Serial change of C1 inhibitor in patients with sepsis--a preliminary report.

OBJECTIVE: C1 inhibitor (C1INH) regulates not only the complement system but also the plasma kallikrein-kinin, fibrinolytic, and coagulation systems. The biologic activities of C1INH can be divided into the regulation of vascular permeability and anti-inflammatory functions. The objective was to clarify the serial change of C1INH in patients with sepsis. METHODS: We serially examined C1INH activity values (reference range, 70%-130%) and quantitative values (reference range, 160-330 µg/mL) in patients with sepsis admitted into the intensive care unit of the Trauma and Acute Critical Care Center at Osaka University Hospital (Osaka, Japan) during the period between December 2012 and February 2013. We also analyzed their clinical course. We defined "refractory shock" as septic shock requiring steroid administration to maintain hemodynamics. RESULTS: The serial change of C1INH was evaluated in 5 patients (4 survivors and 1 nonsurvivor). Two patients were diagnosed as having refractory shock. In the nonsurvivor after refractory shock, C1INH activity on admission was 97.2%, and the quantitative value was 133.1 µg/mL. In the other patient with refractory shock, C1INH activity on admission was 94.4%, and the quantitative value was 126.7 µg/mL. This patient's general condition had improved by day 6, with increases in C1INH activity (139.9%) and quantitative value (250.1 µg/mL). In the 3 nonrefractory shock patients, C1INH activity on admission was 130.6%±8.7%, and the quantitative value was 215±26.5 µg/mL. CONCLUSIONS: Enhancement of C1INH activity was not observed in the refractory shock patients, and the C1INH quantitative values were low. Further evaluation of the serial change of C1INH and the validity of C1INH replacement therapy in patients with septic shock may lead to a new strategy for sepsis management.

Neutrophil extracellular traps in bronchial aspirates: a quantitative analysis.

Neutrophil extracellular traps (NETs) are structures composed of DNA and granular proteins, which rapidly trap and kill pathogens. The formation of NETs has been detected during infection in animal experiments, but their role in humans is unclear. The purposes of this study were to quantitatively evaluate the production of NETs during acute respiratory infection and to study the relationship between the NET length and various inflammatory mediators. We examined bronchial aspirates collected from nine intubated patients in an intensive care unit. Samples were collected at the onset of acute respiratory infection (day 0) and on days 1, 3-5, and 6-8. The NET length was visualised by immunohistochemistry and quantified using computer tracing software. The NET length was measured and compared at each time point. The length differed significantly between time points (p<0.001). NETs were significantly longer on day 1 than on day 0 (p<0.001). Neutrophils released NETs abundantly in response to respiratory infection and regression analysis showed that NET length correlated with six clinical parameters (white blood cells, platelets, lactate, CXC ligand-2, interleukin-8, and procalcitonin) as the explanatory variables. NETs in bronchial aspirates may reflect disease progression of respiratory infections. Quantification of NETs in bronchial aspirates may provide a new indicator of inflammation.

Synbiotics decrease the incidence of septic complications in patients with severe SIRS: a preliminary report.

The purpose of this study was to evaluate if synbiotic therapy can correct the deteriorated gut flora and environment in patients with severe systemic inflammatory response syndrome (SIRS). Twenty-nine SIRS patients, who fulfilled a serum C-reactive protein (CRP) level >10 mg/dl, received synbiotics (Bifidobacterium breve, Lactobacillus casei, and galactooligosaccharides) (S group) and were compared with previous observations in 26 patients without synbiotics (NS group). Analysis of fecal flora confirmed that patients in the S group had significantly greater levels of beneficial Bifidobacterium, Lactobacillus, and total organic acids (particularly short-chain fatty acids) than those in the NS group. The incidence of infectious complications such as enteritis, pneumonia, and bacteremia was significantly lower in the S group than in the NS group. Synbiotics maintain the gut flora and environment and decrease the incidence of septic complications in patients with severe SIRS. Further randomized controlled study is necessary to determine the effects of synbiotics.

Acute lung injury and multiple organ dysfunction syndrome secondary to intra-abdominal hypertension and abdominal decompression in extensively burned patients.

