Bladder instillation and intraperitoneal injection of Escherichia coli lipopolysaccharide up-regulate cytokines and iNOS in rat urinary bladder

Systemic bacterial lipopolysaccharides (LPS) induce inflammatory responses characteristic of sepsis. Instillation of LPS into rat bladder produces a localized inflammatory response similar to that seen in urinary tract infections (UTIs). Four hours after intravesical instillation of LPS, neutrophils infiltrate into the bladder, and mRNA for inducible nitric oxide synthase (iNOS) and the cytokines, interleukin (IL)-6 and IL-10, is detected in rat bladder but not in the kidney. Induction of iNOS protein is inferred because urinary nitrate and cGMP levels are increased 4 hr after LPS intravesical instillation and remain elevated for at least 24 hr. When LPS is injected intraperitoneally, iNOS and IL-6 mRNA are induced both in the bladder and in the kidney. These data are consistent with the effects of intravesical instillation of LPS remaining localized, iNOS activity increases in both particulate and soluble bladder fractions when measured 4 hr after intravesical instillation of LPS. The magnitude of these increases in iNOS activity in the bladder is not as great as when LPS is injected intraperitoneally. Intravesical instillation of LPS induces no increase in lung or kidney NOS activity. The localized inflammatory response produced by intravesical instillation of LPS demonstrates the importance of LPS as a mediator of the host response in UTIs and supports the use of urinary measurements of nitrate and cGMP in humans as indicative of the localized induction of iNOS in UTIs.     

Does hepatocyte transplantation in a chemically induced acute hepatic failure make sense?

PURPOSE: The aim of this study was to compare the effects of topically applied transforming growth factor-beta 2 (TGF-beta 2) and interleukin 6 (IL-6), alone and combined with fibronectin, on the rate of corneal wound healing in rabbits. METHODS: Twenty-eight rabbits were used for the experiment. After the right eye of each rabbit was debrided with n-heptyl alcohol, the animals were divided into four treatment groups (six rabbits per group) and one control group (four rabbits). The debrided eyes were treated, beginning immediately after wounding and continuing every 2 hours from 8 a.m. to 8 p.m. for 48 hours. Group 1 received TGF-beta 2; group 2 IL-6; group 3, TFR-beta 2 and purified fibronectin; group 4, IL-6 and fibronectin; control group, balanced salt solution. At set intervals each eye was stained with fluorescein and photographed; epithelial defects were measured with a computer-assisted digitizer. The healing rate was calculated by linear regression analysis. RESULTS: The mean healing rates in groups 1, 2, 3, 4, and controls were respectively 1.65 +/- 0.16, 1.68 +/- 0.11, 1.99 +/- 0.12, 2.23 +/- 0.09, and 0.93 +/- 0.18 mm2/h. Mean epithelial healing rates for all drug-treatment groups were significantly faster than controls. The healing rates of groups 3 and 4 were significantly faster than groups 1 and 2. CONCLUSIONS: We conclude that cytokines, in combination with extracellular matrix proteins, facilitate corneal epithelial wound healing in vivo, possibly by making corneal epithelial cells more sensitive to fibronectin receptors.     

Helicobacter pylori induces interleukin-8 expression in endothelial cells and the signal pathway is protein tyrosine kinase dependent

Helicobacter pylori (HP) infection has been shown to increase gastric mucosal interleukin 8 (IL-8) expression, and whether HP or its toxin induces endothelial cell IL-8 expression is unknown. We aimed to compare the IL-8 expression in endothelial cells after stimulation with HP toxin, tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS) and to study their signal pathways. HP or its toxin induced significant IL-8 expression in endothelial cells. HP toxin, TNF-alpha, and LPS also showed a time- and dose-dependent increase in IL-8 expression over the control. Both protein kinase C (PKC) and protein kinase A (PKA) inhibitors had no effect on IL-8 response to these stimuli. Protein tyrosine kinase (PTK) inhibitor genistein at concentrations of 150, 300, and 450 microM dose-dependently reduced LPS- and TNF-alpha-induced IL-8 expression by 29.43, 43.8, and 47.3% and 20.5, 49.9, and 61.8% respectively, whereas HP toxin-induced IL-8 secretion could only be reduced at 450 microM by 35.7%. Geldanamycin, a more potent PTK inhibitor, at doses of 0.5, 1, and 2 microM dose-dependently reduced HP toxin induced endothelial cell IL-8 expression by 24.8, 26, and 44.3% respectively. It is concluded that HP and its toxin can increase IL-8 expression in endothelial cells, and the expression of IL-8 elicited by HP toxin, TNF-alpha, and LPS is partially dependent on PTK but not PKA or PKC activation.     

