ABSTRACT
Discussion on how the modulation of Rev-Erb-alpha and the circadian CLOCK proteins remain an interesting but elusive target for modulation of acute inflammatory response in critical illness.
Subject(s)
Nuclear Receptor Subfamily 1, Group D, Member 1 , Pneumonia , Animals , Circadian Rhythm , Klebsiella , Klebsiella pneumoniae , MiceABSTRACT
Injured patients with lung contusion (LC) are at risk of developing bacterial pneumonia (PNA) followed by sepsis and death. A recent genome-wide association study (GWAS) showed FER gene expression positively correlating with survival rates among individuals with above conditions. We sought to determine whether electroporation (EP)-mediated delivery of FER gene could indeed improve survival, in a lethal model of combined LC and PNA. C57BL/6 mice sustained unilateral LC, which preceded a 500 Klebsiella colony forming unit (CFU) inoculation by 6 h. In-between these insults, human FER plasmid (pFER) was introduced into the lungs followed by eight EP pulses applied externally (10 ms at 200 V cm-1). Control groups included EP of empty vector (pcDNA3) or Na+/K+-ATPase genes (pPump) and no treatment (LC+PNA). We recorded survival, histology, lung mechanics, bronchial alveolar lavage (BAL) fluid, FER and inflammatory gene expression and bacteriology. The data show that 7-day survival was significantly improved by pFER compared with control groups. pFER increased BAL monocytes and activated antibacterial response genes (nitric oxide synthase (NOS), Fizz). pFER treatment showed decreased lung and blood Klebsiella counts reaching, in some cases, complete sterilization. In conclusion, FER gene delivery promoted survival in LC+PNA mice via recruitment of activated immune cells, improving efficiency of bacterial clearance within contused lung.
Subject(s)
Contusions/complications , Electroporation , Genetic Therapy , Pneumonia, Bacterial/therapy , Protein-Tyrosine Kinases/genetics , Animals , Bacterial Load , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Humans , Klebsiella/pathogenicity , Male , Mice , Mice, Inbred C57BL , Pneumonia, Bacterial/etiology , Pneumonia, Bacterial/microbiology , Protein-Tyrosine Kinases/metabolism , Sodium-Potassium-Exchanging ATPase/genetics , Sodium-Potassium-Exchanging ATPase/metabolismABSTRACT
To increase the levels of pulmonary gene transfer by nonviral vectors, we have adopted electroporation protocols for use in the lung. A volume of 100-200 microl of purified plasmid DNA suspended in saline was instilled into the lungs of anesthetized mice. Plasmids expressed luciferase, or beta-galactosidase under control of the CMV immediate-early promoter and enhancer. Immediately following delivery, a series of eight square wave electric pulses of 10 ms duration each at an optimal field strength of 200 V/cm were administered to the animals using 10 mm Tweezertrodes (Genetronics, San Diego, CA, USA). The electrodes were placed on either side of the chest, which had been wetted with 70% ethanol. The animals recovered and survived with no apparent trauma until the experiments were terminated at the desired times, between 1 and 7 days post-treatment. Gene expression was detected by 1 day postelectroporation and peaked between 2 and 5 days. By 7 days, expression was back to baseline. By contrast, essentially no gene expression was detected in the absence of electric pulses. Using a beta-galactosidase-expressing plasmid, the distribution of gene expression appeared to be concentrated in the periphery of the lung, but was also present throughout the parenchyma. The primary cell types expressing gene product include alveolar type I and type II epithelial cells. No inflammation or lung injury was detected histologically or by cytokine measurements in lungs at either 1 or 24 h following electroporation treatment. These results provide evidence that electroporation is a safe and effective means for introducing naked DNA into the lung and form the basis for future studies on targeted pulmonary gene therapy.
