Kiyoyasu Kurahashi

Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia.

The pathogenesis of septic shock occurring after Pseudomonas aeruginosa pneumonia was studied in a rabbit model. The airspace instillation of the cytotoxic P. aeruginosa strain PA103 into the rabbit caused a consistent alveolar epithelial injury, progressive bacteremia, and septic shock. The lung instillation of a noncytotoxic, isogenic mutant strain (PA103DeltaUT), which is defective for production of type III secreted toxins, did not cause either systemic inflammatory response or septic shock, despite a potent inflammatory response in the lung. The intravenous injection of PA103 did not cause shock or an increase in TNF-alpha, despite the fact that the animals were bacteremic. The systemic administration of either anti-TNF-alpha serum or recombinant human IL-10 improved both septic shock and bacteremia in the animals that were instilled with PA103. Radiolabeled TNF-alpha instilled in the lung significantly leaked into the circulation only in the presence of alveolar epithelial injury. We conclude that injury to the alveolar epithelium allows the release of proinflammatory mediators into the circulation that are primarily responsible for septic shock. Our results demonstrate the importance of compartmentalization of inflammatory mediators in the lung, and the crucial role of bacterial cytotoxins in causing alveolar epithelial damage in the pathogenesis of acute septic shock in P. aeruginosa pneumonia.

ACTIVE AND PASSIVE IMMUNIZATION WITH THE PSEUDOMONAS V ANTIGEN PROTECTS AGAINST TYPE III INTOXICATION AND LUNG INJURY

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Damage to the lung epithelium is associated with the expression of toxins that are directly injected into eukaryotic cells through a type III-mediated secretion and translocation mechanism. Here we show that the P. aeruginosa homolog of the Yersinia V antigen, PcrV, is involved in the translocation of type III toxins. Vaccination against PcrV ensured the survival of challenged mice and decreased lung inflammation and injury. Antibodies to PcrV inhibited the translocation of type III toxins.

The Effect of Pentoxifylline on Acid-induced Alveolar Epithelial Injury

Background Acid instillation into one lung is known to cause an increase in the permeability of the endothelium to protein in both the instilled and the contralateral lungs. Activated neutrophils are believed to be involved in causing this increased permeability. Pentoxifylline, a drug used in clinical practice, has multiple effects on neutrophils, including inhibition of phagocytosis, degranulation, and superoxide generation. This study investigated whether pretreatment with pentoxifylline would protect the alveolar epithelium or lung endothelium from injury. Methods The effect of acid instillation into one lung of anesthetized rabbits using several quantitative parameters was investigated. The quantification of the bidirectional movement of the alveolar (sup 125 Iodine‐albumin) and the circulating protein tracers (sup 131 Iodine‐albumin) was used as a measurement of the permeabilities of the lung epithelium and the lung endothelium in the acid‐instilled lung. Bronchoalveolar lavage and measurement of the entry of the circulating protein tracer were used to assess the permeabilities of these barriers in the noninstilled lung. Results The instillation of HCl (pH 1.25, 1.2 ml/kg) into the right lung resulted in an increase in the protein permeability of the right lungs alveolar epithelium and endothelium as well as an increase in the permeability to protein of the left lungs endothelium. Pentoxifylline pretreatment attenuated the increase in the endothelial permeability of both lungs by 50% and restored the PaO2 /FI sub O2 to normal in the pretreated animals exposed to acid injury. Conclusions Acid aspiration causes a dramatic increase in the alveolar epithelial permeability of the acid‐instilled lung, but the permeability of the alveolar epithelium of the contralateral lung remains normal. In contrast, unilateral acid instillation causes an increase in the permeability of the endothelium of both lungs. The increase in endothelial permeability can be attenuated by pretreatment with pentoxifylline administration, and this leads to restoration of normal gas exchange.

Influence of blood sample oxygen tension on blood glucose concentration measured using an enzyme-electrode method

OBJECTIVE To determine the accuracy of a bedside glucometer with an enzyme-electrode sensor based on enzyme oxidation by glucose oxidase. DESIGN Prospective, cross-sectional clinical study. SETTING Operating room in a public hospital. PATIENTS Fifty-four patients undergoing surgical procedures for a derivation (n = 17) and a validation (n= 37) study. INTERVENTIONS Arterial blood samples were obtained via a 20-gauge cannula inserted into each patients radial artery. MEASUREMENTS AND MAIN RESULTS Glucose measurements and arterial blood gas analyses were concurrently performed, using 48 blood samples for the derivation study and 45 blood samples for the validation study of this technique. Blood glucose concentrations were measured with both a bedside glucometer using an enzyme-electrode method and a laboratory glucometer based on the colorimetric method. The bedside glucometer consistently underestimated the glucose concentrations and the underestimation was related to the sample oxygen tension but not to hematocrit, plasma protein, creatinine, uric acid, or bilirubin. The present investigation used the following correction formula: (corrected glucose value) = (glucose concentration obtained by a bedside glucometer) + 0.1 x (sample oxygen tension) + 16. The corrected data were in agreement with the laboratory-determined glucose values (i.e., the mean difference and precision were 0.4 and 7.1 mg/dL, respectively). A validation study confirmed the generalization of the present correction formula which facilitates a more accurate estimation of blood glucose concentrations. CONCLUSIONS Blood glucose values measured using a bedside glucometer in this study were influenced by the sample oxygen tension. We used a corrective equation which improved the accuracy of estimating blood glucose values to a clinically acceptable range.