Promotion of second intention wound healing by emu oil lotion: comparative results with furasin, polysporin, and cortisone

Previous studies showed that twice-daily application of emu oil lotion (mixture of emu oil/fat, vitamin E, and botanical oil) immediately after creation of full-thickness skin defects delayed wound healing 6 days later, perhaps owing to its antiinflammatory actions. If administration was delayed for 48 hours, a two-fold promotion of wound contraction, epithelialization, and infiltration of organized granulation tissue was observed. In the present study, emu oil lotion was applied to full-thickness skin defects in rodents 24 hours after surgery. Six days postoperatively, wound contraction and infiltration of fronts of epithelialized and granulation tissue were assessed. Results indicated a two-fold promotion of all of the above parameters with emu oil lotion. No such effects were exerted by pure emu oil, furasin, cortaid, or polysporin. Data obtained indicate promise for emu oil lotion as an aid in treating full-thickness skin defects if applied after the major postinflammatory stages of wound healing have transpired.     

Modulation of cytokine release and neutrophil function by granulocyte colony-stimulating factor during endotoxemia in humans

In this double-blind, cross-over, placebo-controlled, randomized study, two groups of eight healthy male volunteers were challenged with endotoxin (4 ng/kg) on two occasions, once in conjunction with placebo and once with granulocyte colony-stimulating factor (G-CSF; 5 microg/kg). In group 1, G-CSF was administered intravenously 2 hours before endotoxin challenge; in group 2, G-CSF was administered subcutaneously 24 hours before endotoxin challenge. In group 1, G-CSF significantly enhanced the release of tumor necrosis factor (TNF), interleukin-6 (IL-6), IL-8, IL-1 receptor antagonist (IL-1ra), and soluble TNF receptors. In group 2, G-CSF significantly reduced IL-8 concentrations and modestly attenuated TNF and IL-6 levels. In this group, IL-1ra and soluble TNF receptors were enhanced by G-CSF pretreatment and lipopolysaccharide (LPS)-induced soluble TNF receptor release was further augmented, whereas LPS-induced IL-1ra concentrations remained unaltered. Both pretreatments with G-CSF increased LPS-induced peripheral neutrophilia; the expression of CD11b, CD18, and CD67; and the release of elastase and lactoferrin. Both pretreatments also down-regulated neutrophil L-selectin expression and prevented the endotoxin-induced pulmonary neutrophil accumulation during the first 2 hours after endotoxin challenge. These data indicate that two different pretreatments with G-CSF result in differential effects on LPS-induced cytokine release but similar effects on LPS-induced neutrophil activation and changes in expression of cell surface molecules. Finally, regardless of the effects of G-CSF on LPS-induced cytokine release, G-CSF blocks LPS-induced pulmonary granulocyte accumulation.     

Upregulation of xanthine oxidase by lipopolysaccharide, interleukin-1, and hypoxia. Role in acute lung injury

LPS and selected cytokines upregulate xanthine dehydrogenase/xanthine oxidase (XDH/XO) in cellular systems. However, the effect of these factors on in vivo XDH/XO expression, and their contribution to lung injury, are poorly understood. Rats were exposed to normoxia or hypoxia for 24 h after treatment with LPS (1 mg/kg) and IL-1beta (100 microg/kg) or sterile saline. Lungs were then harvested for measurement of XDH/XO enzymatic activity and gene expression, and pulmonary edema was assessed by measurement of the wet/dry lung weight ratio (W/D). Although treatment with LPS + IL-1beta or hypoxia independently produced a 2-fold elevation (p < 0. 05 versus exposure to normoxia and treatment with saline) in lung XDH/XO activity and mRNA, the combination of LPS + IL-1beta and hypoxia caused a 4- and 3.5-fold increase in these values, respectively. XDH/XO protein expression was increased 2-fold by hypoxia alone and 1.3-fold by treatment with LPS + IL-1beta alone or combination treatment. Compared with normoxic lungs, W/D was significantly increased by exposure to hypoxia, LPS + IL-1beta, or combination treatment. This increase was prevented by treatment of the animals with tungsten, which abrogated lung XDH/XO activity. In conclusion, LPS, IL-1beta, and hypoxia significantly upregulate lung XDH/XO expression in vivo. The present data support a role for this enzyme in the pathogenesis of acute lung injury.     

