Mechanical ventilation interacts with endotoxemia to induce extrapulmonary organ dysfunction

INTRODUCTION: Multiple organ dysfunction syndrome (MODS) is a common complication of sepsis in mechanically ventilated patients with acute respiratory distress syndrome, but the links between mechanical ventilation and MODS are unclear. Our goal was to determine whether a minimally injurious mechanical ventilation strategy synergizes with low-dose endotoxemia to induce the activation of pro-inflammatory pathways in the lungs and in the systemic circulation, resulting in distal organ dysfunction and/or injury. METHODS: We administered intraperitoneal Escherichia coli lipopolysaccharide (LPS; 1 μg/g) to C57BL/6 mice, and 14 hours later subjected the mice to 6 hours of mechanical ventilation with tidal volumes of 10 ml/kg (LPS + MV). Comparison groups received ventilation but no LPS (MV), LPS but no ventilation (LPS), or neither LPS nor ventilation (phosphate-buffered saline; PBS). RESULTS: Myeloperoxidase activity and the concentrations of the chemokines macrophage inflammatory protein-2 (MIP-2) and KC were significantly increased in the lungs of mice in the LPS + MV group, in comparison with mice in the PBS group. Interestingly, permeability changes across the alveolar epithelium and histological changes suggestive of lung injury were minimal in mice in the LPS + MV group. However, despite the minimal lung injury, the combination of mechanical ventilation and LPS resulted in chemical and histological evidence of liver and kidney injury, and this was associated with increases in the plasma concentrations of KC, MIP-2, IL-6, and TNF-α. CONCLUSION: Non-injurious mechanical ventilation strategies interact with endotoxemia in mice to enhance pro-inflammatory mechanisms in the lungs and promote extra-pulmonary end-organ injury, even in the absence of demonstrable acute lung injury

Epidermal growth factor mediates detachment from and invasion through collagen I and Matrigel in Capan-1 pancreatic cancer cells

BACKGROUND: Pancreatic adenocarcinoma is a highly invasive neoplasm. Epidermal growth factor (EGF) and its receptor are over expressed in pancreatic cancer, and expression correlates with invasion and metastasis. We hypothesized that EGF receptor and integrin signalling pathways interact in mediating cellular adhesion and invasion in pancreatic cancer, and that invasiveness correlates temporally with detachment from extracellular matrix. METHODS: We tested this hypothesis by investigating the role of EGF in mediating adhesion to and invasion through collagen I and Matrigel in the metastatic pancreatic adenocarcinoma cell line Capan-1. Adhesion and invasion were measured using in vitro assays of fluorescently-labeled cells. Adhesion and invasion assays were also performed in the primary pancreatic adenocarcinoma cell line MIA PaCa-2. RESULTS: EGF inhibited adhesion to collagen I and Matrigel in Capan-1 cells. The loss of adhesion was reversed by AG825, an inhibitor of erbB2 receptor signalling and by wortmannin, a PI3K inhibitor, but not by the protein synthesis inhibitor cycloheximide. EGF stimulated invasion through collagen I and Matrigel at concentrations and time courses similar to those mediating detachment from these extracellular matrix components. Adhesion to collagen I was different in MIA PaCa-2 cells, with no significant change elicited following EGF treatment, whereas treatment with the EGF family member heregulin-alpha elicited a marked increase in adhesion. Invasion through Matrigel in response to EGF, however, was similar to that observed in Capan-1 cells. CONCLUSION: An inverse relationship exists between adhesion and invasion capabilities in Capan-1 cells but not in MIA PaCa-2 cells. EGF receptor signalling involving the erbB2 and PI3K pathways plays a role in mediating these events in Capan-1 cells

Effects of acutely inhibiting PI3K isoforms and mTOR on regulation of glucose metabolism in vivo

In in vitro studies class-I PI3Ks (phosphoinositide 3-kinases), class-II PI3Ks and mTOR (mammalian target of rapamycin) have all been described as having roles in the regulation of glucose metabolism. The relative role each plays in the normal signalling processes regulating glucose metabolism in vivo is less clear. Knockout and knockin mouse models have provided some evidence that the class-I PI3K isoforms p110α, p110β, and to a lesser extent p110γ, are necessary for processes regulating glucose metabolism and appetite. However, in these models the PI3K activity is chronically reduced. Therefore we analysed the effects of acutely inhibiting PI3K isoforms alone, or PI3K and mTOR, on glucose metabolism and food intake. In the present study impairments in glucose tolerance, insulin tolerance and increased hepatic glucose output were observed in mice treated with the pan-PI3K/mTOR inhibitors PI-103 and NVP-BEZ235. The finding that ZSTK474 has similar effects indicates that these effects are due to inhibition of PI3K rather than mTOR. The p110α-selective inhibitors PIK75 and A66 also induced these phenotypes, but inhibitors of p110β, p110δ or p110γ induced only minor effects. These drugs caused no significant effects on BMR (basal metabolic rate), O2 consumption or water intake, but BEZ235, PI-103 and PIK75 did cause a small reduction in food consumption. Surprisingly, pan-PI3K inhibitors or p110α inhibitors caused reductions in animal movement, although the cause of this is not clear. Taken together these studies provide pharmacological evidence to support a pre-eminent role for the p110α isoform of PI3K in pathways acutely regulating glucose metabolism

