Electroporation-mediated delivery of FER gene enhances innate immune response and improves survival in a murine model of pneumonia.

Previously, we reported that electroporation-mediated (EP) delivery of the FER gene improved survival in a combined trauma-pneumonia model. The mechanism of this protective effect is unknown. In this paper, we performed a pneumonia model in C57/BL6 mice with 500 CFU of Klebsiella pneumoniae. After inoculation, a plasmid encoding human FER was delivered by EP into the lung (PNA/pFER-EP). Survival of FER-treated vs. controls (PNA; PNA/EP-pcDNA) was recorded. In parallel cohorts, bronchial alveolar lavage (BAL) and lung were harvested at 24 and 72 h with markers of infection measured. FER-EP-treated animals reduced bacterial counts and had better 5-day survival compared to controls (80 vs. 20 vs. 25%; p < 0.05). Pre-treatment resulted in 100% survival. With FER, inflammatory monocytes were quickly recruited into BAL. These cells had increased surface expression for Toll-receptor 2 and 4, and increased phagocytic and myeloperoxidase activity at 24 h. Samples from FER electroporated animals had increased phosphorylation of STAT transcription factors, varied gene expression of IL1ß, TNFα, Nrf2, Nlrp3, Cxcl2, HSP90 and increased cytokine production of TNF-α, CCL-2, KC, IFN-γ, and IL-1RA. In a follow-up experiment, using Methicillin-resistant Staphylococcus aureus (MRSA) similar bacterial reduction effects were obtained with FER gene delivery. We conclude that FER overexpression improves survival through STAT activation enhancing innate immunity and accelerating bacterial clearance in the lung. This constitutes a novel mechanism of inflammatory regulation with therapeutic potential in the setting of hospital-acquired pneumonia.

Electroporation-mediated delivery of the FER gene in the resolution of trauma-related fatal pneumonia.

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.

Characterization of ultrasound-induced pulmonary capillary hemorrhage in rats.

Routine pulmonary ultrasound for diagnosis of disease or injury relies on interpretation of image features, such as comet-tail artifacts, which can also be indicative of the poorly understood phenomenon of ultrasound-induced pulmonary capillary hemorrhage (PCH). Evans blue extraction and bronchoalveolar lavage (BAL) were evaluated for assessment of PCH induced by ultrasound scanning. Rats anesthetized with ketamine with or without xylazine received sham or scanning for 5 min with a 7.6 MHz linear array. Evans blue extraction and BAL albumin measurements failed to demonstrate significant increases for scanning, even though the induction of comet-tail artifacts was significant. BAL cell counts had an insignificant increase relative to shams at a near-threshold mechanical index (MI) of 0.52 (P=0.07), but a highly significant increase at MI=0.9 (P=0.001). The possibility of xylazine-induced elevated albumin was tested, but no significant decrease was found for sham or scanned rats with ketamine-only anesthesia. Interestingly, without xylazine, the widths of comet-tail artifacts in the ultrasound images were significantly smaller (P=0.001) and cell counts in BAL fluid also were reduced (P=0.014). The BAL cell-count method provides a valuable additional means of PCH quantification.

Increased phospholipase A2 and lyso-phosphatidylcholine levels are associated with surfactant dysfunction in lung contusion injury in mice.