Elevated levels of human salivary epidermal growth factor after oral and juxtaoral surgery

PURPOSE: Saliva provides a natural reservoir of growth factors whose purposes have remained elusive. Animal studies suggest that saliva-derived growth factors play a role in systemic and oral wound healing. In the current study, salivary concentrations of epidermal growth factor (EGF) were monitored in patients before and after oral and juxtaoral surgery. PATIENTS AND METHODS: Whole resting saliva was collected from a group of patients with parotid gland tumors requiring surgical resection. Another group of patients a history of periodontal disease requiring surgical intervention also provided whole salivary samples. Healthy age- and sex-matched persons served as controls. RESULTS: Salivary EGF levels were elevated in both groups of patients within 24 hours after surgery. In the periodontitis patients, a second smaller peak was assayed noted between 36 and 48 hours. After this, EGF concentrations returned to levels comparable to healthy controls in both experimental groups. CONCLUSIONS: Although the local cells have the ability to synthesize and secrete growth factors at a site of injury, these results suggest that surgery stimulates increased synthesis and secretion of growth factors in the saliva as well. This increased level of saliva-derived growth factor may also aid in promoting wound healing.     

Relationship of tissue and cellular interleukin-1 and lipopolysaccharide after endotoxemia and bacteremia

Distributions of immunoreactive interleukin-1 (IL-1) and lipopolysaccharide (LPS) were studied in the tissues of rats after intravenous injection of purified LPS or live Escherichia coli bacteria. IL-1 staining in the spleen peaked at 4-8 h, colocalized with LPS in marginal zone macrophages, and was undetectable 24 h after injection, whereas LPS staining peaked at 24 h and was detectable for 4 weeks. The tissue IL-1 response was similar for LPS and live bacteria. Thus, tissue IL-1 is down-regulated within hours despite maintenance of LPS in the same cells for weeks. Macrophages in liver and lung had only slight IL-1 staining despite intense staining for LPS. Tissue IL-1 production appears to be differentially regulated after gram-negative bacteremia; LPS cleared by liver and lung macrophages elicit minimal IL-1, whereas there is high local IL-1 production in the marginal zone of the spleen that may increase immune responses to bacterial wall antigens.     

Early detection of anastomotic leaks after colorectal surgery by measuring endotoxin in the drainage fluid

BACKGROUND/AIMS: Early detection of anastomotic leaks after colorectal anastomosis is essential for adequate intervention to prevent peritonitis. We investigated whether the measurement of endotoxin (LPS) concentrations in the drainage has any value for the early detection of anastomotic leaks. MATERIALS AND METHODS: Twenty two patients with colorectal anastomosis were enrolled in this study, 3 developed clinically established signs of anastomotic leaks and 19 recovered without complications. LPS concentrations in the drainage, the total daily excreted LPS amounts, leukocyte and thrombocyte counts, plasma urea and creatinine, and body temperature were measured for up to 8 days after surgery and tested for their value to detect anastomotic leaks. RESULTS: LPS concentrations in the drainage fluid and daily excreted LPS amounts of patients with anastomotic leaks were significantly higher compared to the group without anastomotic leaks. On the third postoperative day, LPS concentrations ranged from 5270 to 6750 pg/ml in patients with anastomotic leaks and from 1 to 1848 pg/ml in patients without complications. Total daily excreted LPS amounts were 270-675 ng/day in patients with anastomotic leak and 0-92 ng/day in patients without anastomotic leaks. Both LPS-related parameters allowed reliable detection of anastomotic leaks on day 3 after surgery (Student's t-Test, p < 0.0005), while leukocyte and thrombocyte counts, plasma urea and creatinine, and body temperatures of both patient groups were not significantly different at any time (p > 0.05). CONCLUSION: We found that the measurement of LPS concentrations in the drainage and the daily excreted LPS amount could be valuable parameters for the early detection of anastomotic leaks as early as on the third post-operative day.     

