[Show abstract][Hide abstract] ABSTRACT: Background: Cysteine-rich protein 61 (Cyr61/CCN1) is a multifunctional matricellular protein that has recently emerged as a potential player in injury-repair mechanisms involving the regulation of inflammatory responses. Experimental research has revealed the pro-inflammatory effects and chemotactic capacities of this protein, and clinical investigations have found it to be elevated in patients with chronic inflammatory conditions. However, its regulation at sites of acute tissue injury and inflammation in humans has not been investigated.
Methods: Ten otherwise healthy patients undergoing major orthopedic surgery for idiopathic thoracic scoliosis were recruited to the study. Fluid from the surgical drain and systemic blood was collected at 0, 1, 2, 4, and 6 hours following wound closure and analyzed for CCN1 levels, neutrophil counts, selected cytokines, and markers of primary hemostasis activation.
Results and Conclusions: In the drained fluid, CCN1 levels increased from 1 hour after wound closure, whereas its levels remained unaltered in systemic circulation. The platelet activation marker soluble P-selectin increased in the drained fluid but not in the systemic blood, resulting in a strong correlation between CCN1 and soluble P-selectin in the drained fluid (r=0.649, p=0.042). Levels of interleukin-6 and granulocyte-colony stimulating factor were elevated in the drained fluid only from two hours after wound closure, and neutrophil counts, tumor necrosis factor– alpha, interleukin-8 and interleukin-10 were elevated from 4 hours after wound closure. The present study demonstrated that CCN1 accumulates in fluid drained from surgical wounds following major surgery. It revealed that CCN1 levels increased simultaneously with soluble P-selectin, and prior to those of classical cytokine mediators, which suggests a connection between platelet activation and CCN1 accumulation. Collectively, these data suggest that CCN1 may play a role in the acute phases of human tissue injury and support its role as an early participant in acute inflammation in humans.
[Show abstract][Hide abstract] ABSTRACT: Many preclinical studies in critical care medicine and related disciplines rely on hypothesis-driven research in mice. The underlying premise posits that mice sufficiently emulate numerous pathophysiological alterations produced by trauma/sepsis and can serve as an experimental platform for answering clinically relevant questions. Recently the lay press severely criticized the translational relevance of mouse models in critical care medicine. A series of provocative editorials were elicited by a highly-publicized research report in the Proceedings of the National Academy of Sciences (PNAS; February 2013), which identified an unrecognized gene expression profile mismatch between human and murine leukocytes following burn/trauma/endotoxemia. Based on their data, the authors concluded that mouse models of trauma/inflammation are unsuitable for studying corresponding human conditions. We believe this conclusion was not justified. In conjunction with resulting negative commentary in the popular press, it can seriously jeopardize future basic research in critical care medicine. We will address some limitations of that PNAS report to provide a framework for discussing its conclusions and attempt to present a balanced summary of strengths/weaknesses of use of mouse models. While many investigators agree that animal research is a central component for improved patient outcomes, it is important to acknowledge known limitations in clinical translation from mouse to man. The scientific community is responsible to discuss valid limitations without over-interpretation. Hopefully a balanced view of the strengths/weaknesses of using animals for trauma/endotoxemia/critical care research will not result in hasty discount of the clear need for using animals to advance treatment of critically ill patients.
