Preventive Effects of Suvorexant on Delirium: A Randomized Placebo-Controlled Trial.

OBJECTIVE: No highly effective pharmacologic interventions to prevent delirium have been identified. We examined whether suvorexant, a potent and selective orexin receptor antagonist, is effective for the prevention of delirium. METHODS: We conducted a multicenter, rater-blinded, randomized, placebo-controlled clinical trial in intensive care units and regular acute wards between April 2015 and March 2016. Eligible patients were 65 to 89 years old, newly admitted due to emergency, and able to take medicine orally and had an expected stay or life expectancy of 48 hours or more. Seventy-two patients were randomly assigned using the sealed envelope method to receive suvorexant (15 mg/d; 36 patients) or placebo (36 patients) every night for 3 days. The primary outcome measure was incidence of delirium as determined by the DSM-5. Trained psychiatrists assessed for delirium. RESULTS: We found that delirium developed significantly less often among patients taking suvorexant than among those taking placebo (0% [n/N = 0/36] vs 17% [6/36], respectively, P = .025). Comparison by log-rank test also showed that delirium developed significantly less often among patients taking suvorexant than among those taking placebo (χ² = 6.46, P = .011). Analysis of variance revealed a tendency for main effect of treatment (F = 3.79, P = .053) on the sleep-wake cycle disturbance score (item 1) of the Japanese version of the Delirium Rating Scale-Revised-98 (DRS-R-98-J). There were no significant differences in adverse events. CONCLUSIONS: Suvorexant administered nightly to elderly patients admitted for acute care may provide protection against delirium. Larger studies are needed to show the potential of suvorexant to improve the circadian core domain of delirium. TRIAL REGISTRATION: UMIN Clinical Trials Registry identifier: UMIN000015681​.

Treatment of etoposide combined with 15-deoxy-Δ12,14-prostaglandin J2 exerted synergistic antitumor effects against renal cell carcinoma via peroxisome proliferator-activated receptor-γ-independent pathways.

Renal cell carcinoma (RCC) is characterized by diverse clinical manifestations, few early warning signs and a resistance to radiotherapy and chemotherapy. Although several clinical trials have investigated potential effective therapeutic strategies for RCC, the chemoresistance of RCC has not yet been overcome. An endogenous ligand for the peroxisome proliferator-activated receptor-γ (PPARγ), 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), was shown to induce apoptosis in RCC. The aim of the present study was to investigate the synergistic effects of carcinostatics on the antitumor activity of 15d-PGJ2 in the Caki-2 human RCC cell line with the MTT assay. Our results demonstrated that the topoisomerase-II inhibitor etoposide (VP-16) exhibited cytotoxic effects synergistically with 15d-PGJ2. Furthermore, the presence of the PPARγ antagonist GW9662 did not protect Caki-2 cells against 15d-PGJ2-induced cytotoxicity. Additionally, it was observed that the combined treatment of VP-16 and 15d-PGJ2 activated caspase-3 more efficiently compared to each treatment alone. Therefore, the combined treatment with 15d-PGJ2 and VP-16 exhibited synergistic antitumor activity independently of PPARγ.

Impact of bronchial circulation on bronchial exudates following combined burn and smoke inhalation injury in sheep.

UNLABELLED: We previously reported bronchial circulation contributes to pulmonary edema and increases shunt fraction following smoke inhalation, and bronchial blood flow significantly increases in inhalation injury. We hypothesized reduction of bronchial blood flow reduces exudation to the airway and ameliorates lung injury from combined burn and smoke insults (B&S injury). METHOD: Merino ewes (n=28) randomly divided into three groups: (1) bronchial artery ligated and injured (injury+ligation group); (2) bronchial artery left intact and injured (injury+no ligation group); (3) bronchial artery ligated but not injured (no injury+ligation group) were subjected to a flame burn and inhalation injury under halothane anesthesia. Parameters were analyzed using Scheffe's post hoc test (P<0.05). All Groups were resuscitated with Ringer lactate solution and placed on a ventilator for 48h. RESULTS: Pulmonary gas exchange (PaO(2)/FiO(2)) improved in injury+ligation group. Further, obstruction score, an index of airway cast formation, significantly changed between injury+no ligation group compared to both ligation groups. CONCLUSION: Bronchial circulation plays a significant role in lung injury after B&S injury, and reduction of bronchial blood flow by bronchial artery ligation reduces bronchial exudates, resulting in improved gas exchange.

Activated nuclear factor kappa B and airway inflammation after smoke inhalation and burn injury in sheep.

In a recent study, we have shown a rapid inflammatory cell influx across the glandular epithelium and strong proinflammatory cytokine expression at 4 hours after inhalation injury. Studies have demonstrated a significant role of nuclear factor kappa B in proinflammatory cytokine gene transcription. This study examines the acute airway inflammatory response and immunohistochemical detection of p65, a marker of nuclear factor kappa B activation, in sheep after smoke inhalation and burn injury. Pulmonary tissue from uninjured sheep and sheep at 4, 8, 12, 24, and 48 hours after inhalation and burn injury was included in the study. Following immunostaining for p65 and myeloperoxidase, the cell types and the percentage of bronchial submucosal gland cells staining for p65 and the extent of myeloperoxidase stained neutrophils in the bronchial submucosa were determined. Results indicate absence of detection of P65 before 12 hours after injury. At 12 hours after injury, strong perinuclear staining for p65 was evident in bronchial gland epithelial cells, macrophages, and endothelial cells. Bronchial submucosal gland cells showed a significant increase in the percentage of cells stained for p65 compared with uninjured animals and earlier times after injury, P < .05. At 24 and 48 hours after injury, p65 expression was evident in the bronchiolar epithelium, Type II pneumocytes, macrophages, and endothelial cells. Quantitation of the neutrophil influx into the bronchial submucosa showed a significant increase compared with uninjured tissue at 24 and 48 hours after injury, P < .05. In conclusion, immunohistochemical detection of activated p65 preceded the overall inflammatory response measured in the lamina propria. However, detection of p65 did not correlate with a recent study showing rapid emigration of neutrophils at 4 hours postinjury. Together, these results suggest that p65 immunostaining may identify cells that are activated to produce proinflammatory cytokines after injury; however, the immunoexpression may not adequately reflect the temporal activation of gene transcription that may occur with proinflammatory cytokine production with inhalation injury.

Pulmonary expression of nitric oxide synthase isoforms in sheep with smoke inhalation and burn injury.

Previous studies have indicated increased plasma levels of inducible nitric oxide synthase in lung. This study further examines the pulmonary expression of nitric oxide synthase (NOS) isoforms in an ovine model of acute lung injury induced by smoke inhalation and burn injury (S+B injury). Female range bred sheep (4 per group) were sacrificed at 4, 8, 12, 24, and 48 hours after injury and immunohistochemistry was performed in tissues for various NOS isoforms. The study indicates that in uninjured sheep lung, endothelial (eNOS) is constitutively expressed in the endothelial cells associated with the airways and parenchyma, and in macrophages. Similarly, neuronal (nNOS) is constitutively present in the mucous cells of the epithelium and in neurons of airway ganglia. In uninjured lung, inducible (iNOS) was present in bronchial secretory cells and macrophages. In tissue after S+B injury, new expression of iNOS was evident in bronchial ciliated cells, basal cells, and mucus gland cells. In the parenchyma, a slight increase in iNOS immunostaining was seen in type I cells at 12 and 24 hours after injury only. Virtually no change in eNOS or nNOS was seen after injury.

Activation of sensory neurons reduces ischemia/reperfusion-induced acute renal injury in rats.

BACKGROUND: Prostaglandin I2 (PGI2) produced by endothelial cells improves ischemia/reperfusion-induced acute renal injury by inhibiting leukocyte activation in rats. However, the underlying mechanism(s) of increased PGI2 production is not fully understood. Activation of sensory neurons increases endothelial PGI2 production by releasing calcitonin gene-related peptide (CGRP) in rats with hepatic ischemia or reperfusion. We examined here whether activation of sensory neurons increases PGI2 endothelial production, thereby reducing ischemia/reperfusion-induced acute renal injury. METHODS: Anesthetized rats were subjected to 45 min of renal ischemia/reperfusion. Rats were pretreated with CGRP, capsazepine (a vanilloid receptor-1 antagonist), CGRP(8-37) (a CGRP receptor antagonist), or indomethacin (a cyclooxygenase inhibitor), or subjected to denervation of primary sensory nerves before ischemia/reperfusion. RESULTS: Renal tissue levels of CGRP and 6-keto-prostaglandin F1alpha, a stable metabolite of PGI2, increased after renal ischemia/reperfusion, peaking at 1 h after reperfusion. Overexpression of CGRP was also noted at 1 h after reperfusion. Increases in renal tissue levels of 6-keto-prostaglandin F1alpha at 1 h after reperfusion were significantly inhibited by pretreatment with capsazepine, CGRP(8-37), and indomethacin. Pretreatment with capsazepine, CGRP(8-37), indomethacin, and denervation of primary sensory nerves significantly increased blood urea nitrogen and serum creatinine levels, renal vascular permeability, renal tissue levels of myeloperoxidase activity, cytokine-induced neutrophil chemoattractant, and tumor necrosis factor-alpha, and decreased renal tissue blood flow. However, pretreatment with CGRP significantly improved these changes. CONCLUSIONS: Our results suggest activation of sensory neurons in the pathologic process of ischemia/reperfusion-induced acute renal injury. Such activation reduces acute renal injury by attenuating inflammatory responses through enhanced endothelial PGI2 production.

Neuronal nitric oxide synthase inhibition attenuates cardiopulmonary dysfunctions after combined burn and smoke inhalation injury in sheep.

OBJECTIVE: We hypothesized that nitric oxide derived from the neuronal nitric oxide synthase (NOS) is responsible for much of the injury resulting from skin burn and smoke inhalation. Therefore, we aimed to examine the effects of selective neuronal NOS inhibition on cardiopulmonary functions and cellular injury in sheep with acute respiratory distress syndrome secondary to combined burn and smoke inhalation injury. DESIGN: Prospective, randomized, controlled laboratory experiment. SETTING: Investigational intensive care unit. SUBJECTS: A total of 22 chronically instrumented adult ewes. INTERVENTIONS: Sheep were randomly assigned to either healthy controls (sham), injured controls (40% third-degree flame burn; 48 breaths of cotton smoke), or an injury group treated with the specific neuronal NOS inhibitor 7-nitroindazole (1 mg x kg(-1) x hr(-1)) from 1 hr postinjury to the end of the 48-hr study period. Hypoxic pulmonary vasoconstriction was assessed as decrease in left pulmonary blood flow in response to single-lung hypoxic challenges (100% nitrogen) at baseline, 24 hrs, and 48 hrs. MEASUREMENTS AND MAIN RESULTS: The combination injury contributed to a approximately 90% loss of hypoxic pulmonary vasoconstriction and was associated with significant pulmonary shunting and death of one animal. The increase in nitrate/nitrite plasma levels in injured controls (12 hrs: 17 +/- 2 vs. 6 +/- 1 microM in sham animals; p < .001) was linked to increases in inducible NOS messenger RNA and 3-nitrotyrosine formation in lung tissue (48 hrs: 22 +/- 1 vs. 0.8 +/- 0.3 nM in sham animals; p < .001). 7-Nitroindazole treatment prevented the injury-associated changes in inducible NOS messenger RNA, nitrate/nitrite, and 3-nitrotyrosine, thereby attenuating the loss of hypoxic pulmonary vasoconstriction and improving gas exchange. In addition, 7-nitroindazole decreased lung tissue concentrations of hemoxygenase-1 and ameliorated myocardial depression, airway obstruction, pulmonary edema, ventilatory pressures, and histopathologic changes seen in injured controls. CONCLUSIONS: The present study provides evidence that neuronal NOS-derived nitric oxide plays a pivotal role in the pathogenesis of acute respiratory distress syndrome resulting from combined burn and smoke inhalation injury.

Pulmonary changes in a mouse model of combined burn and smoke inhalation-induced injury.

The morbidity and mortality of burn victims increase when burn injury is combined with smoke inhalation. The goal of the present study was to develop a murine model of burn and smoke inhalation injury to more precisely reveal the mechanistic aspects of these pathological changes. The burn injury mouse group received a 40% total body surface area third-degree burn alone, the smoke inhalation injury mouse group received two 30-s exposures of cotton smoke alone, and the combined burn and smoke inhalation injury mouse group received both the burn and the smoke inhalation injury. Animal survival was monitored for 120 h. Survival rates in the burn injury group, the smoke inhalation injury group, and the combined injury group were 70%, 60%, and 30%, respectively. Mice that received combined burn and smoke injury developed greater lung damage as evidenced by histological changes (septal thickening and interstitial edema) and higher lung water content. These mice also displayed more severely impaired pulmonary gas exchange [arterial PO2 (PaO2)/inspired O2 fraction (FiO2)<200]. Lung myeloperoxidase activity was significantly higher in burn and smoke-injured animals compared with the other three experimental groups. Plasma NO2-/NO3-, lung inducible nitric oxide synthase (iNOS) activity, and iNOS mRNA increased with injury; however, the burn and smoke injury group exhibited a higher response. Severity of burn and smoke inhalation injury was associated with more pronounced production of nitric oxide and accumulation of activated leukocytes in lung tissue. The murine model of burn and smoke inhalation injury allows us to better understand pathophysiological mechanisms underlying cardiopulmonary morbidity secondary to burn and smoke inhalation injury.

L-arginine attenuates acute lung injury after smoke inhalation and burn injury in sheep.

Thermal injury results in reduced plasma levels of arginine (Arg). With reduced Arg availability, NOS produces superoxide instead of NO. We hypothesized that Arg supplementation after burn and smoke inhalation (B + S) injury would attenuate the acute insult to the lungs and, thus, protect pulmonary function. Seventeen Suffolk ewes (n = 17) were randomly divided into three groups: (1) sham injury group (n = 6), (2) B + S injury plus saline treatment (n = 6), and (3) B + S injury plus L-ARG infusion at 57 mg.kg(-1).h(-1) (n = 5). Burn and smoke inhalation injury was induced by standardized procedures, including a 40% area full thickness flame burn combined with 48 breaths of smoke from burning cottons. All animals were immediately resuscitated by Ringer solution and supported by mechanical ventilation for 48 h, during which various variables of pulmonary function were monitored. The results demonstrated that Arg treatment attenuated the decline of plasma Arg concentration after B + S injury. A higher plasma Arg concentration was associated with a less decline in Pao2/Fio2 ratio and a reduced extent of airway obstruction after B + S injury. Histopathological examinations also indicated a remarkably reduced histopathological scores associated with B + S injury. Nitrotyrosine stain in lung tissue was positive after B + S injury, but was significantly reduced in the group with Arg. Therefore, L-Arg supplementation improved gas exchange and pulmonary function in ovine after B + S injury via its, at least in part, effect on reduction of oxidative stress through the peroxynitrite pathway.

Recombinant human activated protein C improves pulmonary function in ovine acute lung injury resulting from smoke inhalation and sepsis.

OBJECTIVE: To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge. DESIGN: Prospective, randomized, controlled, experimental animal study with repeated measurements. SETTING: Investigational intensive care unit at a university hospital. SUBJECTS: Eighteen sheep (37.2 +/- 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2-5 x 10(11) colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 mug/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringer's lactate solution to maintain constant pulmonary artery occlusion pressure. MEASUREMENTS AND MAIN RESULTS: In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in Pao2/Fio2 ratio (control group values were 521 +/- 22 at baseline [BL], 72 +/- 5 at 12 hrs, and 74 +/- 7 at 24 hrs, vs. rhAPC group values of 541 +/- 12 at BL, 151 +/- 29 at 12 hours [p < .05 vs. control], and 118 +/- 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 +/- 0.02, and at 24 hrs, 0.65 +/- 0.08; rhAPC group at BL, 0.24 +/- 0.04, and at 24 hrs, 0.45 +/- 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 +/- 1, and at 24 hrs, 36 +/- 4; rhAPC group at BL, 21 +/- 1, and at 24 hrs, 28 +/- 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 +/- 2; control, 17 +/- 1; rhAPC, 12 +/- 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals). CONCLUSIONS: Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

Vitamin E attenuates acute lung injury in sheep with burn and smoke inhalation injury.

INTRODUCTION: A decrease in alpha-tocopherol (vitamin E) plasma levels in burn patients is typically associated with increased mortality. We hypothesized that vitamin E supplementation (alpha-tocopherol) would attenuate acute lung injury induced by burn and smoke inhalation injury. MATERIALS AND METHODS: Under deep anesthesia, sheep (33 +/- 5 kg) were subjected to a flame burn (40% total body surface area, third degree) and inhalation injury (48 breaths of cotton smoke, < 40 degrees C). Half of the injured group received alpha-tocopherol (1000 IU vitamin E) orally, 24 h prior to injury. The sham group was neither injured nor given vitamin E. All three groups (n = 5 per group) were resuscitated with Ringer's lactate solution (4 ml/kg/%burn/24 h), and placed on a ventilator (PEEP = 5 cmH2O; tidal volume = 15 ml/kg) for 48 h. RESULTS: Plasma alpha-tocopherol per lipids doubled in the vitamin E treated sheep. Vitamin E treatment prior to injury largely prevented the increase in pulmonary permeability index and moderated the increase in lung lymph flow (52.6 +/- 6.2 ml/min, compared with 27.3 +/- 6.0 ml/min, respectively), increased the PaO2/FiO2 ratio, ameliorated both peak and pause airway pressure increases, and decreased plasma conjugated dienes and nitrotyrosine. CONCLUSIONS: Pretreatment with vitamin E ameliorated the acute lung injury caused by burn and smoke inhalation exposure.

The inhibition of inducible nitric oxide synthase in ovine sepsis model.

Excessive NO has been shown to play a major role in the pathogenesis of multiple organ dysfunctions in septic condition. Burn injury, especially if it is associated with smoke inhalation, is often complicated by subsequent development of pneumonia or sepsis that determine the outcome. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that closely mimics human sepsis and pneumonia. We hypothesized that the inhibition of iNOS-derived excessive NO might be beneficial in treating the cardiopulmonary derangement in this model. Female sheep (n = 18) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (< 40 degrees C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa (5 x 10(11) colony forming units) into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of BBS-2 (100 microg/kg/h), an iNOS inhibitor, was started 1 h after insult. The administration of BBS-2 improved pulmonary gas exchange (PaO2/FiO2 and pulmonary shunt fraction) and partially reduced airway obstruction and an increase in ventilatory pressures. The lung water content was not affected by iNOS inhibition. The hypotension seen in nontreated animals was not ameliorated either. The increase in plasma concentration of nitrate and nitrite was inhibited by BBS-2. The results of present study show that iNOS may be partially involved in the pathogenesis of acute lung injury induced by smoke inhalation followed by bacterial instillation in the airway.

Combined burn and smoke inhalation injury impairs ovine hypoxic pulmonary vasoconstriction.

OBJECTIVE: To examine the effects of combined burn and smoke inhalation injury on hypoxic pulmonary vasoconstriction, 3-nitrotyrosine formation, and respiratory function in adult sheep. DESIGN: Prospective, placebo-controlled, randomized, single-blinded trial. SETTING: University research laboratory. SUBJECTS: Twelve chronically instrumented ewes. INTERVENTIONS: Following a baseline measurement, sheep were randomly allocated to either healthy controls (sham) or the injury group, subjected to a 40%, third-degree body surface area burn and 48 breaths of cotton smoke according to an established protocol (n = 6 each). Hypoxic pulmonary vasoconstriction was assessed as changes in pulmonary arterial blood flow (corrected for changes in cardiac index) in response to left lung hypoxic challenges performed at baseline and at 24 and 48 hrs postinjury. MEASUREMENTS AND MAIN RESULTS: Combined burn and smoke inhalation was associated with increased expression of inducible nitric oxide (NO) synthase, elevated NO2/NO3 (NOx) plasma levels (12 hrs, sham, 6.2 +/- 0.6; injury, 16 +/- 1.6 micromol.L; p < .01) and increased peroxynitrite formation, as indicated by augmented lung tissue 3-nitrotyrosine content (30 +/- 3 vs. 216 +/- 8 nM; p < .001). These biochemical changes occurred in parallel with pulmonary shunting, progressive decreases in Pao2/Fio2 ratio, and a loss of hypoxic pulmonary vasoconstriction (48 hrs, -90.5% vs. baseline; p < .001). Histopathology revealed pulmonary edema and airway obstruction as the morphologic correlates of the deterioration in gas exchange and the increases in airway pressures. CONCLUSIONS: This study provides evidence for a severe impairment of hypoxic pulmonary vasoconstriction following combined burn and smoke inhalation injury. In addition to airway obstruction, the loss of hypoxic pulmonary vasoconstriction may help to explain why blood gases are within physiologic ranges for a certain time postinjury and then suddenly deteriorate.

Aerosolized alpha-tocopherol ameliorates acute lung injury following combined burn and smoke inhalation injury in sheep.

Victims of fire accidents who sustain both thermal injury to the skin and smoke inhalation have gross evidence of oxidant injury. Therefore, we hypothesized that delivery of vitamin E, an oxygen superoxide scavenger, directly into the airway would attenuate acute lung injury postburn and smoke inhalation. Sheep (N = 17 female, 35 +/- 5 kg) were divided into 3 groups: (1) injured, then nebulized with vitamin E (B&S, Vitamin E, n = 6); (2) injured, nebulized with saline (B&S, Saline, n = 6); and (3) not injured, not treated (Sham, n = 5). While under deep anesthesia with isoflurane, the sheep were subjected to a flame burn (40% total body surface area, 3rd degree) and inhalation injury (48 breaths of cotton smoke, <40 degrees C). All groups were resuscitated with Ringer lactate solution (4 mL/kg/%burn/24 h) and placed on a ventilator [positive end-expiratory pressure (PEEP) = 5 cm H2O, tidal volume = 15 mL/kg] for 48 h. B&S injury halved the lung alpha-tocopherol concentrations (0.9 +/- 0.1 nmol/g) compared with sham-injured animals (1.5 +/- 0.3), whereas vitamin E treatment elevated the lung alpha-tocopherol concentrations (7.40 +/- 2.61) in the injured animals. B&S injury decreased pulmonary gas exchange (PaO2/FiO2 ratios) from 517 +/- 15 at baseline to 329 +/- 49 at 24 h and to 149 +/- 32 at 48 h compared with sham ratios of 477 +/- 14, 536 +/- 48, and 609 +/- 49, respectively. Vitamin E treatment resulted in a significant improvement of pulmonary gas exchange; ratios were 415 +/- 34 and 283 +/- 42 at 24 and 48 h, respectively. Vitamin E nebulization therapy improved the clinical responses to burn and smoke inhalation-induced acute lung injury.

Olprinone reduces ischemia/reperfusion-induced acute renal injury in rats through enhancement of cAMP.

Activated leukocytes are implicated in development of ischemia/reperfusion (I/R)-induced organ injuries. Phosphodiesterase 3 inhibitors have anti-inflammatory effects by preventing cyclic adenosine monophosphate (cAMP) degradation. We examined the effects of olprinone, a specific phosphodiesterase 3 inhibitor, on I/R-induced acute renal injury model in rats. Forty-five minute renal I/R was induced in uni-nephrectomized rats. Rats were divided into a vehicle group, an olprinone group, and a dibutyril (DB) cAMP group. Olprinone (0.2 microg/kg/minute) infusion began 30 min after reperfusion and continued for 3 h. DBcAMP (5 mg/kg), a stable analog of cAMP, was intraperitoneally administered 5 min after reperfusion to clarify the effect of cAMP in our model. Olprinone reduced the I/R-induced increases in serum levels of blood urea nitrogen and creatinine, and improved histological changes, including acute tubular necrosis in the outer medulla. Hemodynamic status was not affected by olprinone. I/R-induced a decrease in renal tissue blood flow, an increase in renal vascular permeability, and an enhancement of leukocyte activation, reflected by renal tissue levels of myeloperoxidase activity, and the tissue levels of cytokine-induced neutrophil chemoattractant (an equivalent of human interleukin 8) and tumor necrosis factor-alpha were all significantly decreased by olprinone. Olprinone also increased the renal tissue and plasma levels of cAMP in rats subjected to renal I/R. DBcAMP showed similar effects. Our results indicated that olprinone reduced the I/R-induced acute renal injury, probably by inhibiting leukocyte activation. The effects of olprinone could be explained through its action on cAMP levels.

New tumor necrosis factor-alpha-inducing protein released from Helicobacter pylori for gastric cancer progression.

PURPOSE: To investigate the association between Helicobacter pylori infection and its inflammatory reaction in gastritis, gastric ulcer, and gastric cancer, a new tumor necrosis factor-alpha (TNF-alpha)-inducing protein of H. pylori was studied. METHODS: The HP0596 gene of H. pylori was identified as the TNF-alpha-inducing protein (Tipalpha) gene from genome sequence of H. pylori strain 26695. Using recombinant Tipalpha (rTipalpha) and deleted Tipalpha (rdel-Tipalpha) proteins, the latter of which lacks six amino acids containing two cysteines in the N-terminal domain, we examined their activities in TNF-alpha gene expression and NF-kappaB activation in both Bhas 42 (v-H-ras transfected BALB/3T3) cells and mouse gastric epithelial cell line MGT-40, and in vitro transformation of Bhas 42 cells. RESULTS: Tipalpha protein as a homodimer form (38 kDa) was found in both extracts and culture medium of various H. pylori strains. rTipalpha significantly induced TNF-alpha gene expression and NF-kappaB activation in both Bhas 42 cells and MGT-40, and induced in vitro transformation of Bhas 42 cells. However, rdel-Tipalpha did not. Treatment with MG-132, a proteasome inhibitor, inhibited translocation of NF-kappaB p65, and abrogated TNF-alpha induction induced by Tipalpha protein. CONCLUSION: Tipalpha is a new carcinogenic factor released from H. pylori mediated through NF-kappaB activation.

Skin nitric oxide and its metabolites are increased in nonburned skin after thermal injuries.

Local and systemic inflammation can lead to progression of burn wounds, converting second- to third-degree wounds or extending the burn to adjacent areas. Previous studies have suggested that the skin is an important site of production of nitric oxide (NO), synthesized by inducible nitric oxide synthase (iNOS) activation after injury. NO increases in burned wounds, but its formation in noninjured skin has not been investigated. We hypothesized that after severe burns, NO and cytotoxic peroxynitrite would increase in noninjured skin. We also tested the hypothesis that BBS-2, a specific inhibitor of iNOS, would impair NO formation after burn. Thirteen female sheep were randomized into burn injury and smoke inhalation (n = 5, group 1), burn and smoke treated with BBS-2 (n = 3, group 2), and sham (saline treatment, no injury) (n = 5, group 3). All the animals, including the sham-injury group, were mechanically ventilated for 48 h. Samples of nonburned skin and plasma were collected from each animal, and levels of NO and its metabolites were evaluated using a NO chemiluminescent detector. Nitrotyrosine and iNOS expression were determined in the skin by Immunoperoxidase staining, and scoring of masked slides (epidermis, hair follicles, vessels, glands, and stroma) was performed. Skin NO and metabolites significantly increased in the burn and smoke injury group, and this was inhibited by BBS-2. Nitrotyrosine expression also increased significantly in the skin of burned animals. BBS-2 prevented the increase of NOx but not the increase of nitrotyrosine expression in skin. Plasma levels of NO increased in burned animals when compared with sham, but this increase was not significant. The increase of NO and its metabolites after burn in noninjured skin is followed by a significant increase in peroxynitrite, a potent cytotoxic mediator.