The effect of octreotide on wound healing: an in vitro and in vivo experimental study

OBJECTIVE: To investigate the effect of octreotide on wound healing. DESIGN: Experimental studies in vitro and in rats. SETTING: Teaching hospital, Israel. MATERIAL: Cultured human diploid fetal fibroblasts, and 36 male Wistar rats. INTERVENTIONS: Octreotide was added to cultures of fibroblasts in doses of 2, 10, 30, 60 and 120 ng/ml and fibroblasts were counted after 2, 4, and 6 days. Intestinal anastomoses were made in 36 rats. Rats in the octreotide group (n = 18) were given subcutaneous injections of 0.25 microg/kg twice daily and 6 rats were killed at 3, 7, and 14 days. The control group were given injections of saline. Anastomotic bursting pressures and hydroxyproline content were measured at each of the three times. MAIN OUTCOME MEASURES: Fibroblast counts, anastomotic bursting pressures, and hydroxyproline concentrations. RESULTS: Octreotide did not inhibit fibroblast proliferation in any of the doses at any of the time periods. The anastomotic bursting pressure was slightly higher in the octreotide group at each of the time points, but not significantly so, and there was no difference in hydroxyproline content between the octreotide and control groups. Octreotide did not inhibit wound healing either in vitro or in vivo.     

Decrease in contents of pancreatic carboxyl ester lipase, phospholipase A2, and lingual lipase in rats with streptozotocin-induced diabetes

Fibrin glue has been used as a protective seal in normal and high-risk anastomoses to prevent leakage. The influence of fibrin adhesive on the healing colonic anastomosis in a control and high-risk model was tested. Resection and anastomosis of the left colon was performed in rats. In group Ia an end-to-end anastomosis was constructed with 12 7-O polypropylene sutures; in group Ib the anastomosis was sealed with fibrin adhesive. In group II an incomplete anastomosis was constructed with only 4 sutures at 90 degrees, therefore potentially leaking. In group IIb additional sealing with fibrin glue was performed. On Days 2, 4, and 7 body weight, adhesion formation, anastomotic bursting pressure, and collagen concentration were measured. The results showed increased adhesion formation after fibrin sealing. The anastomotic bursting pressure of incomplete anastomoses showed a significant increase after sealing on Day 2 only; on Day 4 and 7 no differences were found. Sealing of control anastomoses caused lower bursting pressures on Day 4. Collagen concentration is significantly reduced after fibrin sealing of normal anastomoses. We conclude that fibrin sealing of control anastomoses inhibits wound healing. Incomplete anastomoses are temporarily protected by fibrin glue sealing. Finally, fibrin sealing of the colon wound does not prevent adhesion formation.     

IL-10 is a major mediator of sepsis-induced impairment in lung antibacterial host defense

To explore the mechanism of immunosuppression associated with sepsis, we developed a murine model of sepsis-induced Pseudomonas aeruginosa pneumonia. CD-1 mice underwent either cecal ligation and 26-gauge needle puncture (CLP) or sham surgery, followed by the intratracheal (i.t.) administration of P. aeruginosa or saline. Survival in mice undergoing CLP followed 24 h later by the i.t. administration of saline or P. aeruginosa was 58% and 10%, respectively, whereas 95% of animals undergoing sham surgery followed by P. aeruginosa administration survived. Increased mortality in the CLP/P. aeruginosa group was attributable to markedly impaired lung bacterial clearance and the early development of P. aeruginosa bacteremia. The i.t. administration of bacteria to CLP-, but not sham-, operated mice resulted in an impressive intrapulmonary accumulation of neutrophils. Furthermore, P. aeruginosa challenge in septic mice resulted in a relative shift toward enhanced lung IL-10 production concomitant with a trend toward decreased IL-12. The i.p., but not i.t., administration of IL-10 Abs given just before P. aeruginosa challenge in septic mice significantly improved both survival and clearance of bacteria from the lungs of septic animals administered P. aeruginosa. Finally, alveolar macrophages isolated from animals undergoing CLP displayed a marked impairment in the ability to ingest and kill P. aeruginosa ex vivo, and this defect was partially reversed by the in vivo neutralization of IL-10. Collectively, these observations indicate that the septic response substantially impairs lung innate immunity to P. aeruginosa, and this effect is mediated primarily by endogenously produced IL-10.     