[Show abstract][Hide abstract] ABSTRACT: Sepsis and sepsis-induced organ dysfunction remain lethal and common conditions among intensive care patients. Accumulating evidence suggests that the matri-cellular Cyr61/CCN1-protein is involved in the regulation of inflammatory responses and possesses organ-protective capabilities in diseases of an inflammatory etiology. However, its regulation in sepsis remains largely unexplored.The present study provides a comprehensive description of CCN1-regulation in the circulation and vital organs during experimentally-induced sepsis with developing organ dysfunction.Female CD-1 mice served as baseline controls or were subjected to cecal ligation and puncture (CLP) for 18 - 96 h and CCN1 regulation was analyzed in selected organs and in the circulation.A 5-, 5-, and 3-fold increase in circulating CCN1-protein were observed at 18, 48, and 96 h post-CLP, respectively. Hepatic and pulmonary CCN1 mRNA expression was down-regulated by 80, 60, and 55% and 85, 80, and 65% at 18, 48, and 96 h post-CLP and undetectable in circulating white blood cells. To identify a potential source for the circulating protein, mouse and human platelets were explored and revealed to contain CCN1. Human platelets were stimulated by thrombin and a specific PAR1 agonist (SFLLRN) in vitro. Both agonists induced an instant CCN1 release and the effect of SFLLRN was blocked by the specific antagonist RWJ56110.The current study demonstrates that experimental sepsis is associated with a robust increase in circulating CCN1-protein levels and a paradoxical down-regulation of CCN1 mRNA expression in vital organs. It provides evidence that CCN1 is released from activated platelets, suggesting that platelets constitute a novel source for CCN1 release to the circulation during sepsis.
[Show abstract][Hide abstract] ABSTRACT: Background:
Both glucocorticosteroids and biologic drugs such as the tumor necrosis factor (TNF)-α antagonist infliximab are used often in the treatment of rheumatoid arthritis or inflammatory bowel disease. In severe disease, or if allergic reactions occur during treatment with infliximab, combined therapy with these drugs often is instituted. Combining infliximab and glucocorticosteroids may increase substantially the risk of severe opportunistic infections or dissemination of malignant tumors because of their additive effects as immunosuppressants.
In a whole-blood in vitro model, we studied the influence of different doses of infliximab and hydrocortisone, either separately or in combination, on the synthesis of selected cytokines after stimulation with lipopolysaccharide (LPS).
Hydrocortisone in therapeutic serum concentrations significantly inhibited the expression of a majority of the cytokines tested. Infliximab, in serum concentrations relevant to clinical situations, inhibited TNF-α activity significantly. This effect was potentiated when infliximab was combined with hydrocortisone. Similar effects were found using a low dose of infliximab combined with hydrocortisone. Infliximab alone inhibited the expression of the cytokines interleukin (IL)-1 receptor antagonist, monocyte chemoattractant protein-1, IL-8, and IL-12. Hydrocortisone in combination with low-dose infliximab potentiated the suppressive effects on TNF-α, IL-1β, IL-8, and macrophage inflammatory protein-1α synthesis.
Immune-modulating effects of infliximab were found both in clinically relevant doses and, most notably, in low doses reflecting serum concentrations found commonly in patients several months after the last injection. Infliximab potentiates the suppressive effects of hydrocortisone on cytokine synthesis.
[Show abstract][Hide abstract] ABSTRACT: Innate immune pro- and anti-inflammatory responses in patients with chronic subdural hematoma (CSDH) were investigated by measuring and comparing the systemic and subdural fluid levels of cytokines.
Cytokine values were analyzed in samples obtained during surgery of 56 adult patients who were operated on for unilateral CSDHs using a Multiplex antibody bead kit.
There were significantly higher levels of the pro-inflammatory IL-2R (p = 0.004), IL-5 (p < 0.001), IL-6 (p < 0.001), and IL-7 (p < 0.001), and anti-inflammatory mediators IL-10 (p < 0.001) and IL-13 (p = 0.002) in CSDH fluid compared with systemic levels. The pro-inflammatory TNF-alpha (p < 0.001), IL-1beta (p < 0.001), IL-2 (p = 0.007) and IL-4 (p < 0.001) were significantly lower in hematoma fluid compared with systemic levels. The ratios between pro- versus anti-inflammatory cytokines were statistically significant higher in CSDH (7.8) compared with systemic levels (1.3).
The innate immune responses occur both locally at the site of CSDH, as well as systematically in patients with CSDH. The local hyper-inflammatory and low anti-inflammatory responses exist simultaneously. The findings suggest poorly coordinated innate immune responses at the site of CSDH that may lead to propagating of local inflammatory process and basically contribute to formation and progression of CSDH.
