Heparin and non-anticoagulant heparin attenuate histone-induced inflammatory responses in whole blood.

Cytotoxic and pro-inflammatory histones are present in neutrophil extracellular traps (NETs) and are elevated in blood in several inflammatory conditions, sepsis being a major example. Compounds which can attenuate activities of histones are therefore of interest, with heparin being one such material that has previously been shown to bind to histones. Heparin, a successful anticoagulant for nearly a century, has been shown experimentally to bind to histones and exhibit a protective effect in inflammatory conditions. In the present study carried out in whole blood, heparin and selectively desulfated heparin reduced histone induced inflammatory markers such as interleukin 6 (IL 6), interleukin 8 (IL 8) and tissue factor and C3a, a complement component. The selectively desulfated heparins, with reduced anticoagulant activities, retained a high degree of effectiveness as an anti-histone agent, whereas fully desulfated heparin was found to be ineffective. The results from this study indicate that the presence of sulfate and other specific structural features are required for heparin to attenuate the inflammatory action of histones in whole blood.

The Identification of Immune-Related Plasma Proteins Associated with Spontaneous Preterm Delivery and Intra-Amniotic Infection in Women with Premature Cervical Dilation or an Asymptomatic Short Cervix.

BACKGROUND: We aimed to investigate whether various immune-related plasma proteins, alone or in combination with conventional clinical risk factors, can predict spontaneous preterm delivery (SPTD) and intra-amniotic infection in women with premature cervical dilation or a short cervix (≤ 25 mm). METHODS: This retrospective study included 80 asymptomatic women with premature cervical dilation (n = 50) or a short cervix (n = 30), who underwent amniocentesis at 17-29 weeks. Amniotic fluid (AF) was cultured, and maternal plasma was assayed for interleukin (IL)-6, matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinases (TIMP)-1, and complements C3a and C5a, using enzyme-linked immunosorbent assay (ELISA) kits. The primary outcome measures were SPTD at < 32 weeks and positive AF cultures. RESULTS: The plasma levels of IL-6, C3a, and C5a, but not of MMP-9 and TIMP-1, were significantly higher in women with SPTD at < 32 weeks than in those who delivered at ≥ 32 weeks. The women who delivered at < 32 weeks had more advanced cervical dilatation, and higher rates of antibiotic and tocolytic administration and were less likely to be given vaginal progesterone than those who delivered at ≥ 32 weeks. Using a stepwise regression analysis, a combined prediction model was developed, which included the plasma IL-6 and C3a levels, and cervical dilatation (area under the curve [AUC], 0.901). The AUC for this model was significantly greater than that for any single variable included in the predictive model. In the univariate analysis, plasma IL-6 level was the only significant predictor of intra-amniotic infection. CONCLUSION: In women with premature cervical dilation or a short cervix, maternal plasma IL-6, C3a, and C5a levels could be useful non-invasive predictors of SPTD at < 32 weeks. A combination of these biomarkers and conventional clinical factors may clearly improve the predictability for SPTD, as compared with the biomarkers alone. An increased plasma level of IL-6 predicted intra-amniotic infection.

Complement activation products in the circulation and urine of primary membranous nephropathy.

BACKGROUND: Complement activation plays a substantial role in the pathogenesis of primary membranous nephropathy (pMN). C5b-9, C3c, MBL, and factor B have been documented in the subepithelial immune deposits. However, the changing of complement activation products in circulation and urine is not clear. METHODS: We measured the circulating and urinary levels of C1q, MBL, C4d, Bb, properdin, C3a, C5a, and sC5b-9, in 134 patients with biopsy-proven pMN, by enzyme-linked immunosorbent assay. All the plasma values were corrected by eGFR and all the urinary values were corrected by urinary creatinine and urinary protein excretion. Anti-PLA2R antibodies were measured in all patients. RESULTS: The plasma complement activation products were elevated both in the patients with and without anti-PLA2R antibodies. C3a levels were remarkably increased in the circulation and urine, much higher than the elevated levels of C5a. C5b-9 was in normal range in plasma, but significantly higher in urine. The urinary C5a had a positive correlation with anti-PLA2R antibody levels and urinary protein. The plasma level of C4d was elevated, but C1q and MBL were comparable to healthy controls. Positive correlations were observed between plasma C4d/MBL and urinary protein, only in the patients with positive anti-PLA2R antibodies but not in those without. The plasma level of Bb was elevated and had positive correlation with urinary protein only in the patients without anti-PLA2R antibodies. CONCLUSION: Complement activation products were remarkable increased in pMN and may serve as sensitive biomarkers of disease activity. The complement may be activated through lectin pathway with the existence of anti-PLA2R antibodies, while through alternative pathway in the absence of antibody.