An economic analysis of teleconsultation in otorhinolaryngology

Meningococcal sepsis is a good model to study the dynamic response of cytokines and other soluble factors in vivo in the early stages of the disease. Levels of soluble CD14, interleukin-6 (IL-6), IL-6 receptor (IL-6R), and C-reactive protein (CRP) have been measured in plasma from 26 children with septic shock (nine of whom had disseminated intravascular coagulation) and from ten control children. All samples were collected at the onset, before treatment, and, when possible, 24 and 48 hours later. At admission, patients had significantly higher levels of IL-6 (p < 0.001) and CRP (p < 0.001), and lower levels of IL-6R (p < 0.005) than normal controls. After 24 hours, there was a significant increase of sCD24 (p < 0.05) and CRP (p < 0.001). Although IL-6 showed a progressive decline since the onset, its levels were always higher than controls. There was an inverse correlation between IL-6 and both IL-6R (p < 0.001) and CRP (p < 0.001), probably due to the later increase of CRP. Nevertheless, sCD14 did not correlate with IL-6 levels. We have confirmed the finding of IL-6 as a sensitive and reliable inflammatory marker in septic shock. Moreover, the ratio IL-6/IL-6R may have a prognostic value, given a putative role of IL-6R in modulating the effects of IL-6 in meningococcal sepsis.     

Pulmonary and hepatic gene expression following cecal ligation and puncture: monophosphoryl lipid A prophylaxis attenuates sepsis-induced cytokine and chemokine expression and neutrophil infiltration

Polymicrobial sepsis induced by cecal ligation and puncture (CLP) reproduces many of the pathophysiologic features of septic shock. In this study, we demonstrate that mRNA for a broad range of pro- and anti-inflammatory cytokine and chemokine genes are temporally regulated after CLP in the lung and liver. We also assessed whether prophylactic administration of monophosphoryl lipid A (MPL), a nontoxic derivative of lipopolysaccharide (LPS) that induces endotoxin tolerance and attenuates the sepsis syndrome in mice after CLP, would alter tissue-specific gene expression post-CLP. Levels of pulmonary interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), IL-1 receptor antagonist (IL-1ra), and IL-10 mRNA, as well as hepatic IL-1beta, IL-6, gamma interferon (IFN-gamma), G-CSF, inducible nitric oxide synthase, and IL-10 mRNA, were reduced in MPL-pretreated mice after CLP compared to control mice. Chemokine mRNA expression was also profoundly mitigated in MPL-pretreated mice after CLP. Specifically, levels of pulmonary and hepatic macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 (MCP-1) mRNA, as well as hepatic IFN-gamma-inducible protein 10 and KC mRNA, were attenuated in MPL-pretreated mice after CLP. Attenuated levels of IL-6, TNF-alpha, MCP-1, MIP-1alpha, and MIP-2 in serum also were observed in MPL-pretreated mice after CLP. Diminished pulmonary chemokine mRNA production was associated with reduced neutrophil margination and pulmonary myeloperoxidase activity. These data suggest that prophylactic administration of MPL mitigates the sepsis syndrome by reducing chemokine production and the recruitment of inflammatory cells into tissues, thereby attenuating the production of proinflammatory cytokines.     