Agents and Actions 04/2012; 61(8):845-52. DOI:10.1007/s00011-012-0476-0 · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Organ failure is a severe complication in sepsis for which the pathophysiology remains incompletely understood. Recently, the matri-cellular cysteine-rich, angiogenic induced, 61 (Cyr61/CCN1); connective tissue growth factor (Ctgf/CCN2); and nephroblastoma overexpressed gene (Nov/CCN3) (CCN)-protein family have been attributed organ-protective properties. Their expression is sensitive to mediators of sepsis pathophysiology but a potential role in sepsis remains elusive. To provide an initial assessment, 50 rats were subjected to 18 h of cecal-ligation and puncture or sham operation. Hepatic and pulmonary CCN1 mRNA displayed an average 7.4- and 3.3-fold induction, while its cardiac expression was unchanged. The changes coincided with excessive hepatic and pulmonary inflammatory gene activation and a restricted cardiac inflammation. Furthermore, hepatocytes displayed a dosage-dependent CCN1 mRNA response in vitro, supporting a cytokine-mediated CCN1 regulation in sepsis. CCN2 mRNA was 2.2-fold induced in the liver, while 2.0-fold and 1.4-fold repressed in the heart and lung. Meanwhile, it did not respond to TNF-α exposure in vitro, which indicates different means of regulation than for CCN1. Taken together, this study provides the first evidence for multi-organ regulation of CCN1 and CCN2 in early stages of sepsis, and implies the eruption of inflammatory mediators as a potential mechanism behind the observed CCN1 regulation.
[Show abstract][Hide abstract] ABSTRACT: The goal of this study was to investigate the chemokines CCL2, CXCL8, CXCL9 and CXCL10 as markers of the inflammatory responses in chronic subdural hematoma (CSDH).
Samples of peripheral venous blood and CSDH fluid (obtained during surgery) in 76 adult patients were prospectively analyzed. Chemokine values were assessed by a Multiplex antibody bead kit.
We found significantly higher levels of chemokines CCL2, CXCL8, CXCL9 and CXCL10 in hematoma fluid compared with serum.
Chemokines are elevated in the hematoma cavity of patients with CSDH. It is likely that these signaling modulators play an important role in promoting local inflammation. Furthermore, biological activity of CCL2 and CXCL8 may promote neovascularization within the outer CSDH membrane, and a compensatory angiostatic activity of CXCL9 and CXCL10 may contribute to repairing this disorder. This phenomenon was restricted to the hematoma site, and the systemic chemokine levels might not reflect local immune responses.
[Show abstract][Hide abstract] ABSTRACT: Liver X receptor (LXR) is a transcription factor of the nuclear receptor family, regulating genes involved in metabolism, inflammation, and apoptosis. In the present investigation, we examined the role of LXR in organ injury and systemic inflammation in rodent models of polymicrobial peritonitis caused by cecal ligation and puncture (CLP).
Rats were subjected to CLP sepsis or a sham operation. Some were treated with the synthetic LXR agonist GW3965 0.3 mg/kg 30 min prior to the CLP procedure, and organs and plasma were harvested at 3, 10, 18, or 24 h. Organs were analyzed for RNA expression by quantitative polymerase chain reaction or for morphologic differences by histologic review. Organ injury and inflammatory markers were measured in plasma. Results: Expression of the LXRα gene was decreased in the livers of CLP rats compared with sham-operated rats. Administration of a synthetic agonist of LXR (GW3965) reduced biochemical indices of liver injury in the blood of CLP rats. We also demonstrated that liver injury associated with CLP is aggravated in LXRα- and LXRαβ-deficient mice compared with wild-type and LXRβ-deficient mice, indicating a role for LXRα in protecting the liver. The enhanced liver injury in LXR-deficient mice was associated with elevated plasma concentrations of high mobility group box 1, a late mediator of inflammation and a known factor in the pathology of this model.
Collectively, these results argue in favor of a role for LXRα in protection against liver injury in experimental sepsis induced by CLP.