Differentiation of Gram-Negative Bacterial Aerosol Exposure Using Detected Markers in Bronchial-Alveolar Lavage Fluid

The identification of biosignatures of aerosol exposure to pathogens has the potential to provide useful diagnostic information. In particular, markers of exposure to different types of respiratory pathogens may yield diverse sets of markers that can be used to differentiate exposure. We examine a mouse model of aerosol exposure to known Gram negative bacterial pathogens, Francisella tularensis novicida and Pseudomonas aeruginosa. Mice were subjected to either a pathogen or control exposure and bronchial alveolar lavage fluid (BALF) was collected at four and twenty four hours post exposure. Small protein and peptide markers within the BALF were detected by matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and analyzed using both exploratory and predictive data analysis methods; principle component analysis and degree of association. The markers detected were successfully used to accurately identify the four hour exposed samples from the control samples. This report demonstrates the potential for small protein and peptide marker profiles to identify aerosol exposure in a short post-exposure time frame

Roles of the Amino Terminal Region and Repeat Region of the Plasmodium berghei Circumsporozoite Protein in Parasite Infectivity

The circumsporozoite protein (CSP) plays a key role in malaria sporozoite infection of both mosquito salivary glands and the vertebrate host. The conserved Regions I and II have been well studied but little is known about the immunogenic central repeat region and the N-terminal region of the protein. Rodent malaria Plasmodium berghei parasites, in which the endogenous CS gene has been replaced with the avian Plasmodium gallinaceum CS (PgCS) sequence, develop normally in the A. stephensi mosquito midgut but the sporozoites are not infectious. We therefore generated P. berghei transgenic parasites carrying the PgCS gene, in which the repeat region was replaced with the homologous region of P. berghei CS (PbCS). A further line, in which both the N-terminal region and repeat region were replaced with the homologous regions of PbCS, was also generated. Introduction of the PbCS repeat region alone, into the PgCS gene, did not rescue sporozoite species-specific infectivity. However, the introduction of both the PbCS repeat region and the N-terminal region into the PgCS gene completely rescued infectivity, in both the mosquito vector and the mammalian host. Immunofluorescence experiments and western blot analysis revealed correct localization and proteolytic processing of CSP in the chimeric parasites. The results demonstrate, in vivo, that the repeat region of P. berghei CSP, alone, is unable to mediate sporozoite infectivity in either the mosquito or the mammalian host, but suggest an important role for the N-terminal region in sporozoite host cell invasion

Efficacy and Safety of Inhaled Carbon Monoxide during Pulmonary Inflammation in Mice

Background: Pulmonary inflammation is a major contributor to morbidity in a variety of respiratory disorders, but treatment options are limited. Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation. Methodology: Mice were exposed to 0–500 ppm inhaled CO for periods of up to 24 hours prior to and following intratracheal instillation of 10 ng LPS. Animals were sacrificed and assessed for intraalveolar neutrophil influx and cytokine levels, flow cytometric determination of neutrophil number and activation in blood, lung and lavage fluid samples, or neutrophil mobilisation from bone marrow. Principal Findings: When administered for 24 hours both before and after LPS, inhaled CO of 100 ppm or more reduced intraalveolar neutrophil infiltration by 40–50%, although doses above 100 ppm were associated with either high carboxyhemoglobin, weight loss or reduced physical activity. This anti-inflammatory effect of CO did not require pre-exposure before induction of injury. 100 ppm CO exposure attenuated neutrophil sequestration within the pulmonary vasculature as well as LPS-induced neutrophilia at 6 hours after LPS, likely due to abrogation of neutrophil mobilisation from bone marrow. In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space

Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices

Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions

The Role of Alveolar Epithelial Cells in Initiating and Shaping Pulmonary Immune Responses: Communication between Innate and Adaptive Immune Systems

Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC) have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM) isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG) growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes) were able to induce expression of chemokines (IP-10 and RANTES) by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli