IL-1β depletion and IL-1 receptor antagonist treatment alleviated the neutrophilic inflammation.

<p>The characteristic pulmonary histopathological images (A) of <i>M</i>. <i>marinum</i>-infected <i>Ncf1</i>^<i>-/-</i> mice and WT mice on day-5 with and without IL-1β depletion by a monoclonal antibody directed against IL-1β (150μg on day-1). The immunofluorescent stain of neutrophil elastase (NE) (B) also showed an apparently decrease of NE-positive cells after the depletion of IL-1β. The representative histological images (C) of WT and <i>Ncf1</i>^-/-mice treated with and without IL-1 receptor antagonist (Anakinra, 100mg/kg/day for 5days). Percentages of infiltration area (D) were quantified by using Image J. The experiments were analyzed with Kruskal-Wallis test and repeated with similar results. *p < 0.05; **p < 0.005.</p

Expression of neutrophil elastase and IL-1β in the caseous granulomatous inflammation in the human lung tissues of pulmonary tuberculosis.

<p>Formalinized lung tissue (A) of one patient with TB infection. The solid part (B) of the lung tissue showed characteristic caseous granulomatous inflammation with Langhans giant cells, and numerous neutrophils were found within the caseous part (C) of the lung tissues in high power field tissue. Immunohistochemical stain of neutrophil elastase (D) and IL-1β (E) showed abundant neutrophil elastase-positive cells and IL-1β in the granulomatous inflammation.</p

A Higher level of pulmonary inflammation in <i>Ncf1</i>^<i>-/-</i> mice after <i>M</i>. <i>marinum</i> infection in comparison with the inflammation in WT mice.

<p>Representative gross pictures (A) and cross-sectional histological examinations (B) of <i>M</i>. <i>marinum</i>-infected lungs from WT and <i>Ncf1</i>^<i>-/-</i> mice at day 7 and day 14 after infection. Acid-fast stains (AFS) (B) at day14 showed abundant AFS-positive bacilli in <i>Ncf1</i>^-/- mice, whereas sparse AFS-positive bacilli were found in WT mice. These experiments were repeated with similar results.</p

Gemcitabine Plus Erlotinib for Advanced Pancreatic Cancer: A Systematic Review with Meta-Analysis

<div><p>Background</p><p>This study aims to comprehensively summarize the currently available evidences on the efficacy and safety of gemcitabine plus erlotinib for treating advanced pancreatic cancer.</p> <p>Methodology/Principal Findings</p><p>PubMed, EMBASE, The Cochrane Library and abstracts of recent major conferences were systematically searched to identify relevant publications. Studies that were conducted in advanced pancreatic cancer patients treated with gemcitabine plus erlotinib (with or without comparison with gemcitabine alone) and reporting objective response rate, disease control rate, progression-free survival, time-to-progression, overall survival, 1-year survival rate and/or adverse events were included. Data on objective response rate, disease control rate, 1-year survival rate and adverse events rate, respectively, were combined mainly by using Meta-Analyst software with a random-effects model. Data on progression-free survival, time-to-progression and overall survival were summarized descriptively. Sixteen studies containing 1,308 advanced pancreatic cancer patients treated with gemcitabine plus erlotinib were included. The reported median progression-free survival (or time-to-progression), median overall survival, 1-year survival rates, objective response rates and disease control rates were 2–9.6 months, 5–12.5 months, 20%–51%, 0%–28.6% and 25.0%–83.3%, respectively. The weighted 1-year survival rate, objective response rate and disease control rate based on studies reporting robust results were 27.9%, 9.1% and 57.0%, respectively. According to the studies with relevant data, the incidences of total and severe adverse events were 96.3% and 62.9%, respectively. The most frequently reported adverse events were leucopenia, rash, diarrhea, vomitting, neutropenia, thrombocytopenia, anaemia, stomatitis, drug-induced liver injury, fatigue and fever. Compared with gemcitabine alone, the progression-free survival and overall survival with gemcitabine plus erlotinib were significantly longer, but there were also more deaths and interstitial lung disease-like syndrome related to this treatment.</p> <p>Conclusions/Significance</p><p>Gemcitabine plus erlotinib represent a new option for the treatment of advanced pancreatic cancer, with mild but clinically meaningful additive efficacy compared with gemcitabine alone. Its safety profile is generally acceptable, although careful management is needed for some specific adverse events.</p> </div