[Show abstract][Hide abstract] ABSTRACT: The question of specific immunomodulating qualities of hypertonic saline (HTS) has not been settled. It has proven difficult to distinguish between immunomodulation directly attributable to HTS and influence because of favorable circulatory effects. The nature of immune activator may also play a role. In a whole-blood model, we have investigated these relations further, with special emphasize on osmolalities usually found after recommended dosing. Blood from 10 healthy donors was exposed to osmolalities ranging from 295 to 480 mOsm/kg and stimulated with the two clinically relevant stimulators peptidoglycan (1 µg/mL) or LPS (10 ng/mL) for 6 h at 37°C. Leukocyte response was evaluated by measuring selected cytokines in the supernatant. Moderate hyperosmolality alone boosted the release of CXCL8/IL-8. The peptidoglycan-stimulated synthesis of pivotal proinflammatory cytokines was inhibited in an osmolality-dependent way, but statistically significant only at osmolalities above those attained after routine use of HTS, i.e., 310 mOsm/kg or greater: IL-6 (P < 0.05 at 315 mOsm/kg), IL-1ß, and TNF-α (P < 0.05 at 335 mOsm/kg). Similar effects were seen for the chemokine CCL3 and the anti-inflammatory cytokine IL-10. In contrast, the effects in cells stimulated with LPS were either lower or absent. Thus, osmolalities usually found after clinical use of HTS only modestly influenced the selected immune parameters, regardless of stimulator.
[Show abstract][Hide abstract] ABSTRACT: Acute pancreatitis was induced in anesthetized pigs by injection of Na-taurocholate into the pancreatic duct. The pigs were allocated to 4 groups. One group remained untreated while the other groups received either C1-inhibitor, aprotinin or methyl-prednisolone intravenously as pretreatment. Extensive necroses of the pancreatic parenchyma, peripancreatic oedema and accumulation of large amounts of fluid in the abdominal cavity developed within a few hours in all experimental groups. Pretreatment significantly improved hemodynamics and increased the survival rate at 6 hours. It is concluded that the most essential effect of the pretreatments were reduction of proteolytic activities which are secondary to the pancreatic lesions.
[Show abstract][Hide abstract] ABSTRACT: The ultrastructure of developing lung lesions in two groups of dogs exposed to a combination of haemorrhagic hypotension and liver trauma was studied with particular attention to changes at the alveolar level and lung micro-vessels. Lung samples were obtained every four hours and at collapse in one group and 12 hrs after initiation of the trauma in the other. An interstitial oedema was recognized in biopsies obtained 4 hrs after initiation of the trauma, and before marked lesions were observed at the ultrastructural level in endothelial cells. Endothelial damage was, however, evident in biopsies obtained at 8 hrs and at collapse. Aggregates of degranulated and degenerated leucocytes and platelets were occasionally found to obstruct respiratory capillaries together with erythrocytes, some of which seemed to be haemolysing. A considerable amount of protein-rich oedema, cellular debris and fibrinoid material was found in alveolar lumina at collapse. The present experiments indicate that increased vascular permeability in lung micro-vessels is of importance for the development of the characteristic lesions seen in shock lungs. Possible pathogenetic mechanisms, initiating the lung lesions, are discussed with special emphasis on the significance of kinin activation and the presence of polymorphonuclear leucocytes and microthrombi.
[Show abstract][Hide abstract] ABSTRACT: Both the coagulation and fibrinolytic cascades generate proteolytic enzymes which appear to be essential for tumor invasion. In the present investigation adenocarcinomas and normal colon from 14 patients with colorectal cancer were studied by immunohistochemistry. The most striking observation was an enrichment of plasminogen activator inhibitor in the tumor tissue, whereas no such immunoreactivity was detected in the biopsies of normal colon. The tumor-host interface was characterized by a massive accumulation of inflammatory cells, macrophages and T lymphocytes. In this area fibrin(ogen) immunoreactivity as a sign of local activation of the coagulation cascade was also seen. The transition zone between the tumor and normal tissue was furthermore characterized by a marked enrichment of urokinase plasminogen activator immunoreactivity. The study strongly indicates that proteases and inhibitors of the fibrinolytic system may be of great importance in tumor invasion.
