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SummaryIntracellular bacteria have been shown to cause autophagy, which impacts infectious outcomes, whereas extracellular bacteria have not been reported to activate autophagy. Here, we demonstrate that Pseudomonas aeruginosa, a Gram-negative extracellular bacterium, activates autophagy with considerably increased LC3 punctation in both an alveolar macrophage cell line (MH-S) and primary alveolar macrophages. Using the LC3 Gly120 mutant, we successfully demonstrated a hallmark of autophagy, conjugation of LC3 to phosphatidylethanolamine (PE). The accumulation of typical autophagosomes with double membranes was identified morphologically by transmission electron microscopy (TEM). Furthermore, the increase of PE-conjugated LC3 was indeed induced by infection rather than inhibition of lysosome degradation. P. aeruginosa induced autophagy through the classical beclin-1-Atg7-Atg5 pathway as determined by specific siRNA analysis. Rapamycin and IFN-c (autophagy inducers) augmented bacterial clearance, whereas beclin-1 and Atg5 knockdown reduced intracellular bacteria. Thus, P. aeruginosa-induced autophagy represents a host protective mechanism, providing new insight into the pathogenesis of this infection.

Caveolin-1 (Cav-1), an important composition protein within the flask-shaped membrane invaginations termed caveolae, may play a role in host defense against infections. However, the phenotype in Pseudomonas aeruginosa-infected cav1 knock-out (KO) mice is still unresolved, and the mechanism involved is almost entirely unknown. Using a respiratory infection model, we confirmed a crucial role played by Cav-1 in host defense against this pathogen because Cav-1 KO mice showed increased mortality, severe lung injury, and systemic dissemination as compared with wild-type (WT) littermates. In addition, cav1 KO mice exhibited elevated inflammatory cytokines (IL-6, TNF-␣, and IL-12a), decreased phagocytic ability of macrophages, and increased superoxide release in the lung, liver, and kidney. We further studied relevant cellular signaling processes and found that STAT3 and NF-B are markedly activated. Our data revealed that the Cav-1/STAT3/NF-B axis is responsible for a dysregulated cytokine response, which contributes to increased mortality and disease progression. Moreover, downregulating Cav-1 in cell culture with a dominant negative strategy demonstrated that STAT3 activation was essential for the translocation of NF-B into the nucleus, confirming the observations from cav1 KO mice. Collectively, our studies indicate that Cav-1 is critical for inflammatory responses regulating the STAT3/NF-B pathway and thereby impacting P. aeruginosa infection.Pseudomonas aeruginosa accounts for 25% of Gram-negative bacteria isolated from hospitals and is associated with high morbidity and mortality (1). P. aeruginosa frequently infects immunocompromised individuals, such as those affected by ventilator-associated infection, severe burns, and cancer (2). More than 80% of cystic fibrosis patients suffer severe P. aeruginosa infection (3). This bacterium becomes increasingly resistant to various antibiotics. Further understanding the mechanism of the host-pathogen interaction may result in effective approaches to preventing this infection. Thus, P. aeruginosa has been a focus of airway infectious diseases (1, 4 -6).Recent studies suggest that P. aeruginosa also invades the lung epithelial cells through lipid raft-mediated endocytosis (7-9), which is a possible reason why this bacterium develops such formidable resistance to antibiotics (10). The invasion process of P. aeruginosa may involve certain lipid raft-associated proteins, including the caveolin family of proteins (9). Using caveolin-1 (cav1) 3 KO mice, two recent studies investigated the role of Cav-1 during P. aeruginosa infection (11, 12); however, their observations were very different. Thus, the role of Cav-1 in this infection needs to be clearly characterized.Caveolins are a family of integral membrane proteins involved in caveola formation and receptor-dependent endocytosis (13-15). Cav-1 and -2 are co-expressed in various cells, such as endothelial cells, airway epithelial cells, and type I pneumocytes. Cav-1 is the major component of caveolae, flaskshaped plasma membran...

Background and Aims Little is known about functions of microRNA (miR) passenger strands (miR*), or their roles in tumor development or progression. We screened for miRs and miR* whose levels were altered in metastatic colorectal cancer (CRC) cells and human tumor samples, and investigated their targets and effects on cell function and tumor progression in mice. Methods We performed array-based profile analysis to identify miRs whose levels were increased more than 2-fold in metastatic (SW620) CRC cells, compared with non-metastatic (SW480) cells. Quantitative PCR, immunoblot, and in situ hybridization analyses were used to measure miRNA levels in CRC cell lines and human tumor samples. We used miRNA duplex mimics or inhibitors to increase and decrease levels of miRNA in CRC cells, and assessed their activities and ability to form metastatic xenograft tumors in nude mice. Results Levels of miR-221* and miR-224 were reduced in metastatic, compared with non-metastatic, CRC cells; levels in human tumor samples correlated inversely with tumor stage and metastasis to lymph nodes, as well as patient survival times. SW480 cells transfected with miR-221* or miR-224 inhibitors had increased motility in vitro, compared with SW480 control cells, and formed larger, more metastatic tumors following into mice. SW620 cells transfected with miR-221* or miR-224 mimics had reduced migration and motility in vitro and formed smaller tumors with fewer metastases in mice, compared with control SW620 cells. We identified the 3'UTR of MBD2 mRNA as a target of miR-221* and 281 miR-224. MBD2 silences the gene encoding Maspin, a suppressor of metastasis. In CRC cells, we found that miR-221* and miR-224 increase the expression of Maspin through MBD2 downregulation. Conclusion In metastatic CRC cells, reduced levels of miR-221* and miR-224 increase levels of MBD2, thereby decreasing expression of the metastasis suppressor Maspin. Increased activities of miR-221* and miR-224 reduce growth and metastasis of CRC xenograft tumors in mice; these miRs might be developed as therapeutic reagents or biomarkers of CRC progression.

8-oxoguanine-DNA glycosylase (OGG-1) is a base excision DNA repair enzyme; however, its function in modulating allergic diseases remains undefined. Using OGG-1 knockout (KO) mice, we show that this protein impacts allergic airway inflammation following sensitization and challenge by ovalbumin (OVA). OGG-1 KO mice exhibited less inflammatory cell infiltration and reduced oxidative stress in the lungs after OVA challenge compared to WT mice. The KO phenotype included decreased IL-4, IL-6, IL-10, and IL-17 in lung tissues. In addition, OGG-1 KO mice showed decreased expression and phosphorylation of STAT6 as well as NF-κB. Down-regulation of OGG-1 by siRNA lowered ROS and IL-4 levels but increased INF-γ production in cultured epithelial cells following exposure to house dust mite (HDM) extracts. OGG-1 may affect the levels of oxidative stress and proinflammatory cytokines during asthmatic conditions. OGG-1-deficiency negatively regulates allergen-induced airway inflammatory response.

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DCE-MRI identified a subgroup of breast cancer patients with multifocal/centric disease not evident on standard triple assessment. MRI of the breast should be considered for the preoperative planning of surgery for primary breast cancer.

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