Effect of preoperative inhaled budesonide on pulmonary injury after cardiopulmonary bypass: A randomized pilot study.

BACKGROUND: Cardiopulmonary bypass can result in lung injury. This prospective, double-blinded, randomized trial aimed to evaluate the protective effect of inhaled budesonide on lung injury after cardiopulmonary bypass. METHODS: Sixty patients, aged 25 to 65 years, requiring cardiopulmonary bypass were randomized to groups treated with saline or budesonide inhalation preoperatively. The respiratory mechanics were recorded. Bronchoalveolar lavage fluid was collected before cardiopulmonary bypass and after sternal closure. Serum and bronchoalveolar lavage fluid levels of proinflammatory and anti-inflammatory factors were analyzed. The primary end point was the lowest ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen after cardiopulmonary bypass. The durations of ventilation and postoperative recovery time were noted. RESULTS: Budesonide significantly improved respiratory mechanics after cardiopulmonary bypass. Budesonide improved the partial pressure of arterial oxygen to the fraction of inspired oxygen ratio from 8 to 48 hours after the operation. Budesonide shortened the durations of mechanical ventilation and postoperative recovery time. Budesonide decreased the levels of proinflammatory factors while increasing the levels of anti-inflammatory factors in bronchoalveolar lavage fluid and serum (all P < .05). The macrophage and neutrophil counts, and protein and elastase concentrations were decreased by budesonide treatment. CONCLUSIONS: Budesonide treatment shortened the durations of mechanical ventilation, inhibited local and systemic inflammation, and improved respiratory function after cardiopulmonary bypass.

Plasma inflammatory and immune proteins as predictors of intra-amniotic infection and spontaneous preterm delivery in women with preterm labor: a retrospective study.

BACKGROUND: We investigated whether various inflammatory and immune proteins in plasma predict intra-amniotic infection and imminent preterm delivery in women with preterm labor and compared their predictive ability with that of amniotic fluid (AF) interleukin (IL)-6 and serum C-reactive protein (CRP). METHODS: This retrospective cohort study included 173 consecutive women with preterm labor who underwent amniocentesis for diagnosis of infection and/or inflammation in the AF. The AF was cultured, and assayed for IL-6. CRP levels and cervical length by transvaginal ultrasound were measured at the time of amniocentesis. The stored maternal plasma was assayed for IL-6, matrix metalloproteinase (MMP)-9, and complements C3a and C5a using ELISA kits. The primary and secondary outcome criteria were positive AF cultures and spontaneous preterm delivery (SPTD) within 48 h, respectively. Univariate, multivariate, and receiver operating characteristic analysis were used for the statistical analysis. RESULTS: In bivariate analyses, elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery, whereas elevated plasma levels of MMP-9, C3a, and C5a were not associated with these two outcomes. On multivariate analyses, an elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery after adjusting for confounders, including high serum CRP levels and short cervical length. In predicting intra-amniotic infection, the area under the curve (AUC) was significantly lower for plasma IL-6 than for AF IL-6 but was similar to that for serum CRP. Differences in the AUCs between plasma IL-6, AF IL-6, and serum CRP were not statistically significant in predicting imminent preterm delivery. CONCLUSIONS: Maternal plasma IL-6 independently predicts intra-amniotic infection in women with preterm labor; however, it has worse diagnostic performance than that of AF IL-6 and similar performance to that of serum CRP. To predict imminent preterm delivery, plasma IL-6 had an overall diagnostic performance similar to that of AF IL-6 and serum CRP. Plasma MMP-9, C3a, and C5a levels could not predict intra-amniotic infection or imminent preterm delivery.