Heparin-coated circuits reduce the formation of TNFα during cardiopulmonary bypass

Background: Cardiopulmonary bypass (CPB) causes a systemic intlammatory response. TNFα, which is a major inflammatory mediator, has been found in the circulation during and after CPB. Although previous studies have shown that heparin coating of the extracorporeal circuits reduces complement and granulocyte activation, and the inflammatory response, the possible effect of heparin coating on TNFα formation and the inflammatory response has not been fully investigated.

Febrile-Range Hyperthermia Augments Lipopolysaccharide-Induced Lung Injury by a Mechanism of Enhanced Alveolar Epithelial Apoptosis

Fever is common in critically ill patients and is associated with worse clinical outcomes, including increased intensive care unit mortality. In animal models, febrile-range hyperthermia (FRH) worsens acute lung injury, but the mechanisms by which this occurs remain uncertain. We hypothesized that FRH augments the response of the alveolar epithelium to TNF-α receptor family signaling. We found that FRH augmented LPS-induced lung injury and increased LPS-induced mortality in mice. At 24 h, animals exposed to hyperthermia and LPS had significant increases in alveolar permeability without changes in inflammatory cells in bronchoalveolar lavage fluid or lung tissue as compared with animals exposed to LPS alone. The increase in alveolar permeability was associated with an increase in alveolar epithelial apoptosis and was attenuated by caspase inhibition with zVAD.fmk. At 48 h, the animals exposed to hyperthermia and LPS had an enhanced lung inflammatory response. In murine lung epithelial cell lines (MLE-15, LA-4) and in primary type II alveolar epithelial cells, FRH enhanced apoptosis in response to TNF-α but not Fas ligand. The increase in apoptosis was caspase-8 dependent and associated with suppression of NF-κB activity. The FRH-associated NF-κB suppression was not associated with persistence of IκB-α, suggesting that FRH-mediated suppression of NF-κB occurs by means other than alteration of IκB-α kinetics. These data show for the first time that FRH promotes lung injury in part by increasing lung epithelial apoptosis. The enhanced apoptotic response might relate to FRH-mediated suppression of NF-κB activity in the alveolar epithelium with a resultant increase in susceptibility to TNF-α–mediated cell death.

Keratinocyte Growth Factor Gene Transduction Ameliorates Pulmonary Fibrosis Induced by Bleomycin in Mice

Pulmonary fibrosis has high rates of mortality and morbidity, but there is no established therapy at present. We demonstrate here that bleomycin-induced pulmonary fibrosis in mice is ameliorated by intratracheal administration of keratinocyte growth factor (KGF)-expressing adenovirus vector. Progressive pulmonary fibrosis was created by continuous subcutaneous administration of 120 mg/kg of bleomycin subcutaneously using an osmotic pump twice from Day 1 to 7 and Day 29 to 35. The mice initially exhibited subpleural fibrosis and then exhibited advanced fibrosis in the parenchyma of the lungs. These histopathological changes were accompanied by reduced lung compliance (0.041 ± 0.011 versus 0.097 ± 0.004; P < 0.001), reduced messenger expression of surfactant proteins, and reduced KGF messenger expression in the lungs at 4 weeks compared with naive group. Intratracheal instillation of Ad-KGF at 1 week after the first administration of bleomycin increased KGF mRNA expression in the lungs compared with the fibrosis-induced mice that received saline alone. The phenotype was associated with alveolar epithelial cell proliferation, increased pulmonary compliance (0.062 ± 0.005 versus 0.041 ± 0.011; P = 0.023), and decreased mortality (survival rate on Day 56: 68.8% versus 0%; P = 0.002), compared with mice receiving only the saline vehicle. These observations suggest the therapeutic utility of a KGF-expressing adenoviral vector for pulmonary fibrosis.

Induction of Cyclooxygenase-2 in Alveolar Macrophages after Acid Aspiration Selective Cyclooxygenase-2 Blockade Reduces Interleukin-6 Production