BACKGROUND: Secondary abdominal compartment syndrome (ACS) is a lethal complication after resuscitation from burn shock, even after abdominal decompression (AD) is performed. This study investigated increased susceptibility to multiple organ dysfunction syndrome (MODS) in extensively burned patients with ACS. METHODS: Patients admitted to our burn unit between 2002 and 2005 with burns affecting 40% or more of the total body surface area without severe inhalation injury were analyzed. Hemodynamic parameters, blood gas analysis, and intrabladder pressure as intra-abdominal pressure were recorded. Serum interleukin (IL)-8 and IL-6 concentrations were measured in 20 of these patients. Lung injury score and Sequential Organ Failure Assessment scores were serially determined. RESULTS: Fourteen of 38 patients developed intra-abdominal hypertension in 22.9 +/- 8.9 hours postburn. Hemodynamic parameters in these 14 patients, including peak intra-abdominal pressure (46.6 +/- 11.2 to 19.8 +/- 9.9 cm H2O), peak inspiratory pressure (51.4 +/- 10.5 to 31.8 +/- 7.0 cm H2O), and abdominal perfusion pressure (51.3 +/- 18.3 to 73.9 +/- 13.6 mm Hg), were improved immediately after AD. Despite AD, lung injury score and Sequential Organ Failure Assessment scores increased significantly 2 and 3 days postburn in patients who required AD. Plasma concentration of IL-8 was elevated in intra-abdominal hypertension patients 3 days postburn. CONCLUSION: Intra-abdominal hypertension induced acute lung injury and MODS with IL-8 elevation, even though AD improved hemodynamic parameters in extensively burned patients.

Synergistic effects of recombinant human soluble thrombomodulin and fluid-volume resuscitation in a rat lethal crush injury model.

Severe crush injury results in a high mortality rate because of acute circulatory failure and hyperkalemia. The purpose of this study was to evaluate whether administration of prophylactic-recombinant human soluble thrombomodulin (rhsTM) and/or fluid-volume resuscitation before reperfusion attenuates severe crush injury in rats. Both hindlimbs of anesthetized rats were compressed for 6 h under blocks weighing 3.5 kg each, followed by 3 h of reperfusion. In the first group, fluid resuscitation with normal saline (1 mL/kg/h) was performed throughout the experiment. In the second group, volume resuscitation treatment with normal saline (10 mL/kg/h) was initiated 60 min before the end of the crush period and was continued until the end of the experiment. In the third group, normal saline-resuscitation treatment plus rhsTM (3 mg/kg) was performed. In the fourth group, volume resuscitation treatment plus rhsTM was performed. Blood samples were collected 6 h after the end of the crush period. Complete blood count and platelets were measured. In addition, serum lactate, base deficit, serum potassium, creatine phosphokinase, blood urea nitrogen, creatinine, myoglobin, and some cytokines were evaluated. In another experiment, survival of each group was monitored for 72 h after the end of the crush period. Combined administration of rhsTM and volume resuscitation significantly decreased hemoconcentration and hyperkalemia. The serum interleukin-6 level and mortality were also significantly improved in the combination group compared with those in the other groups. We conclude that prophylactic combination of rhsTM administration and volume resuscitation may be an effective therapy for severe crush injury.

Early activation of gammadelta T lymphocytes in patients with severe systemic inflammatory response syndrome.

Innate immunity plays an important role in host defense after severe insult. gammadelta T lymphocytes are recognized as the first line of defense against microbial invasion. In this study, we evaluated gammadelta T lymphocytes in the peripheral blood of patients with severe systemic inflammatory response syndrome (SIRS), and examined on role of these cells. Thirty-seven patients with severe SIRS (SIRS criteria and serum C-reactive protein > or = 10 mg/dL) and 27 healthy volunteers were studied. Severe SIRS was caused by trauma in 14 patients (Injury Severity Score of 30.1 +/- 10.8) and by sepsis in 23 patients. The counts of gammadelta and alphabeta T lymphocytes were determined by flow cytometry of cells stained with monoclonal antibodies to gammadelta and alphabeta T lymphocyte receptors. The activation of these cells was evaluated by flow cytometry of cells stained with monoclonal antibodies to CD69 and HLA-DR. Serial counts and activation of gammadelta and alphabeta T lymphocytes were also determined in eight trauma patients (Injury Severity Score of 31.0 +/- 13.5) during a 2-week observation period. The count of gammadelta T lymphocytes in the peripheral blood of SIRS patients (30.1 +/- 6.0/microL) was significantly lower (P < 0.05) than that of the healthy volunteers (104.3 +/- 10.9/microL). The expression of CD69, an index of early activation of T lymphocytes, was significantly greater on gammadelta T lymphocytes from SIRS patients (patients 23.9% +/- 3.4%, healthy controls 4.8% +/- 0.6%, P < 0.05). In trauma patients, the expression of CD69 on gammadelta T lymphocytes increased rapidly within 48 h after injuries. In conclusion, gammadelta T lymphocytes are activated and decreased in the peripheral blood of severe SIRS patients. In trauma patients, the activation of gammadelta T lymphocytes occurs in the fairly acute phase after injuries. These results suggest a significant role for gammadelta T lymphocytes as early responders after severe insult.

Presence of neutrophil extracellular traps and citrullinated histone H3 in the bloodstream of critically ill patients.

Neutrophil extracellular traps (NETs), a newly identified immune mechanism, are induced by inflammatory stimuli. Modification by citrullination of histone H3 is thought to be involved in the in vitro formation of NETs. The purposes of this study were to evaluate whether NETs and citrullinated histone H3 (Cit-H3) are present in the bloodstream of critically ill patients and to identify correlations with clinical and biological parameters. Blood samples were collected from intubated patients at the time of ICU admission from April to June 2011. To identify NETs, DNA and histone H3 were visualized simultaneously by immunofluorescence in blood smears. Cit-H3 was detected using a specific antibody. We assessed relationships of the presence of NETs and Cit-H3 with the existence of bacteria in tracheal aspirate, SIRS, diagnosis, WBC count, and concentrations of IL-8, TNF-α, cf-DNA, lactate, and HMGB1. Forty-nine patients were included. The median of age was 66.0 (IQR: 52.5-76.0) years. The diagnoses included trauma (7, 14.3%), infection (14, 28.6%), resuscitation from cardiopulmonary arrest (8, 16.3%), acute poisoning (4, 8.1%), heart disease (4, 8.1%), brain stroke (8, 16.3%), heat stroke (2, 4.1%), and others (2, 4.1%). We identified NETs in 5 patients and Cit-H3 in 11 patients. NETs and/or Cit-H3 were observed more frequently in "the presence of bacteria in tracheal aspirate" group (11/22, 50.0%) than in "the absence of bacteria in tracheal aspirate" group (4/27, 14.8%) (p<.01). Multiple logistic regression analysis showed that only the presence of bacteria in tracheal aspirate was significantly associated with the presence of NETs and/or Cit-H3. The presence of bacteria in tracheal aspirate may be one important factor associated with NET formation. NETs may play a pivotal role in the biological defense against the dissemination of pathogens from the respiratory tract to the bloodstream in potentially infected patients.

Identification of neutrophil extracellular traps in the blood of patients with systemic inflammatory response syndrome.

OBJECTIVE: Neutrophils are able to form 'neutrophil extracellular traps' (NETs), which they use to trap and kill pathogens such as bacteria and fungi at the foci of infection. This observational study investigated the presence of NETs in the blood from critically ill patients and healthy volunteers. METHODS: Fluorescent triple-colour immunocytochemical analysis of blood smears collected from patients with systemic inflammatory response syndrome (SIRS; associated with various clinical conditions) who had been hospitalized in the intensive care unit, and healthy volunteers, was undertaken to identify NETs in the blood. Blood smears were stained for DNA, histone H1 and neutrophil elastase. RESULTS: NETs were identified in 10 of 21 (47.6%) blood samples from the study group compared with none of the blood samples from eight healthy volunteers. CONCLUSION: These data suggest that fluorescent triple-colour immunocytochemical staining of NETs in the blood could be used to simplify the early identification of critically ill patients with SIRS. Larger studies are required to clarify the pathophysiological role of NETs in this specific patient population.

Systemic involvement of high-mobility group box 1 protein and therapeutic effect of anti-high-mobility group box 1 protein antibody in a rat model of crush injury.

Patients with crush injury often present systemic inflammatory response syndrome and fall into multiple organ failure. The mechanism by which the local tissue damage induces distant organ failure is still unclear. We focused on high-mobility group box 1 protein (HMGB1) as one of the damage-associated molecular pattern molecules that cause systemic inflammation in crush injury. We investigated involvement of HMGB1 and the effects of treatment with anti-HMGB1 antibody in a rat model of crush injury. Both hindlimbs of rats were compressed for 6 h and then released. In the crush injury group, the level of serum HMGB1 peaked at 3 h after releasing compression, followed by the increasing in the serum levels of interleukin 6 and tumor necrosis factor α. Hematoxylin-eosin staining showed substantial damage in the lung 24 h after the crush injury, with upregulation of the expression of receptor for advanced glycation end products, as revealed by immunohistochemical analysis. Intravenous administration of anti-HMGB1 antibody improved survival (n = 20 each group) and significantly suppressed serum levels of HMGB1, interleukin 6, and tumor necrosis factor α compared with the untreated crush injury group (n = 6-9 each group). Histological findings of lung damage were ameliorated, and the expression of receptor for advanced glycation end products was hampered by the treatment. These results indicate that HMGB1 is released in response to damage immediately after crush injury and acts as a proinflammatory mediator. Administration of anti-HMGB1 antibody reduced inflammatory reactions and improved survival by blocking extracellular HMGB1. Thus, HMGB1 appears to be a therapeutic target, and anti-HMGB1 antibody may become a promising novel therapy against crush injury to prevent the progression to multiple organ failure.

The effect of antithrombin on pulmonary endothelial damage induced by crush injury.

After crush injury, patients often experience multiple organ dysfunction syndrome. In this study, we focused on vascular endothelial damage, which is believed to be a possible cause of multiple organ dysfunction syndrome, and revealed a pathological condition of distant organ failure. In particular, the lung is an especially prone target organ at the time of systemic inflammatory invasion after crush injury. We ascertained the effect of antithrombin (AT), which has recently attracted attention for its endothelial protective effects. Using a rat model of crush syndrome, we assessed severity of systemic inflammation and vascular endothelial damage through a blood test and degree of lung injury and centrally focused on morphological analysis of endothelium over time. Crush injury significantly elevated the blood concentration of tissue plasminogen activator-plasminogen activator inhibitor 1 complex, monocyte chemoattractant protein 1, and IL-6. Accumulation of active inflammatory cells (OX-42-positive cells) and expression of von Willebrand factor and vascular cell adhesion molecule 1 significantly increased in the lung 24 h after releasing crush. After 48 h, disarray of alveolar structure and alveolar hemorrhage appeared. Antithrombin administration significantly suppressed accumulation of inflammatory cells, expression of von Willebrand factor and vascular cell adhesion molecule 1, and mortality rate. Our research demonstrates that crush injury induces acute lung injury as distant organ failure, and it would seem that AT administration diminishes vascular endothelial damage and is effective against crush injury.

A neutrophil elastase inhibitor, sivelestat, improves leukocyte deformability in patients with acute lung injury.

BACKGROUND: The objective of this study was to evaluate whether the neutrophil elastase (NE) inhibitor, sivelestat, improves leukocyte deformability and pulmonary function in patients with acute lung injury (ALI). PATIENTS AND METHODS: Twenty-four patients with systemic inflammatory response syndrome (SIRS) were divided into two groups: those with ALI (ALI group, n = 14), and those without ALI (non-ALI group, n = 10). Within 72 hours after the diagnosis, we measured the total leukocyte count (TLC), C-reactive protein (CRP) level, NE concentration, APACHE II score, Goris multiple organ failure (MOF) index, respiratory index (RI), lung injury score (LIS), and oxygenation index (P/F ratio). Leukocyte deformability was examined with a microchannel array etched on a single-crystal silicon tip that simulates the microvasculature. The number of obstructed microchannels (NOM) because of stiffened neutrophils and transit time (TT), defined as the time needed for 100 microL of whole blood to pass through the microchannels, were determined. We then administered sivelestat (4.8 mg/kg/d) to nine ALI patients (sivelestat group) for 5 days and compared with seven ALI patients treated previously without sivelestat (conventional group). The factors described above were measured before and 5 days after treatment. RESULTS: There were no significant differences in age, TLC, CRP, APACHE II score, and MOF index between ALI and non-ALI group. RI and LIS were higher and the P/F ratio was significantly lower in the ALI group than in the non-ALI group. NE concentration, NOM, and TT were significantly higher in the ALI group than in the non-ALI group (p < 0.05). After 5 days of treatment with sivelestat, the APACHE II score, MOF index, RI, LIS, NE concentration, TT, and NOM were lower and the P/F ratio was significantly higher than baseline values and those in the conventional group (p < 0.05). CONCLUSION: NE concentration and neutrophil rigidity are significantly increased in SIRS patients with ALI. Sivelestat appears to reduce NE concentration and neutrophil stiffness and improve pulmonary oxygenation in patients with ALI.

Enhanced expression of intracellular heme oxygenase-1 in deactivated monocytes from patients with severe systemic inflammatory response syndrome.

BACKGROUND: Monocyte deactivation is an important contributor to infectious susceptibility in critically ill patients. However, the mechanism of monocyte deactivation has not been fully elucidated. Recently, intracellular heme oxygenese-1 (HO-1), an anti-inflammatory heat-shock protein, was reported to be activated by Toll-like receptors (TLRs), and to inhibit inflammatory cytokine production such as that of TNF-alpha. In the present study, we evaluated the expression of intracellular HO-1 and TLRs in monocytes from patients with severe systemic inflammatory response syndrome (SIRS) and examined the role of HO-1 in monocyte deactivation. PATIENTS: Twenty-seven patients who fulfilled the criteria for severe SIRS and had a serum C-reactive protein (CRP) level >10 mg/dL were included in this study. The cause of SIRS was sepsis in 16 patients, trauma in 7, and other in 4. Expression of intracellular HO-1, surface TLR2 and TLR4, and intracellular cytokines (TNF-alpha, Interleukin-6) stimulated via TLR activation were measured in circulating monocytes by flow cytometry. Intracellular HO-1 expression was evaluated in normal monocytes stimulated with patient serum. Serum cytokine levels were also measured. Patient data were compared with data from healthy volunteers (n = 16). RESULTS: Cytoplasmic HO-1 was clearly detected by fluorescence microscopy. Expression of HO-1, TLR2, and TLR4 in monocytes was significantly enhanced in patients with severe SIRS compared with that in healthy volunteers, whereas intracellular TNF-alpha expression with peptidoglycan was significantly decreased (p < 0.05) in patients compared with that in healthy volunteers. HO-1 expression was significantly enhanced in normal monocytes stimulated with patient serum. Intracellular HO-1 levels were positively related to serum TNF-alpha levels in patients (r = 0.46). CONCLUSIONS: Expression of intracellular HO-1 and of TLRs was enhanced in deactivated monocytes from patients with SIRS. Increased production of intracellular HO-1 in response to serum factors may play a role in monocyte deactivation after systemic inflammation.

New experimental model of crush injury of the hindlimbs in rats.

BACKGROUND: Crush injury (CI) remains a life-threatening condition. Because there is a shortage of animal models of CI, we purposed to develop a reproducible model of CI of hindlimbs in rats and to evaluate correlation between the volume of muscles traumatized and the severity of CI. METHODS: The right or both hindlimbs of anesthetized rats were compressed for 6 hours under blocks weighing 3 kg. This was followed by 3 hours of reperfusion. Serum lactate, base excess (BE), and potassium (K) were measured at 10 minutes after cannulaton (baseline), immediately before release (compression), and 3 hours after release (reperfusion). Serum creatine phosphokinase (CK), lactate dehydrogenase (LDH), aspartate transferase (AST) and alanine transferase (ALT) were measured at baseline and reperfusion. Muscles and kidneys were evaluated morphologically. In a separate group of animals treated in the same way, survival rate was monitored for 168 hours. RESULTS: Unilateral CI did not induce serious systemic impairment. Bilateral CI resulted in severe lactic acidosis. Serum K levels increased similarly and significantly in both groups. Serum CK levels correlated strongly with the volume of muscles traumatized. Bilateral CI produced a sharp increase in serum LDH, AST and ALT levels by the end of experiment. Signs of direct cellular damage and ischemia-reperfusion injury were found in histology specimens. In bilaterally crushed rats there were patent signs of acute tubular necrosis at 24 hours after insult. All rats with unilateral CI survived, whereas mortality rate reached 58.3% in rats with bilateral CI. The majority of these animals died within 24 hours after compression. CONCLUSIONS: We developed a valid experimental model of severe CI of the hindlimbs in rats. Systemic responses to CI and the severity of CI appeared to correlate strongly with the volume of muscle traumatized.

The balance between expression of intranuclear NF-kappaB and glucocorticoid receptor in polymorphonuclear leukocytes in SIRS patients.