OBJECTIVE: Surfactant dysfunction is an important pathologic disturbance in various forms of acute inflammatory lung injury. Previously we reported the presence of marked alterations in the composition and activity of pulmonary surfactant in bilateral lung contusions (LC) injury induced by blunt trauma in rats. This is extended here to a mouse model of unilateral LC with a focus on compositional and functional changes in surfactant associated with permeability injury and increases in activity of secretory phospholipase A2. RESULTS: Surfactant-associated gene expression was not altered in mice with unilateral LC injury on the basis of Affymetrix analysis. LC mice had significant permeability injury with increased albumin and total protein in bronchoalveolar lavage at 5, 24, 48, and 72 hours after insult compared with uninjured controls. The percent content of large surfactant aggregates was depleted at all postinjury times, and pulmonary pressure-volume (P-V) mechanics and compliance were abnormal during this period. Surfactant dysfunction was evaluated in 24 hours, when permeability injury and P-V changes were most prominent. At this time, activity levels of secretory phospholipase A2 were increased in bronchoalveolar lavage, and chromatographic analysis showed that large surfactant aggregates had decreased levels of phosphatidylcholine and increased levels of lyso-phosphatidylcholine. These changes were accompanied by severe detriments in large aggregate surface activity by pulsating bubble surfactometry. Large aggregates from LC mice at 24 hours had minimum surface tensions of only 12.6 ± 1.1 mN/m after prolonged bubble pulsation (20 min) compared with 0.7 ± 0.03 mN/m for uninjured controls. CONCLUSION: These results document important detriments in the composition and activity of pulmonary surfactant in LC injury in mice and suggest that active synthetic phospholipase-resistant exogenous surfactants may have utility in treating surfactant dysfunction in this clinically important condition.

Electroporation-mediated in vivo gene delivery of the Na+/K+-ATPase pump reduced lung injury in a mouse model of lung contusion.

BACKGROUND: Lung contusion (LC) is an independent risk factor for acute respiratory distress syndrome. The final common pathway in ARDS involves accumulation of fluid in the alveoli. In this study, we demonstrate the application of a potential gene therapy approach by delivering the Na+/K+-ATPase pump subunits in a murine model of LC. We hypothesized that restoring the activity of the pump will result in removal of excess alveolar fluid and additionally reduce inflammation. METHODS: Under anesthesia, C57/BL6 mice were struck along the right posterior axillary line 1 cm above the costal margin with a cortical contusion impactor. Immediately afterward, 100 µg of plasmid DNA coding for the α,ß of the Na+/K+-ATPase pump were instilled into the lungs (LC-electroporation-pump group). Contusion only (LC-only) and a sham saline instillation group after contusion were used as controls (LC-electroporation-sham). By using a BTX 830 electroporator, eight electrical pulses of 200 V/cm field strength were applied transthoracically. Mice were killed at 24 hours, 48 hours, and 72 hours after delivery. Bronchial alveolar lavage was recollected to measure albumin and cytokines by enzyme-linked immunosorbent assay. Pulmonary compliance was measured, and lungs were subject to histopathologic analysis. RESULTS: After the electroporation and delivery of genes coding for the α,ß subunits of the Na+/K+-ATPase pump, there was a significant mitigation of acute lung injury as evidenced by reduction in bronchial alveolar lavage levels of albumin, improved pressure volume curves, and reduced inflammation seen on histology. CONCLUSION: Electroporation-mediated gene transfer of the subunits of the Na+/K+-ATPase pump enhanced recovery from acute inflammatory lung injury after LC.

Regulatory roles for histone deacetylation in IL-1beta-induced nitric oxide release in pancreatic beta-cells.

Histone (de)acetylases control gene transcription via modification of the chromatin structure. Herein, we investigated potential roles for histone deacetylation (or hypoacetylation) in interleukin-1beta (IL-1beta)-mediated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release in insulin-secreting INS 832/13 (INS) cells. Western blot analysis suggested localization of members of Class 1 and Class 2 families of histone deacetylases (HDACs) in these cells. Trichostatin A (TSA), a known inhibitor of HDACs, markedly reduced IL-1beta-mediated iNOS expression and NO release from these cells in a concentration-dependent manner. TSA also promoted hyperacetylation of histone H4 under conditions in which it inhibited IL-1beta-mediated effects on isolated beta cells. Rottlerin, a known inhibitor of protein kinase Cdelta, also increased histone H4 acetylation, and inhibited IL-1beta-induced iNOS expression and NO release in these cells. It appears that the putative mechanism underlying the stimulatory effects of rottlerin on acetylation status of histone H4 are distinct from the HDAC inhibitory property of TSA, since rottlerin failed to inhibit HDAC activity in nuclear extracts isolated from INS cells. These data are suggestive of potential regulatory effects of rottlerin at the level of increasing the histone acetyltransferase activity in these cells. Together our studies present the first evidence to suggest a PKCdelta-mediated signalling step, which promotes hypoacetylation of candidate histones culminating in IL-1beta-induced metabolic dysfunction of the isolated beta cell.

