Healing efficacy of sea buckthorn (Hippophae rhamnoides L.) seed oil in an ovine burn wound model.

To investigate the efficacy of sea buckthorn (SBT) seed oil - a rich source of substances known to have anti-atherogenic and cardioprotective activity, and to promote skin and mucosa epithelization - on burn wound healing, five adult sheep were subjected to 3rd degree flame burns. Two burn sites were made on the dorsum of the sheep and the eschar was excised down to the fascia. Split-thickness skin grafts were harvested, meshed, and fitted to the wounds. The autograft was placed on the fascia and SBT seed oil was topically applied to one recipient and one donor site, respectively, with the remaining sites treated with vehicle. The wound blood flow (LASER Doppler), and epithelization (ultrasound) were determined at 6, 14, and 21 days after injury. 14 days after grafting, the percentage of epithelization in the treated sites was greater (95 ± 2.2% vs. 83 ± 2.9%, p<0.05) than in the untreated sites. Complete epithelization time was shorter in both treated recipient and donor sites (14.20 ± 0.48 vs. 19.60 ± 0.40 days, p<0.05 and 13.40 ± 1.02 vs. 19.60 ± 0.50 days, p<0.05, respectively) than in the untreated sites, confirmed by ultrasound. In conclusion, SBT seed oil has significant wound healing activity in full-thickness burns and split-thickness harvested wounds.

Energy return on investment for algal biofuel production coupled with wastewater treatment.

This study presents a second-order energy return on investment analysis to evaluate the mutual benefits of combining an advanced wastewater treatment plant (WWTP) (with biological nutrient removal) with algal biofuel production. With conventional, independently operated systems, algae production requires significant material inputs, which require energy directly and indirectly, and the WWTP requires significant energy inputs for treatment of the waste streams. The second-order energy return on investment values for independent operation of the WWTP and the algal biofuels production facility were determined to be 0.37 and 0.42, respectively. By combining the two, energy inputs can be reduced significantly. Consequently, the integrated system can outperform the isolated system, yielding a second-order energy return on investment of 1.44. Combining these systems transforms two energy sinks to a collective (second-order) energy source. However, these results do not include capital, labor, and other required expenses, suggesting that profitable deployment will be challenging.

Pulmonary microvascular hyperpermeability and expression of vascular endothelial growth factor in smoke inhalation- and pneumonia-induced acute lung injury.

INTRODUCTION: Acute lung injury (ALI) and sepsis are major contributors to the morbidity and mortality of critically ill patients. The current study was designed further evaluate the mechanism of pulmonary vascular hyperpermeability in sheep with these injuries. METHODS: Sheep were randomized to a sham-injured control group (n=6) or ALI/sepsis group (n=7). The sheep in the ALI/sepsis group received inhalation injury followed by instillation of Pseudomonas aeruginosa into the lungs. These groups were monitored for 24 h. Additional sheep (n=16) received the injury and lung tissue was harvested at different time points to measure lung wet/dry weight ratio, vascular endothelial growth factor (VEGF) mRNA and protein expression as well as 3-nitrotyrosine protein expression in lung homogenates. RESULTS: The injury induced severe deterioration in pulmonary gas exchange, increases in lung lymph flow and protein content, and lung water content (P<0.01 each). These alterations were associated with elevated lung and plasma nitrite/nitrate concentrations, increased tracheal blood flow, and enhanced VEGF mRNA and protein expression in lung tissue as well as enhanced 3-nitrotyrosine protein expression (P<0.05 each). CONCLUSIONS: This study describes the time course of pulmonary microvascular hyperpermeability in a clinical relevant large animal model and may improve the experimental design of future studies.

Beneficial pulmonary effects of a metalloporphyrinic peroxynitrite decomposition catalyst in burn and smoke inhalation injury.

During acute lung injury, nitric oxide (NO) exerts cytotoxic effects by reacting with superoxide radicals, yielding the reactive nitrogen species peroxynitrite (ONOO(-)). ONOO(-) exerts cytotoxic effects, among others, by nitrating/nitrosating proteins and lipids, by activating the nuclear repair enzyme poly(ADP-ribose) polymerase and inducing VEGF. Here we tested the effect of the ONOO(-) decomposition catalyst INO-4885 on the development of lung injury in chronically instrumented sheep with combined burn and smoke inhalation injury. The animals were randomized to a sham-injured group (n = 7), an injured control group [48 breaths of cotton smoke, 3rd-degree burn of 40% total body surface area (n = 7)], or an injured group treated with INO-4885 (n = 6). All sheep were mechanically ventilated and fluid-resuscitated according to the Parkland formula. The injury-related increases in the abundance of 3-nitrotyrosine, a marker of protein nitration by ONOO(-), were prevented by INO-4885, providing evidence for the neutralization of ONOO(-) action by the compound. Burn and smoke injury induced a significant drop in arterial Po(2)-to-inspired O(2) fraction ratio and significant increases in pulmonary shunt fraction, lung lymph flow, lung wet-to-dry weight ratio, and ventilatory pressures; all these changes were significantly attenuated by INO-4885 treatment. In addition, the increases in IL-8, VEGF, and poly(ADP-ribose) in lung tissue were significantly attenuated by the ONOO(-) decomposition catalyst. In conclusion, the current study suggests that ONOO(-) plays a crucial role in the pathogenesis of pulmonary microvascular hyperpermeability and pulmonary dysfunction following burn and smoke inhalation injury in sheep. Administration of an ONOO(-) decomposition catalyst may represent a potential treatment option for this injury.

Time course of nitric oxide synthases, nitrosative stress, and poly(ADP ribosylation) in an ovine sepsis model.

INTRODUCTION: Different isoforms of nitric oxide synthases (NOS) and determinants of oxidative/nitrosative stress play important roles in the pathophysiology of pulmonary dysfunction induced by acute lung injury (ALI) and sepsis. However, the time changes of these pathogenic factors are largely undetermined. METHODS: Twenty-four chronically instrumented sheep were subjected to inhalation of 48 breaths of cotton smoke and instillation of live Pseudomonas aeruginosa into both lungs and were euthanized at 4, 8, 12, 18, and 24 hours post-injury. Additional sheep received sham injury and were euthanized after 24 hrs (control). All animals were mechanically ventilated and fluid resuscitated. Lung tissue was obtained at the respective time points for the measurement of neuronal, endothelial, and inducible NOS (nNOS, eNOS, iNOS) mRNA and their protein expression, calcium-dependent and -independent NOS activity, 3-nitrotyrosine (3-NT), and poly(ADP-ribose) (PAR) protein expression. RESULTS: The injury induced severe pulmonary dysfunction as indicated by a progressive decline in oxygenation index and concomitant increase in pulmonary shunt fraction. These changes were associated with an early and transient increase in eNOS and an early and profound increase in iNOS expression, while expression of nNOS remained unchanged. Both 3-NT, a marker of protein nitration, and PAR, an indicator of DNA damage, increased early but only transiently. CONCLUSIONS: Identification of the time course of the described pathogenetic factors provides important additional information on the pulmonary response to ALI and sepsis in the ovine model. This information may be crucial for future studies, especially when considering the timing of novel treatment strategies including selective inhibition of NOS isoforms, modulation of peroxynitrite, and PARP.

Human CD8(+) T cells clear Cryptosporidium parvum from infected intestinal epithelial cells.

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, especially in immunocompromised individuals. CD4(+) T cells and interferon-gamma are key factors in the control of cryptosporidiosis in human and murine models. Previous studies led us to hypothesize that CD8(+) T cells contribute to clearance of intestinal epithelial Cryptosporidium infection in humans. We report here that antigen expanded sensitized CD8(+) T cells reduce the parasite load in infected intestinal epithelial cell cultures and lyse infected intestinal epithelial cells. These effects are most likely mediated by the release of cytotoxic granules. Elimination of parasites seems to require antigen presentation through both human leukocyte antigen (HLA)-A and HLA-B. These data suggest that cytotoxic CD8(+) T cells play a role in clearing Cryptosporidium from the intestine, a previously unrecognized feature of the human immune response against this parasite.

Molecular biological effects of selective neuronal nitric oxide synthase inhibition in ovine lung injury.

Neuronal nitric oxide synthase is critically involved in the pathogenesis of acute lung injury resulting from combined burn and smoke inhalation injury. We hypothesized that 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, blocks central molecular mechanisms involved in the pathophysiology of this double-hit insult. Twenty-five adult ewes were surgically prepared and randomly allocated to 1) an uninjured, untreated sham group (n = 7), 2) an injured control group with no treatment (n = 7), 3) an injury group treated with 7-nitroindazole from 1-h postinjury to the remainder of the 24-h study period (n = 7), or 4) a sham-operated group subjected only to 7-nitroindazole to judge the effects in health. The combination injury was associated with twofold increased activity of neuronal nitric oxide synthase and oxidative/nitrosative stress, as indicated by significant increases in plasma nitrate/nitrite concentrations, 3-nitrotyrosine (an indicator of peroxynitrite formation), and malondialdehyde lung tissue content. The presence of systemic inflammation was evidenced by twofold, sixfold, and threefold increases in poly(ADP-ribose) polymerase, IL-8, and myeloperoxidase lung tissue concentrations, respectively (each P < 0.05 vs. sham). These molecular changes were linked to tissue damage, airway obstruction, and pulmonary shunting with deteriorated gas exchange. 7-Nitroindazole blocked, or at least attenuated, all these pathological changes. Our findings suggest 1) that nitric oxide formation derived from increased neuronal nitric oxide synthase activity represents a pivotal reactive agent in the patho-physiology of combined burn and smoke inhalation injury and 2) that selective neuronal nitric oxide synthase inhibition represents a goal-directed approach to attenuate the degree of injury.

Pulmonary vascular permeability changes in an ovine model of methicillin-resistant Staphylococcus aureus sepsis.

INTRODUCTION: Endothelial dysfunction is a hallmark of sepsis, associated with lung transvascular fluid flux and pulmonary dysfunction in septic patients. We tested the hypothesis that methicillin-resistant Staphylococcus aureus (MRSA) sepsis following smoke inhalation increases pulmonary transvascular fluid flux via excessive nitric oxide (NO) production. METHODS: Ewes were chronically instrumented, and randomised into either a control or MRSA sepsis (MRSA and smoke inhalation) group. RESULTS: Pulmonary function remained stable in the control group, whereas the MRSA sepsis group developed impaired gas exchange and significantly increased lung lymph flow, permeability index and bloodless wet-to-dry weight-ratio (W/D ratio). The plasma nitrate/nitrite (NOx) levels, lung inducible nitric oxide synthases (iNOS) and endothelial nitric oxide synthases (eNOS), vascular endothelial growth factor (VEGF) protein expressions and poly-(ADP)-ribose (PAR) were significantly increased by MRSA challenge. CONCLUSIONS: These results provide evidence that excessive NO production may mediate pulmonary vascular hyperpermeability in MRSA sepsis via up regulation of reactive radicals and VEGF.

Inhibition of neuronal nitric oxide synthase in ovine model of acute lung injury.

OBJECTIVE: Acute respiratory distress syndrome/acute lung injury is a serious complication of burn patients with concomitant smoke inhalation injury. Nitric oxide has been shown to play a major role in pulmonary dysfunction from thermal damage. In this study, we have tested the hypothesis that inhibition of neuronal nitric oxide synthase could ameliorate the severity of acute lung injury using our well-established ovine model of cutaneous burn and smoke inhalation. DESIGN: Prospective, randomized, controlled, experimental animals study. SETTING: Investigational intensive care unit at university hospital. SUBJECTS: Adult female sheep. INTERVENTIONS: Female sheep (n = 16) were surgically prepared for the study. Seven days after surgery, all sheep were randomly allocated into three study groups: sham (noninjured, nontreated, n = 6); control (injured, treated with saline, n = 6); and neuronal nitric oxide synthase (injured, treated with specific neuronal nitric oxide synthase inhibitor, ZK 234238 (n = 4). Control and neuronal nitric oxide synthase groups were given a cutaneous burn (40% of total body surface, third degree) and insufflated with cotton smoke (48 breaths, <40 degrees C) under halothane anesthesia. Animals in sham group received fake injury also under halothane anesthesia. After injury or fake injury procedure, all sheep were placed on ventilators and resuscitated with lactated Ringer's solution. Neuronal nitric oxide synthase group was administered with continuous infusion of ZK 234238 started 1 hr postinjury with a dose of 100 microg/kg/hr. Sham and control groups received same amount of saline. MEASUREMENTS AND MAIN RESULTS: Cardiopulmonary hemodynamics monitored during the 24-hr experimental time period was stable in the sham group. Control sheep developed multiple signs of acute lung injury. This pathophysiology included decreased pulmonary gas exchange and lung compliance, increased pulmonary edema, and inflammatory indices, such as interleukin-8. Treatment of injured sheep with neuronal nitric oxide synthase inhibitor attenuated all the observed pulmonary pathophysiology. CONCLUSIONS: The results provide definitive evidence that inhibition of neuronal nitric oxide synthase-derived excessive nitric oxide may be a novel and beneficial treatment strategy for pulmonary pathology in burn victims with smoke inhalation injury.

Combined neuronal and inducible nitric oxide synthase inhibition in ovine acute lung injury.

OBJECTIVE: Acute lung injury with subsequent pneumonia and sepsis represents a major cause of morbidity and mortality in thermally injured patients. Production of nitric oxide by the neuronal and inducible nitric oxide synthase may be critically involved in the pathophysiology of the disease process at different time points, and thus specific inhibition at different times may represent an effective treatment regimen. DESIGN: Prospective, controlled, randomized trial. SETTING: University research laboratory. SUBJECTS: Eighteen chronically instrumented, adult, female sheep. INTERVENTIONS: Following baseline measurements, the animals were allocated to either sham-injured, nontreated controls (sham), injured, nontreated controls (control), or injured animals treated with continuous infusion of 7-nitroindazole, a specific neuronal nitric oxide synthase inhibitor, during the first 12 hrs postinjury and infusion of BBS-2, a specific inducible nitric oxide synthase inhibitor, during the next 12 hrs. Injury was induced by 48 breaths of cotton smoke and subsequent instillation of Pseudomonas aeruginosa into the lungs. All sheep were mechanically ventilated and fluid resuscitated for the entire duration of the 24-hr experiment. MEASUREMENTS AND MAIN RESULTS: The injury induced severe pulmonary dysfunction, which was associated with increases in lung edema formation, airway obstruction, and vascular endothelial growth factor, 3-nitrotyrosine, and poly(adenosine diphosphate ribose) expression in lung tissue. The treatment reduced the degree of airway obstruction and improved pulmonary gas exchange, whereas the development of lung edema was not affected. The increases in lung tissue vascular endothelial growth factor, 3-nitrotyrosine, and poly(ribose) expression were attenuated by the treatment. CONCLUSIONS: The combination of early neuronal nitric oxide synthase and delayed inducible nitric oxide synthase inhibition shows potential benefit in ovine acute lung injury by reducing nitrosative stress in the lung and limiting the degree of airway obstruction.

Assessment of vascular permeability in an ovine model of acute lung injury and pneumonia-induced Pseudomonas aeruginosa sepsis.

OBJECTIVE: To assess the time changes and mechanism of pulmonary and peripheral vascular permeability in sheep with acute lung injury and sepsis. DESIGN: Prospective, controlled, randomized trial. SETTING: University research laboratory. SUBJECTS: A total of 21 chronically instrumented, adult female sheep. INTERVENTIONS: Sheep were instrumented with lung and prefemoral lymph fistulas and allocated to either an uninjured control group (n = 5) or sepsis group (n = 5). The sheep in the sepsis group received cotton smoke inhalation injury followed by instillation of Pseudomonas aeruginosa into the lungs. All sheep were mechanically ventilated and fluid resuscitated for the entire duration of the 24-hr experiment. Additional sheep (n = 11) received injury and were killed at different time points for the measurement of vascular endothelial growth factor in lung tissue. MEASUREMENTS AND MAIN RESULTS: The injury induced a hypotensive-hyperdynamic circulation; increases in pulmonary capillary pressure, net fluid balance, lung and prefemoral lymph flow and protein content, lung water content, abdominal and thoracic fluid and protein content, neutrophil accumulation in the lung, and vascular endothelial growth factor expression in lung tissue; and decreases in PaO2/FiO2 ratio, plasma protein concentration, plasma oncotic pressure, and myocardial contractility. CONCLUSIONS: Lung edema formation in this model was the result of marked increases in both pulmonary microvascular permeability and pressure. Pulmonary vascular hyperpermeability peaked 12 hrs postinjury and was related to vascular endothelial growth factor overexpression. Early myocardial failure was a potential contributor to the constant increase in pulmonary capillary pressure. The sepsis-induced increase in peripheral microvascular permeability was associated with significant accumulation of fluid and protein in the third space.

Novel ovine model of methicillin-resistant Staphylococcus aureus-induced pneumonia and sepsis.

Methicillin-resistant Staphylococcus aureus (MRSA)-related pneumonia and/or sepsis are a frequent serious menace. The aim of the study was to establish a standardized and reproducible model of MRSA-induced septic pneumonia to evaluate new therapies. Sheep were operatively prepared for chronic study. After 5 days' recovery, tracheostomy was performed under anesthesia, and smoke injury was induced by inhalation of cotton smoke (48 breaths, <40 degrees C). Methicillin-resistant S. aureus (AW6) (approximately 2.5x10(11) colony-forming units) was instilled into the airway by a bronchoscope. After the injury, animals were awakened and maintained on mechanical ventilation by 100% oxygen for first 3 h, and thereafter, oxygen concentration was adjusted according to blood gases. The sheep were resuscitated by lactated Ringer solution with an initial rate of 2 mL kg(-1) h(-1) that was further adjusted according to hematocrit. Study groups include (1) sham (noninjured, nontreated; n=6), (2) S+MRSA (exposed to smoke inhalation and MRSA, nontreated; n=6), and (3) smoke (exposed to smoke inhalation alone; n=6). Injured (S+MRSA) animals showed the signs of severe sepsis-related multiple organ failure 3 h after insult. Cardiovascular morbidity was evidenced by severe hypotension, with increased heart rate, cardiac output, left atrial pressure and severely decreased systemic vascular resistance index, and left ventricle stroke work index. Pulmonary dysfunction was characterized by deteriorated gas exchange (PaO2/FIO2 and pulmonary shunt) and increased ventilatory pressures. The S+MRSA group showed significantly greater lung tissue water content, myeloperoxidase activity, and cytokine production compared with uninjured sham animals. Microvascular hyperpermeability was evidenced by marked fluid retention (fluid net balance), decreased plasma protein with decreased plasma oncotic pressure, and increased pulmonary microvascular pressure. All these changes were accompanied by 6- to 7-fold increase in plasma nitrite/nitrate and increased production of reactive nitrogen species in lung. The smoke inhalation alone had a little or no effect on these variables. This model closely mimics hyperdynamic human sepsis. The excessive production of NO may be extensively involved in the pathogenic process.