BACKGROUND: We previously reported enhanced expression of nuclear factor kappa B (NF-kappaB) in activated polymorphonuclear leukocytes (PMNLs) from patients with systemic inflammatory response syndrome (SIRS). Inflammatory response, however, is not regulated only by stimulatory transcription factors. Glucocorticoid receptor (GR) has been recently reported to play an important role in anti-inflammatory signal transduction. The objective of our study was to evaluate the balance between expression of intranuclear NF-kappaB and GR in PMNLs from SIRS patients. METHODS: In study 1, 29 patients with severe SIRS, who fulfilled the criteria for SIRS and had a serum C-reactive protein level of more than 10 mg/dL, were included. Expression of intranuclear NF-kappaB and GR in PMNLs was measured by flow cytometry with antibodies specific for NF-kappaB subunit p65 and GR. PMNL oxidative activity and serum cytokine levels were also measured. Study 2 included 13 patients with severe trauma (Injury Severity Score 24.6 +/- 12.2). We measured serial changes in expression of intranuclear NF-kappaB and GR in days 0 to 2, 3 to 6, and 7 to 14 after injury. RESULTS: In study 1, expression of both intranuclear NF-kappaB and GR in PMNLs was significantly higher in SIRS patients than in healthy controls. There was a strong correlation between expression of these two transcription factors (r = 0.78). Positive correlations were also found between PMNL oxidative activity and both transcription factors. In study 2, expression of both NF-kappaB and GR in PMNLs was markedly elevated on days 3 to 6 after injury and changed serially with strong mutual correlation. CONCLUSIONS: In activated PMNLs from SIRS patients, levels of both intranuclear NF-kappaB and GR were elevated and strongly correlated. In trauma patients, NF-kappaB and GR in PMNLs changed serially with strong mutual correlation. Further studies are needed to clarify the effect of the balance of NF-kappaB and GR on PMNL activation and systemic inflammatory process.

Severe Chlamydophila psittaci pneumonia rapidly diagnosed by detection of antigen in sputum with an immunochromatography assay.

We report a case of severe Chlamydophila (Chlamydia) psittaci pneumonia rapidly diagnosed by detection of antigen in sputum with an immunochromatography assay. The patient was admitted to our hospital because of shock, disturbance of consciousness, accidental hypothermia, and multiple organ dysfunction syndrome, and he recovered after administration of intravenous erythromycin and high-dose methylpredonisolone therapy. Psittacosis was confirmed by detection of 16S rRNA gene of C. psittasi in sputum with multiplex-polymerase chain reaction analysis. Serological responses to C. psittasi, C. trachomatis, and C. pneumoniae were also evaluated, and serological cross-reactivity was observed between each species. We consider that the commercially available immunochromatography assay for Chlamydia species can be helpful for rapid diagnosis of Chlamydia infection of the respiratory tract. Hereafter, further examination will be necessary regarding pretreatment of specimens or detection sensitivity and specificity.

Activated polymorphonuclear leukocytes enhance production of leukocyte microparticles with increased adhesion molecules in patients with sepsis.

BACKGROUND: Leukocyte microparticles (MPs) derived from polymorphonuclear leukocytes (PMNLs) have been recently found to be activators of vascular endothelium in vitro. The precise role of leukocyte MPs has not been clarified in patients suffering severe insult. The objective of this study was to evaluate production of leukocyte MPs and expression of adhesion molecules on the MP surface in patients with sepsis. METHODS: Twenty-one patients with severe infection (fulfilling the criteria of sepsis with serum C-reactive protein > 10 mg/dL) and 21 healthy volunteers were included as study subjects. Production of leukocyte MPs, expression of CD11b on the MPs, and oxidative activity of PMNLs were measured by flow cytometry in the presence and absence of formyl-methionyl-leucyl-phenylalanine. CD11b expression was evaluated according to the MP size (more than, equal to, or less than 1.0 microm). Soluble E-selectin, thrombomodulin, and PMNL elastase were also measured in blood. RESULTS: Production of leukocyte MPs and superoxide production in PMNLs with and without formyl-methionyl-leucyl-phenylalanine increased significantly in patients with sepsis in comparison with production in normal volunteers. In patients with sepsis, expression of CD11b was also markedly enhanced on MPs less than 1.0 microm in diameter in comparison with expression in control subjects. Levels of soluble E-selectin, thrombomodulin, and PMNL elastase in blood were significantly increased in patients with sepsis. We succeeded in detecting leukocyte MPs visually by fluorescence microscopy. CONCLUSION: Activated PMNLs enhance production of leukocyte MPs with increased adhesion molecules in patients with sepsis. Activated leukocyte MPs may play a role in the pathogenesis of endothelial activation and leukocyte-endothelium interaction in the presence of sepsis.