Aerosolized tissue plasminogen inhibitor improves pulmonary function in sheep with burn and smoke inhalation.

Acute respiratory distress syndrome is a major complication in patients with thermal injury. The obstruction of the airway by cast material, composed in part of fibrin, contributes to deterioration of pulmonary gas exchange. We tested the effect of aerosol administration of tissue plasminogen activator, which lyses fibrin clots, on acute lung injury in sheep that had undergone combined burn/smoke inhalation injury. Anesthetized sheep were given a 40% total body surface, third degree burn and were insufflated with cotton smoke. Tissue plasminogen activator (TPA) was nebulized every 4 h at 1 or 2 mg for each nebulization, beginning 4 h after injury. Injured but untreated control sheep developed multiple symptoms of acute respiratory distress syndrome: decreased pulmonary gas exchange, increased pulmonary edema, and extensive airway obstruction. These control animals also showed increased pulmonary transvascular fluid flux and increased airway pressures. These variables were all stable in sham animals. Nebulization of saline or 1 mg of TPA only slightly improved measures of pulmonary function. Treatment of injured sheep with 2 mg of TPA attenuated all the pulmonary abnormalities noted above. The results provide evidence that clearance of airway obstructive cast material is crucial in managing acute respiratory distress syndrome resulting from combined burn and smoke inhalation injury.

Acute effects of combined burn and smoke inhalation injury on carboxyhemoglobin formation, tissue oxygenation, and cardiac performance.

The objective of this study was to determine the relationship between carboxyhemoglobin (COHb) formation, global oxygen transport, and cardiac performance in the acute phase of combined burn and smoke inhalation injury. Following a third degree burn of 20% of the total body surface area, adult sheep were subjected to cotton smoke (4x12 breaths) according to an established protocol. Compared with baseline (BL), the burn injury led to an immediate and sustained COHb-independent depression in myocardial contractility. Despite a progressive increase in COHb formation, up to a maximum of 78+/-3% (P < 0.001 vs BL), smoke inhalation did not further impair these hemodynamic changes. This study demonstrated that in the early stage of combined burn and smoke inhalation injury, the depression in cardiac function is basically triggered by the burn injury, whereas COHb generation secondary to cotton smoke exposure primarily contributes to pulmonary shunting.

Inhibition of poly (ADP-ribose) polymerase attenuates acute lung injury in an ovine model of sepsis.

It is known that in various pathophysiological conditions, reactive oxidants cause DNA strand breakage and subsequent activation of the nuclear enzyme poly(ADP ribose) polymerase (PARP). Activation of PARP results in cellular dysfunction. We hypothesized that pharmacological inhibition of PARP reduces the damage in the ovine model of acute lung injury (ALI). After smoke inhalation, Pseudomonas aeruginosa (5 x 109 cfu/kg) was instilled into both lungs. All of the animals were mechanically ventilated with 100% O2. The infusion of the PARP inhibitor (INO-1001, n = 6) began 1 h after the injury and thereafter through 24 h (3 mg bolus + 0.3 mg/kg/h, i.v.). Control animals (n = 6) were treated with saline. Sham injury animals (n = 8) received sham smoke and were mechanically ventilated in the same fashion. One-half of those sham animals (n = 4) were given the same dose of INO-1001. PaO2/FiO2 ratio at 24 h in saline and in the INO-1001-treated groups were 95 +/- 22 and 181 +/- 22, respectively (P < 0.05). Peak airway pressure at 24 h in the saline- and INO-1001-treated groups was 32.6 +/- 3.0 and 24.4 +/- 2.2, respectively (P < 0.05). Pulmonary shunt fraction was also significantly attenuated. INO-1001 treatment reduced pulmonary histological injury and attenuated poly (ADP-ribose) accumulation in the lung. In conclusion, inhibition of PARP improved the ALI after smoke inhalation and pneumonia. The results suggest that the activation of PARP plays a role in the pathophysiology of ALI in sheep.