[Show abstract][Hide abstract] ABSTRACT: Modulation of the host inflammatory response to infection may be a key approach to improve the outcome of patients with sepsis and organ injury. We previously reported that pretreatment of rats with the liver X receptor (LXR) agonist GW3965 reduced the liver injury associated with endotoxemia and attenuated the production of TNF-alpha by rat Kupffer cells. Here, we examine the dose-dependent effect of GW3965 on liver injury and cytokine production in a rat model of endotoxemia and explore the mechanisms underlying TNF-alpha attenuation in Kupffer cells. Low doses of GW3965 (0.1 or 0.3 mg/kg) administered 30 min before infusion of LPS and peptidoglycan significantly attenuated the increase in plasma levels of the liver injury markers alanine aminotransferase and bilirubin (6 h) as well as the inflammatory mediators TNF-alpha (1 h) and prostaglandin E2 (6 h) associated with endotoxemia. In contrast, pretreatment with a higher dose of GW3965 (1.0 mg/kg) had no such effect. Studies in primary cultures of rat Kupffer cells demonstrated that LXR agonist treatment attenuated both the secreted and cell-associated levels of TNF-alpha, whereas TNF-alpha mRNA levels were not altered. Phosphorylated p38 mitogen-activated protein kinase, which plays a major role in production of TNF-alpha at the posttranscriptional level, was attenuated by GW3965 treatment in Kupffer cells. Experiments in murine LXR-deficient Kupffer cells demonstrated enhanced production of TNF-alpha in Kupffer cells from LXR-alpha(-/-) mice when challenged with LPS compared with LXR-beta(-/-) and wild-type Kupffer cells. Taken together, these results argue in favor of a novel mechanism for LXR-mediated attenuation of liver injury by interfering with posttranscriptional regulation of TNF-alpha in Kupffer cells.
[Show abstract][Hide abstract] ABSTRACT: Herein we review some of the existing knowledge on how the intracellular signaling mediator cyclic adenosine monophosphate (cAMP) regulates mechanisms involved in sepsis and septic shock, focusing on inflammation and hemodynamics. Elevating cAMP by inhibition of the cAMP-hydrolyzing phosphodiesterases has been reported to protect organ function in animal models of systemic inflammation and endotoxic shock and, likewise, increased survival from septic shock has been reported in mice deficient in specific cAMP phosphodiesterase isoforms. The cAMP signaling pathway modulates inflammation by attenuating proinflammatory cytokine release, neutrophil infiltration, degranulation and cytotoxic responses. cAMP also controls hemodynamics by regulating cardiovascular function, endothelial barrier function and platelet activation. There is a need to obtain a more detailed understanding of cAMP signaling, and to further evaluate the future potential of sepsis treatment targeting this pathway.
Journal of Organ Dysfunction 03/2009; 5(1):38-50. DOI:10.1080/17471060802549517
[Show abstract][Hide abstract] ABSTRACT: Penetrating injuries are frequently combined with polybacterial soiling. Clearance of the microorganisms depends on the ability to activate immune responses, but post-traumatic hyporeactivity of immune cells is almost universal. The aim of this study was to map the early time course of this altered leukocyte reactivity, and to compare the reactions to subsequent Gram-positive or Gram-negative challenges.
Twelve juvenile pigs sustained two standardized rounds, one through the right femur and one through the left upper abdomen. First aid treatment and acute surgery were started immediately. Blood samples were drawn before trauma and after 10, 30, 60, and 90 min, and thereafter stimulated in ex vivo whole blood for 3 h with lipopolysaccharide (LPS, 10 ng/ml), peptidoglycan (PepG, 1 microg/ml), or an equivalent amount of normal saline. The leukocyte response was evaluated by measurement of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6, IL-8, and IL-10 in the supernatant.