Combined effect of ascorbic acid and selenium supplementation on alcohol-induced oxidative stress in guinea pigs.

Oxidative stress is a key step in the pathogenesis of ethanol associated liver injury. Ethanol administration induces an increase in lipid peroxidation either by enhancing the production of oxygen reactive species or by decreasing the level of endogenous antioxidants. In this present study, four groups of male guinea pigs (Cavia porcellus) were maintained for 45 days as follows: Control group (1 mg ascorbic acid (AA)/100 g body wt./day); Ethanol group (1 mg AA/100 g body wt./day+900 mg ethanol/100 g body wt./day); Selenium+AA group (25 mg AA+0.05 mg sodium selenite/100 g body wt./day); Ethanol+Se+AA group (25 mg AA+0.05 mg sodium selenite/100 g body wt.+900 mg ethanol/100 g body wt./day). Malondialehyde (MDA), hydroperoxides (HP) and conjugated dienes (CD) were significantly increased, while the activities of scavenging enzymes superoxide dismutase (SOD) and catalase were reduced in the alcohol administered groups. Co-administration of Se+AA along with alcohol increased the activities of scavenging enzymes and reduced the lipid peroxidation products level in hepatic tissues of guinea pigs. Activities of glutathione peroxidase (GPX) and glutathione reductase (GR) were enhanced in co-administered group. gamma-Glutamyl transpeptidase (GGT), a marker enzyme of alcohol induced toxicity, was also reduced, as was the glutathione content. This study suggests that the combined effect of Se+AA, provides protection against alcohol-induced oxidative stress as evidenced from the decreased levels of lipid peroxidation products and enhanced activities of scavenging enzymes.

Effect of exposure to a country liquor (Toddy) during gestation on lipid metabolism in rats.

The objective of this study was to determine the effects of country liquor Toddy and its equivalent quantity of ethanol on lipid metabolism during gestation in rats. Female rats weighing an average of 125 g were exposed to Toddy (24.5 ml/body weight/day) and ethanol (0.52 ml/kg body weight/day) for 15 days before conception and throughout gestation. On the 19th day of gestation, altered liver function and hyperlipidemia was seen in both the treated groups. Altered liver function was evidenced by the increased activity of alcohol dehydrogenase, aldehyde dehydrogenase, glutamic oxaloacetic transaminase or aspartate amino transferase (GOT), glutamic pyruvic transaminase or alanine amino transferase (GPT) and gamma glutamyl transpeptidase (GGT). Hyperlipidemia was caused by increased biosynthesis and decreased degradation of lipids. The incorporation of 14C acetate in lipids and activities of HMG CoA reductase and lipogenic enzymes were elevated and activity of LPL and bile acids contents were decreased. Toddy treated rats were more severely affected than those receiving an equivalent quantity of ethanol. Toddy seemed to potentiate the toxicity induced by alcohol indicating the role of the nonethanolic portion. Hepatic functions were also affected.

Prenatal exposure of an alcoholic beverage (Arrack) on fetal lipid metabolism in rats.

The objective of this study was to determine the effects of a country liquor (Arrack) and the equivalent quantity of ethanol on liver function and lipid metabolism in utero. Female rats of average weight 125 g were exposed to Arrack (12 ml/kg body weight/day) and ethanol (3.2 ml/kg body weight/day) for 15 days before conception and throughout gestation. On 13th day and 19th day of gestation, altered liver function and hyperlipidemia was seen in the fetus of both the treated groups. Altered liver function was evidenced by the increased activity of alcohol dehydrogenase and glutamic pyruvic transaminase or alanine amino transferase (GPT). Hyperlipidemia was caused by increased biosynthesis since the incorporation of 14C acetate to lipids and activities of HMG CoA reductase and lipogenic enzymes were elevated. Arrack seemed to potentiate the toxicity induced by alcohol indicating the role of non ethanolic portion. Hepatic functions of the 13th day fetuses were effected to a lesser degree than the 19th day hepatic liver.