Background Gastric acid aspiration can result in acute lung injury. In this study, the authors determined whether alveolar macrophages express cyclooxygenase‐2 as a source of inflammatory mediators after acid aspiration. Methods Seventy‐five microliters of hydrochloric acid solution, pH 1.15, was instilled into one lung in mice. After exposure, alveolar macrophages were harvested, and competitive polymerase chain reaction and enzyme‐linked immunosorbent assay were performed to measure expression of cyclooxygenase‐1 and ‐2, interleukin‐1beta and ‐6, tumor necrosis factor‐alpha, and inducible nitric oxide synthase (iNOS). The authors used immunocytochemistry to demonstrate expression of cyclooxygenase‐2 in alveolar macrophages. Selective cyclooxygenase‐2 blockade using N‐2(‐cyclohexyloxy‐4‐nitrophenyl) methane‐sulphonamide was done to characterize prostaglandin‐cytokine interaction. Results Acid aspiration induced upregulation of cyclooxygenase‐2 and interleukin‐6. Tumor necrosis factor‐alpha and iNOS were not upregulated. Interleukin‐1beta was upregulated even with saline instillation but could not be detected in the supernatant of the cell culture. Alveolar macrophages harvested from mice instilled with acid showed a trend toward more production of prostaglandin E2 and produced higher concentrations of interleukin‐6 compared with alveolar macrophages from mice instilled with saline. Selective cyclooxygenase‐2 blockade significantly decreased release of interleukin‐6 from alveolar macrophages harvested from mice instilled with acid. Conclusions Acid aspiration induces strong expression of cyclooxygenase‐2 and production of interleukin‐6 in alveolar macrophages. Selective cyclooxygenase‐2 blockade reduced production of interleukin‐6 by acid‐stimulated alveolar macrophages. These studies suggest that the induction of cyclooxygenase‐2 plays an important role in the systemic inflammatory response induced by acid aspiration.

Depletion of phagocytes in the reticuloendothelial system causes increased inflammation and mortality in rabbits with Pseudomonas aeruginosa pneumonia

Phagocytes of the reticuloendothelial system are important in clearing systemic infection; however, the role of the reticuloendothelial system in the response to localized infection is not well-documented. The major goals of this study were to investigate the roles of phagocytes in the reticuloendothelial system in terms of bacterial clearance and inflammatory modulation in sepsis caused by Pseudomonas pneumonia. Macrophages in liver and spleen were depleted by administering liposome encapsulated dichloromethylene diphosphonate (clodronate) intravenously 36 h before the instillation of Pseudomonas aeruginosa into the lungs of anesthetized rabbits. Blood samples were analyzed for bacteria and cytokine concentrations. Lung injury was assessed by the bidirectional flux of albumin and by wet-to-dry weight ratios. Blood pressure and cardiac outputs decreased more rapidly and bacteremia occurred earlier in the clodronate-treated rabbits compared with the nondepleted rabbits. Plasma TNF-alpha (1.08 +/- 0.54 vs. 0.08 +/- 0.02 ng/ml) and IL-8 (6.8 +/- 1.5 vs. 0.0 +/- 0.0 ng/ml) were higher in the depleted rabbits. The concentration of IL-10 in liver of the macrophage-depleted rabbits was significantly lower than in normal rabbits at 5 h. Treatment of macrophage-depleted rabbits with intravenous IL-10 reduced plasma proinflammatory cytokine concentrations and reduced the decline in blood pressure and cardiac output. These results show that macrophages in the reticuloendothelial system have critical roles in controlling systemic bacteremia and reducing systemic inflammation, thereby limiting the systemic effects of a severe pulmonary bacterial infection.

Day or night administration of ketamine and pentobarbital differentially affect circadian rhythms of pineal melatonin secretion and locomotor activity in rats.

BACKGROUND:Surgery with general anesthesia disturbs circadian rhythms, which may lead to postoperative sleep disorders and delirium in patients. However, it is unclear how circadian rhythms are affected by different anesthetics administered at different times during the rest-activity cycle. We hypothesized that pentobarbital (an agonist at the &ggr;-aminobutyric acid A receptors) and ketamine (an antagonist at the N-methyl-D-aspartate receptors) would have differential effects on circadian rhythms, and these effects would also be influenced by the time of their administration (the active versus resting phase). METHODS:Rats were divided into 4 groups according to the anesthetic administered (pentobarbital or ketamine) and the timing of intraperitoneal administration (active/night phase or resting/day phase). Using online pineal microdialysis, we analyzed pineal melatonin secretion and locomotor activity rhythms in rats under a light/dark (12/12-hour) cycle for 5 days after anesthesia and microdialysis catheter implantation. The data were analyzed for rhythmicity by cosinor analysis. RESULTS:Ketamine administered during the resting phase produced 65- and 153-minute phase advances, respectively, in melatonin secretion and locomotor activity rhythms on the first day after anesthesia. In contrast, ketamine administered during the active phase produced 43- and 235-minute phase delays. Pentobarbital had no effect on the phase of either melatonin secretion or locomotor activity, irrespective of the timing of administration. When administered during the active phase, both anesthetics decreased the amplitude of melatonin secretion on the day after anesthesia; when administered during the resting phase, however, neither anesthetic affected the amplitude. The amplitude of locomotor activity decreased in all animals for 3 days after anesthesia. CONCLUSION:Ketamine has opposite phase-shifting effects on circadian rhythms according to the time of administration, whereas pentobarbital has no effect. Furthermore, both anesthetics decrease the postoperative amplitude of pineal melatonin secretion if administered during the active, but not the resting, phase of the 24-hour rest-activity cycle.