In the post-traumatic in vivo serum, the concentration of TNF-alpha increased steadily (significant after 60 min). A reduced ex vivo reaction to LPS was evident after 10 min, and was statistically significant after 30 min. The lowest levels were reached after 90 min. The ex vivo synthesis of TNF-alpha after stimulation with PepG remained unaltered. A similar development was seen for IL-6. IL-1 beta levels did not change, while IL-8 increased significantly only after 60 and 90 min.
Trauma almost instantaneously reprogrammed circulating leukocytes. As measured with TNF-alpha, a profound hyporeactivity to LPS, but not to PepG, was induced. In addition, no global down-regulation of leukocyte function was found after stimulation with LPS.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to test the hypothesis that inhibitory substances circulating in the patient's serum after trauma might impair leukocyte function by evaluating the effect of such serum on cytokine release in a whole blood model.
Hip replacement surgery was considered a standardized musculoskeletal trauma, and seven women and three men undergoing elective total hip replacement were included in the study. Ex vivo lipopolysaccharide (LPS) and peptidoglycan (PepG) induced tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL-10) releases were measured in whole blood sampled preoperatively and added serum taken before, at the end of operation and at postoperative day 1 and 6. Saline was used as negative control to serum.
LPS and PepG induced a significant release of TNF-alpha and IL-10 in whole blood. Addition of preoperative serum, postoperative serum or day 1 postoperative serum did not alter the LPS-induced release of TNF-alpha as compared with saline control. Addition of preoperative serum significantly increased the PepG-induced release of TNF-alpha as compared with saline control (p = 0.011). This increase was not significantly changed with addition of postoperative serum or day 1 postoperative serum. When serum from postoperative day 6 was added, both LPS and PepG induced expression of TNF-alpha was significantly reduced as compared with preoperative serum (p = 0.018 and 0.008, respectively). Preoperative serum also increased the PepG induced expression of IL-10 (p = 0.007) in relation to saline control, and this increase was not significantly changed by addition of postoperative serum or day 1 and day 6 postoperative serum. Neither of the serum samples altered the LPS induced expression of IL-10 as compared with saline control (p = 0.212).
Our data show that in trauma patients, serum expresses activity that inhibits LPS and PepG induced release of TNF-alpha in a whole blood model, and our study, then, corroborates the hypothesis that inhibitory substances circulating in the patients' serum after trauma impair leukocyte function.
The Journal of trauma 10/2008; 67(3):624-7. DOI:10.1097/TA.0b013e3181813581 · 2.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Trauma is associated with immune paresis which may predispose to postoperative sepsis. We characterized the ex vivo cytokine responses to bacterial cell wall components in whole blood from 8 patients undergoing a major musculoskeletal trauma in the form of total hip replacement. Preoperatively, at the end of operation, and at days 1 and 6 postoperatively, patient blood was obtained, anticoagulated, and incubated at 37 degrees C in the presence of peptidoglycan (PepG) or lipopolysaccharide (LPS). Plasma cytokines were measured by enzyme immunoassay. The numbers of leucocytes, monocytes, and neutrophils were unchanged at the end of surgery, while there were significant increases at postoperative days 1 and 6. We observed significant reductions in tumor necrosis factor-alpha (TNF-alpha) and interleukin 10 (IL-10) responses to PepG at the end of surgery, which was disproportional to the nonsignificant reductions in circulating monocytes, suggesting a functional suppression. However, at postoperative day 1 the responses were recovered. There were no significant changes in responses of TNF-alpha to LPS stimulation at the end of surgery, while there were significant depressions at postoperative days 1 and 6. The expression of IL-10 was significantly depressed at the end of surgery and at day 6. There were modest changes in PepG- and LPS-induced expression of interleukin 8 (IL-8) during the experiments. On the basis of these observations, we conclude that a musculoskeletal trauma is associated with reduced expression of TNF-alpha and IL-10 by whole blood leucocytes when exposed to endotoxin, but there is a difference between gram-positive endotoxin (PepG) and gram-negative endotoxin (LPS).
Journal of Investigative Surgery 09/2008; 21(5):255-60. DOI:10.1080/08941930802180128 · 1.16 Impact Factor