LDL apheresis activates the complement system and the cytokine network, whereas PCSK9 inhibition with evolocumab induces no inflammatory response.

BACKGROUND: Low-density lipoprotein (LDL) apheresis is an extracorporeal treatment modality used in high-risk coronary patients. It may, however, induce complement activation and downstream inflammation due to bio-incompatibility. OBJECTIVE: We explored changes in soluble inflammatory markers when changing from LDL apheresis to the novel PCSK9 inhibitor evolocumab. METHODS: Three patients with familial hypercholesterolemia participated. Blood samples (EDTA plasma) for complement activation and markers of inflammation were obtained before (baseline) and after LDL apheresis week at 0 and before biweekly administration of evolocumab at weeks 1, 3, 5, and 7. Complement activation was measured by ELISA and cytokines by multiplex technology. RESULTS: Complement activation products C3a and Bb were both significantly higher after LDL apheresis compared to baseline (P = .01), returned to baseline levels before administration of evolocumab and remained low through week 7. C4d was unchanged during LDL apheresis, whereas TCC was slightly higher after apheresis compared to baseline and week 7 without statistical difference. MCP-1 was higher after LDL apheresis compared to baseline (P = .04), returned to baseline levels before administration of evolocumab and remained low through week 7. There were minor changes for other cytokines including TNF, IFN-γ, MIP-1α, MIP-1ß, with some higher and some lower after apheresis; however, none of these changes were statistically significant. Fibrinogen and CRP were lower after LDL apheresis and had returned to levels comparable to baseline at week 7, statistically significant however only for fibrinogen. CONCLUSIONS: LDL apheresis activated the alternative complement system significantly as reflected by an increase in C3a and Bb. PCSK9 inhibition did not affect complement or cytokines during 7 weeks follow-up.

Correlations between complement system's activation factors and anti-angiogenesis factors in plasma of patients with early/late-onset severe preeclampsia.

OBJECTIVE: To investigate the expression of complement system's activation factors in plasma of patients with severe preeclampsia and their correlations with anti-angiogenesis factors. METHODS: A case-control study was performed. The study group consisted of 30 cases of early-onset severe preeclampsia (EOSPE) and 30 cases of late-onset severe preeclampsia (LOSPE). Thirty cases were selected as the early-onset control group (E-control) and 30 as the late-onset control group (L-control), with the weeks of gestation matched. Enzyme-linked immunosorbent assay (ELISA) was used to test C3a, C5a, MAC, sEng, and sflt-1 in the maternal peripheral circulation. RESULTS: The complement system's activation factors C3a, C5a, and MAC were increased significantly in EOSPE and LOSPE (all P < 0.01) compared with E/L-control. Plasma levels of C3a correlated inversely with plasma sEng (r = -0.454, P < 0.001) and sflt-1 (r = -0.326, P = 0.011) in preeclampsia patients, while MAC correlated with soluble endoglin (sEng; r = 0.343, P = 0.007) and soluble fms-like tyrosine kinase-1 (sflt-1; r = 0.318, P = 0.013). There were no significant correlations between complement system's activation-related factors and the anti-angiogenesis factors in healthy control group. CONCLUSIONS: Abnormal activation of the complement system exists in the maternal circulation of patients with E/L-onset severe preeclampsia. There were correlations between the abnormal activation of the complement system and the abnormal expression of anti-angiogenesis factors in patients with severe preeclampsia, but the correlation was not strong.

[Association between protective effect of Liuwei Wuling tablets against acute liver injury and its inhibitory effect on cytoplasmic translocation of high-mobility group box-1 in hepatocytes in mice].