Increased production of leukocyte microparticles with enhanced expression of adhesion molecules from activated polymorphonuclear leukocytes in severely injured patients.

BACKGROUND: Polymorphonuclear leukocyte (PMNL)-derived microparticles (MPs) have been recently reported as activators of vascular endothelium in vitro. The objectives of the present study were to evaluate the production of MPs in severely injured patients and to clarify the role of these MPs. METHODS: Thirty severely injured patients (mean Injury Severity Score of 27 +/- 11) and 21 healthy volunteers participated in the study. Blood samples were obtained serially at three time points: days 0 to 1, days 2 to 5, and days 6 to 12 after the trauma event. MP production, CD11b and CD62L expression on MPs, and oxidative activity in PMNLs were measured by flow cytometry in both the presence and absence of formylmethionyl-leucyl-phenylalanine. Expressions of CD11b and CD62L were differentially evaluated according to the size of the MPs (>or= or < 1.0 microm). Soluble E-selectin and thrombomodulin levels in blood, variables representative of systemic vascular endothelial damage, were also measured. RESULTS: Production of MPs with and without formylmethionyl-leucyl-phenylalanine and the oxidative activity in PMNLs (O ) were prominently increased on days 2 to 5 after trauma. CD62L expression was enhanced on MPs at all three time points, and CD11b expression was enhanced on MPs < 1.0 microm in diameter at all three time points. Soluble E2-selectin and thrombomodulin in blood did not change significantly between time points. CONCLUSION: Activated PMNLs enhance production of PMNL-derived MPs with increased adhesion molecule expression on days 2 to 5 after severe trauma. This response per se, however, may not progress to systemic vascular endothelial damage.

Hepatocyte growth factor in polymorphonuclear leukocytes is increased in patients with systemic inflammatory response syndrome.

BACKGROUND: Hepatocyte growth factor (HGF) has a significant effect on the regeneration of epithelial and endothelial cells. Studies have also shown an important role of HGF in wound healing and organ regeneration. Because recent studies indicate that polymorphonuclear leukocytes (PMNLs) store HGF in their specific granules and that HGF can be degranulated in the inflammatory tissue in which activated PMNLs migrate, we evaluated the storage and release of HGF in PMNLs from patients with systemic inflammatory response syndrome (SIRS) and attempted to examine the role of HGF from PMNLs in the systemic inflammatory process. METHODS: Twenty-four patients with SIRS (serum C-reactive protein, 20.2 +/- 12.4 mg/dL [mean +/- SD]) and 18 healthy volunteers were studied. HGF in PMNLs was measured by flow cytometry by using a monoclonal antibody to HGF. The oxidative activity in PMNLs was also measured by flow cytometry. Serum HGF, interleukin (IL)-6, and IL-8 levels in each patient were measured by enzyme-linked immunosorbent assay. HGF degranulation from PMNLs was evaluated in 10 patients. RESULTS: Immunocytochemistry under fluorescence microscopy revealed enhanced expression of HGF in the granules of PMNLs. HGF in PMNLs significantly increased in patients with SIRS compared with PMNLs from healthy volunteers (SIRS, 171.0 +/- 6.6 fluorescence/cell; control, 130.7 +/- 3.8 fluorescence/cell). N-formylmethionyl-leucyl-phenylalanine and lipopolysaccharide stimulation induced further increase of HGF fluorescence in PMNLs from patients. HGF degranulation from PMNLs was also significantly enhanced in patients. Moreover, oxidative activity in PMNLs was significantly enhanced in patients with SIRS. Plasma HGF (pHGF) correlated positively with IL-6 and IL-8 levels in patients (pHGF and IL-6, gamma = 0.635, p < 0.05; pHGF and IL-8, gamma = 0.827, p < 0.01), but these values did not correlate with HGF in PMNLs. CONCLUSION: Activated PMNLs in SIRS patients increased HGF in their granules and demonstrate enhanced degranulation of HGF. The release of HGF from migrated PMNLs in the inflammatory tissue may play an important role in wound healing and organ regeneration under those conditions.