Keratocyte loss after corneal deepithelialization in primates and rabbits

PURPOSE: To evaluate the response of stromal keratocytes to central corneal deepithelialization. METHODS: Rabbits and monkeys underwent unilateral mechanical deepithelialization with a blunt instrument and were killed at intervals ranging from 15 minutes to 24 hours after surgery. Two rabbits underwent unilateral deepithelialization under a fluid bath containing corneal preservation medium. Two rabbits were treated unilaterally with corneal preservation medium topically applied every 15 minutes for 16 hours after epithelial removal. Four rabbits underwent linear keratotomy immediately after deepithelialization of the cornea or on normal unoperated corneas and were killed 1 day (two animals) and 14 days (two animals) after surgery. RESULTS: Deepithelialization resulted in severe ultrastructural changes in keratocytes within 30 minutes after surgery. After 24 hours, the number of keratocytes in the anterior stroma underneath the deepithelialized area had decreased significantly in rabbits (P = .0001) and in monkeys (P = .0007) compared with controls. The wound healing was altered and delayed when the epithelium was not present after keratotomy. The use of storage media during and after deepithelialization minimized the early keratocyte changes and appeared to stimulate reepithelialization. CONCLUSIONS: Removal of corneal epithelium causes loss of superficial stromal keratocytes in rabbits and monkeys. Keratocyte death may results from osmotic changes that alter the corneal wound healing response.     

Exogenous surfactant and positive end-expiratory pressure in the treatment of endotoxin-induced lung injury

OBJECTIVE: To evaluate the efficacy of treating endotoxin-induced lung injury with single dose exogenous surfactant and positive end-expiratory pressure (PEEP). DESIGN: Prospective trial. SETTING: Laboratory at a university medical center. SUBJECTS: Nineteen certified healthy pigs, weighing 15 to 20 kg. INTERVENTIONS: Pigs were anesthetized and surgically prepared for hemodynamic and lung function measurements. Animals were randomized into four groups: a) Control pigs (n = 4) received an intravenous infusion of saline without Escherichia colilipopolysaccharide (LPS); b) the LPS group (n = 5) received an intravenous infusion of saline containing LPS (100 microg/kg); c) the PEEP plus saline group (n = 5) received an intravenous infusion of saline containing LPS. Two hours after LPS infusion, saline was instilled into the lung as a control for surfactant instillation, and the animals were placed on 7.5 cm H2O of PEEP; d) the PEEP plus surfactant group (n = 5) received an intravenous infusion of saline containing LPS. Two hours following LPS infusion, surfactant (50 mg/kg) was instilled into the lung and the animals were placed on 7.5 cm H2O of PEEP. PEEP was applied first and surfactant or saline was instilled into the lung while maintaining positive pressure ventilation. All groups were studied for 6 hrs after the start of LPS injection. At necropsy, bronchoalveolar lavage was performed and the right middle lung lobe was fixed for histologic analysis. MEASUREMENTS AND MAIN RESULTS: Compared with LPS without treatment, PEEP plus surfactant significantly increased PaO2 (PEEP plus surfactant = 156.6 +/- 18.6 [SEM] torr [20.8 +/- 2.5 kPa]; LPS = 79.2 +/- 21.9 torr [10.5 +/- 2.9 kPa]; p<.05), and decreased venous admixture (PEEP plus surfactant = 12.5 +/- 2.0%; LPS = 46.9 +/- 14.2%; p< .05) 5 hrs after LPS infusion. These changes were not significant 6 hrs after LPS infusion. PEEP plus surfactant did not alter ventilatory efficiency index (VEI = 3800/[peak airway pressure - PEEP] x respiratory rate x PacO2), or static compliance as compared with LPS without treatment at any time point. Cytologic analysis of bronchoalveolar lavage fluid showed that surfactant treatment significantly increased the percentage of alveolar neutrophils as compared with LPS without treatment (PEEP plus surfactant = 39.1 +/- 5.5%; LPS = 17.4 +/- 6.6%; p< .05). Histologic analysis showed that LPS caused edema accumulation around the airways and pulmonary vessels, and a significant increase in the number of sequestered leukocytes (LPS group = 3.4 +/- 0.2 cells/6400 micro2; control group = 1.3 +/- 0.1 cells/6400 micro2; p < .05). PEEP plus saline and PEEP plus surfactant significantly increased the total number of sequestered leukocytes in the pulmonary parenchyma (PEEP plus surfactant = 8.2 +/- 0.7 cells/6400 micro2; PEEP plus saline = 3.9 +/- 0.2 cells/6400 micro2; p <.05) compared with the control and LPS groups. CONCLUSIONS: We conclude that PEEP plus surfactant treatment of endotoxin-induced lung injury transiently improves oxygenation, but is unable to maintain this salutary effect indefinitely. Thus, repeat bolus dosing of surfactant or bolus treatment followed by continuous aerosol delivery may be necessary for a continuous beneficial effect.