Card9 Broadly Regulates Host Immunity against Experimental Pulmonary Cryptococcus neoformans 52D Infection.

The ubiquitous soil-associated fungus Cryptococcus neoformans causes pneumonia that may progress to fatal meningitis. Recognition of fungal cell walls by C-type lectin receptors (CLRs) has been shown to trigger the host immune response. Caspase recruitment domain-containing protein 9 (Card9) is an intracellular adaptor that is downstream of several CLRs. Experimental studies have implicated Card9 in host resistance against C. neoformans; however, the mechanisms that are associated with susceptibility to progressive infection are not well defined. To further characterize the role of Card9 in cryptococcal infection, Card9em1Sq mutant mice that lack exon 2 of the Card9 gene on the Balb/c genetic background were created using CRISPR-Cas9 genome editing technology and intratracheally infected with C. neoformans 52D. Card9em1Sq mice had significantly higher lung and brain fungal burdens and shorter survival after C. neoformans 52D infection. Susceptibility of Card9em1Sq mice was associated with lower pulmonary cytokine and chemokine production, as well as reduced numbers of CD4+ lymphocytes, neutrophils, monocytes, and dendritic cells in the lungs. Histological analysis and intracellular cytokine staining of CD4+ T cells demonstrated a Th2 pattern of immunity in Card9em1Sq mice. These findings demonstrate that Card9 broadly regulates the host inflammatory and immune response to experimental pulmonary infection with a moderately virulent strain of C. neoformans.

Gram-Negative Pneumonia Augments Non-Small Cell Lung Cancer Metastasis through Host Toll-like Receptor 4 Activation.

INTRODUCTION: Surgery is essential for cure of early-stage non-small cell lung cancer (NSCLC). Rates of postoperative bacterial pneumonias, however, remain high, and clinical data suggests that post-operative infectious complications confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory response to infection by recognizing evolutionarily conserved bacterial structures at the surface of numerous pulmonary cell types; yet, little is known about how host TLR activation influences NSCLC metastasis. TLR4 recognizes gram-negative bacterium lipopolysaccharide activating the innate immune system. METHODS: C57BL/6 and TLR4 knockout murine airways were inoculated with Escherichia coli or lipopolysaccharide. Hepatic metastasis assays and intravital microscopy were performed. Bronchoepithelial conditioned media was generated through coincubation of bronchoepithelial cells with TLR4 activating Escherichia coli or lipopolysaccharide. Subsequently, H59 NSCLC were stimulated with conditioned media and subject to various adhesion assays. RESULTS: We demonstrate that gram-negative Escherichia coli pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through TLR4 activation. Additionally, infected C57BL/6 mice demonstrate increased H59 NSCLC in vivo hepatic sinusoidal adhesion compared with negative controls, a response that is significantly diminished in TLR4 knockout mice. Similarly, intratracheal injection of purified TLR4 activating lipopolysaccharide increases in vivo adhesion of H59 cells to murine hepatic sinusoids. Furthermore, H59 cells incubated with bronchoepithelial conditioned medium show increased cell adhesion to in vitro extracellular matrix proteins and in vivo hepatic sinusoids through a mechanism dependent on bronchoepithelial TLR4 activation and interleukin-6 secretion. CONCLUSION: TLR4 is a viable therapeutic target for NSCLC metastasis augmented by gram-negative pneumonia.

Gram-positive pneumonia augments non-small cell lung cancer metastasis via host toll-like receptor 2 activation.

Surgical resection of early stage nonsmall cell lung cancer (NSCLC) is necessary for cure. However, rates of postoperative bacterial pneumonias remain high and may confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory cascade by recognizing microbial products at the surface of numerous cell types in the lung; however, little is known about how host TLRs influence NSCLC metastasis. TLR2 recognizes gram-positive bacterial cell wall components activating innate immunity. We demonstrate that lower respiratory tract infection with Streptococcus pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through host TLR2 activation. Infected mice demonstrate increased H59 and human A549 NSCLC adhesion to hepatic sinusoids in vivo compared with noninfected controls, a response that is significantly diminished in TLR2 knock-out mice. Intra-tracheal injection of purified TLR2 ligand lipoteichoic acid into mice similarly augments in vivo adhesion of H59 cells to hepatic sinusoids. Additionally, H59 and A549 NSCLC cells incubated with bronchoepithelial conditioned media show increased cell adhesion to extracellular matrix components in vitro and hepatic sinusoids in vivo in a manner that is dependent on bronchoepithelial TLR2 activation and interleukin-6 secretion. TLR2 is therefore a potential therapeutic target for gram-positive pneumonia-driven NSCLC metastasis.

Pharmacokinetics of an extended 4-hour infusion of piperacillin-tazobactam in critically ill patients undergoing continuous renal replacement therapy.

STUDY OBJECTIVE: To evaluate the pharmacokinetic and pharmacodynamic profiles of piperacillin-tazobactam administered as a 4-hour infusion in critically ill patients undergoing continuous renal replacement therapy (CRRT). DESIGN: Prospective, observational, pharmacokinetic study. SETTING: Intensive care unit of a tertiary care hospital in Montréal, Canada. PATIENTS: Twenty critically ill adults who were undergoing continuous venovenous hemodiafiltration and receiving a 4-hour infusion of piperacillin 4 g-tazobactam 0.5 g every 8 hours for a documented or suspected infection. INTERVENTION: Blood samples were collected every hour over an 8-hour dosing interval. Prefilter and postfilter blood samples, and effluent and urine samples were also collected. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the proportion of patients who achieved an unbound piperacillin plasma concentration above a target minimum inhibitory concentration (MIC) of 64 mg/L (MIC that inhibits 90% of isolates for Pseudomonas aeruginosa) for at least 50% of the dosing interval; 18 (90%) of the 20 patients achieved this outcome. In all patients, the free piperacillin concentrations were above the Pseudomonas aeruginosa breakpoint of 16 mg/L for the entire time interval. Regarding piperacillin pharmacokinetic parameters, the median (interquartile range) minimum unbound plasma concentration was 65.15 mg/L (51.30-89.30), maximum unbound plasma concentration was 141.3 mg/L (116.75-173.90), sieving coefficient was 0.809 (0.738-0.938), total clearance was 65.82 ml/minute (53.79-102.87), and renal clearance was 0.16 ml/minute (0.05-3.04). The median CRRT dose was 32.0 ml/kg/h (25.0-39.8). CONCLUSIONS: Administration of a 4-hour infusion of piperacillin-tazobactam was associated with a favorable pharmacodynamic profile in patients undergoing CRRT. Concentrations associated with maximal activity were attained in our patients.