Effects of exogenous vitamin C on ethanol toxicity in rats.

The effect of a mega dose of ascorbic acid (200 mg/100 g body wt.) on alcohol-induced toxicity in rats was evaluated. In rats administered alcohol and ascorbic acid, malondialdehyde (MDA), hydroperoxide and conjugated dienes decreased in comparison with that given alcohol alone. The reduced activities of scavenging enzymes, e.g. superoxide dismutase (SOD) and catalase, in ethanol-administered rats were also enhanced by the co-administration of ascorbic acid and ethanol. Co-administration of ethanol and ascorbic acid reduced phospholipids and MDA levels of the erythrocyte membrane in comparison with that of the ethanol fed rats. The reduction in the activities of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), gamaglutamyl transpeptidase (GGT) and the decrease in triglycerides levels also clearly showed the protective action of ascorbic acid in reducing ethanol induced toxicity.

Impact of massive ascorbic acid supplementation on alcohol induced oxidative stress in guinea pigs.

The effects of a mega dose of ascorbic acid (AA) on alcohol induced peroxidative damages were investigated in guinea pigs. In the present study, four groups of male guinea pigs were maintained for 30 days as follows. (1) Control group (1 mg AA/100 g body wt); (2) Ethanol group (1 mg AA/100 g body wt. + 9 g ethanol/kg body wt); (3) AA group (25 mg AA/100 g body wt); (4) AA + ethanol group (25 mg AA/100 g body wt. + 9 g ethanol/kg). Results revealed that alcohol induced significant lipid peroxidation, since the lipid peroxidation products malondialdehyde (MDA), hydroperoxides and conjugated dienes were elevated. The activities of scavenging enzymes superoxide dismutase (SOD), catalase were reduced. However, supplementation of AA along with alcohol reduced the lipid peroxidation products in the liver and enhanced the activities of scavenging enzymes. Activities of glutathione peroxidase and reductase were also greater in guinea pigs given alcohol + AA in comparison with those given alcohol alone. Administration of ascorbic acid also reduced the activity of gamma-glutamyl transpeptidase (GGT), the marker enzyme of alcohol induced toxicity. The vitamin E level, which was reduced by alcohol intake, was raised by the co-administration of AA and alcohol. These studies suggest that a mega dose of AA helps in the prevention of alcohol induced oxidative stress by enhancing the antioxidant capacity and also by reducing the lipid peroxidation products.

Ascorbic acid metabolism in rats and guinea pigs after the administration of ethanol.

Ascorbic acid metabolism was studied in guinea pigs and rats after the administration of ethanol and a high dose of ascorbic acid (AA). Male guinea pigs were maintained for 30 days as follows: (1) controls (1 mg AA/100 g body wt.); (2) ethanol (1 mg AA/100 g body wt. + 900 mg ethanol/100 g body wt); (3) ascorbic acid (25 mg AA/100 g body wt.); (4) ascorbic acid + ethanol (25 mg AA/100 g body wt. + 900 mg ethanol/100 g body wt.). Rats were also grouped into four groups as in the case of guinea pigs, but the dose of AA was 200 mg/100 g body weight. Rats adjusted to ethanol intoxication by enhancing the biosynthesis of ascorbate as evidenced by elevated activity of L-gulono lactone oxidase (GLO). Hence ascorbate levels were not lowered in rats after administration of alcohol. However, alcohol administration lowered tissue levels of ascorbate in guinea pigs. But the supplementation of ascorbate along with alcohol raised the tissue level of this vitamin. Guinea pigs responded to the ascorbate deficiency during alcohol administration by lowering the degradation of ascorbate, as seen by the lower activity of the degrading enzyme gulono lactone hydrolase. It is concluded that on the administration of alcohol, guinea pigs are dependent upon additional exogenous supplies of ascorbic acid, whereas rats are not.

Effect of ethanol/arrack on the lipid metabolism of mammary gland during pregnancy and lactation in rats.

Female rats were exposed to arrack (12.0 ml/kg body weight/day) and ethanol (4.0 g/kg body weight/day) before conception and throughout gestation and lactation. On 19th day of gestation and 21st day of lactation there was increase in the cholesterol phospholipids, triglycerides and free fatty acids in the mammary gland of rats administered arrack/ethanol in comparison with the controls. The lipoprotein lipase activity showed significant increase in the treated groups, in which the activity decreased on 21st day in comparison with 19th day. The absolute and relative weight of mammary gland also showed a significant decrease in ethanol/arrack treated group. The biochemical alterations produced in the mammary gland by arrack and its equivalent alcohol were different showing that non-alcoholic portion of arrack interferes with the toxicity induced by alcohol. Arrack was found to be a potent hyperlipidemic agent than ethanol.

Effect of in utero exposure of Toddy (coconut palm wine) on liver function and lipid metabolism in rat fetuses.

The objective of this study was to determine the effects of a country liquor Toddy (Coconut palm wine) and an equivalent quantity of ethanol on liver function and lipid metabolism in utero. Female albino rats with an average weight of 125 +/- 5 g were exposed to Toddy from coconut palm (24.5 ml/kg body weight/day) and ethanol (0.52 ml/kg body weight/day) for 15 days before conception and during pregnancy. On day 13 and day 19 of gestation, altered liver function and hyperlipidemia were seen in the fetuses of both the treated groups. Altered liver function was evidenced by the increased activity of alcohol dehydrogenase, aldehyde dehydrogenase, glutamic oxaloacetic transaminase (aspartate amino transferase (GOT)), glutamic pyruvic transaminase (alanine amino transferase (GPT)). Hyperlipidemia was caused by increased biosynthesis since the incorporation of 14C acetate into lipids and activities of HMG CoA reductase and lipogenic enzymes were elevated. Toddy treated fetuses were more severely affected than those exposed to an equivalent quantity of ethanol. Toddy seemed to potentiate the toxicity induced by alcohol suggesting the role of non alcoholic components. Hepatic functions of the day 13 fetuses were effected to a lesser degree than those in the day 19 hepatic liver.

Effect of coconut palm wine (Toddy) on carbohydrate metabolism in pregnant rats and fetuses.

The objective of this study was to determine the effects of an alcoholic beverage (Toddy) and the equivalent quantity of ethanol on carbohydrate metabolism in utero. Female rats were exposed to Toddy from coconut palm (24.5 ml/kg body weight/day) and ethanol (0.52 ml/kg body weight/day) for 15 days before conception and throughout gestation. On the 19th day of gestation, hypoglycemia was seen in both the treated groups, but it was more in the Toddy-treated group. Synthesis of glycogen was elevated on exposure to ethanol/Toddy but its degradation was enhanced only in alcohol-exposed rats. Key enzymes of citric acid cycle and gluconeogenesis were inhibited on administration of both alcohol and Toddy. Activity of glycolytic enzymes were increased. Toddy seemed to potentiate the toxicity induced by alcohol, indicating the additive effects of congeners.

Interaction of ethanol and ascorbic acid on lipid metabolism in guinea pigs.

Influence of excessive intake of ascorbic acid (AA) on alcohol induced hyperlipidemia was investigated. In the present study four groups of male guinea pigs were maintained for 30 days as follows: (1) Control group (1 mg AA/100 g body wt). (2) Ethanol group (I mg AA + 9 g ethanol/100 g body wt). (3) AA group (25 mg AA/100 g body wt). (4) AA + Ethanol group (25 mg AA/100 g body wt + 9 g ethanol 100 g body wt). It was shown that tissue ascorbic acid concentration increased with the intake of mega dose of AA. Alcohol administration depleted tissue ascorbic acid content. But coadministration of AA and alcohol enhanced AA levels in comparison with the ethanol group. Alcohol induced hyperlipidemia was reduced in almost all the tissues by the intake of ascorbic acid. This was observed to be due to increased hepatic catabolism of cholesterol to bile acids. However cholesterogenesis was enhanced as evidenced by the increased HMG CoA activity. Thus the results indicate that the mega dose of AA ingestion is beneficial in reducing alcohol induced hyperlipidemia and AA deficiency.