OBJECTIVE: To investigate the effect of Liuwei Wuling tablets on the cytoplasmic translocation and release of high-mobility group box-1 (HMGB1) in hepatocytes in mice with acute live injury induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). METHODS: A Balb/c mouse model of acute liver injury was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (5 ug/kg). A total of 24 healthy mice were randomly and equally divided into acute liver injury control group and Liuwei Wuling tablet treatment group. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in both groups at each time point within one week. Liver tissues were collected at 36 hours to perform pathological examination. The serum levels of HMGB1, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), complement 3a (C3a), and complement 5a (C5a) were measured. Immunohistochemistry was used to determine the expression and cytoplasmic translocation of HMGB1 in hepatocytes. RESULTS: At 6, 12, and 24 hours, the Liuwei Wuling tablet treatment group had significantly lower serum levels of ALT than the control group (225.33±181.64 U/L vs 471.17±174.72 U/L, t = 3.38, P < 0.01; 1509.53±182.51 U/L vs 7149.52±734.25 U/L, t = 25.82, P < 0.01; 162.89±86.51 U/L vs 1318.16±557.71 U/L, t = 7.09, P < 0.01), as well as significantly lower serum levels of AST than the control group (179.22±94.57 U/L vs 561.91±209.6 U/L, t = 5.76, P < 0.01; 590.92±190.92 U/L vs 2266.48±705.64 U/L, t = 7.94, P < 0.01; 231.24±87.7 U/L vs 444.32±117.01 U/L, t = 5.05, P < 0.01). The treatment group had significantly lower levels of HMGB1 than the control group at 6 and 12 hours (54.21±11.89 ng/ml vs 72.07±13.65 ng/ml, t = 3.41, P < 0.01; 49.23±5.97 ng/ml vs 68.71±13.07 ng/ml, t = 4.70, P < 0.01). The treatment group had significantly lower levels of TNF-α, IL-1ß, and IL-6 than the control group at 12 hours (163.62±9.12 pg/ml vs 237.09±51.47 pg/ml, t = 4.86, P < 0.01; 15.66±13.13 pg/ml vs 37.43±18.68 pg/ml, t = 3.30, P < 0.01; 7.10±3.06 pg/ml vs 21.42±8.23 pg/ml, t = 5.65, P < 0.01). The treatment group had significantly lower levels of C3a and C5a than the control group at 12 hours (2.52±1.27 pg/ml vs 9.83±2.96 ng/ml, t = 7.86, P < 0.01; 2.16±1.03 ng/ml vs 7.23±1.55 ng/ml, t = 9.67, P < 0.01). Compared with the control group, the treatment group had significantly reduced liver inflammation and necrosis, and a significantly lower cytoplasmic translocation rate of HMGB1 in hepatocytes (38.76%±7.37% vs 8.15%±2.11%, P < 0.01). CONCLUSION: Liuwei Wuling tablets can reduce the cytoplasmic translocation of HMGB1 in hepatocytes and relieve liver inflammation in mice with acute liver injury.

Neutrophil Depletion Attenuates Placental Ischemia-Induced Hypertension in the Rat.

Preeclampsia is characterized by reduced placental perfusion with placental ischemia and hypertension during pregnancy. Preeclamptic women also exhibit a heightened inflammatory state and greater number of neutrophils in the vasculature compared to normal pregnancy. Since neutrophils are associated with tissue injury and inflammation, we hypothesized that neutrophils are critical to placental ischemia-induced hypertension and fetal demise. Using the reduced uteroplacental perfusion pressure (RUPP) model of placental ischemia-induced hypertension in the rat, we determined the effect of neutrophil depletion on blood pressure and fetal resorptions. Neutrophils were depleted with repeated injections of polyclonal rabbit anti-rat polymorphonuclear leukocyte (PMN) antibody (antiPMN). Rats received either antiPMN or normal rabbit serum (Control) on 13.5, 15.5, 17.5, and 18.5 days post conception (dpc). On 14.5 dpc, rats underwent either Sham surgery or clip placement on ovarian arteries and abdominal aorta to reduce uterine perfusion pressure (RUPP). On 18.5 dpc, carotid arterial catheters were placed and mean arterial pressure (MAP) was measured on 19.5 dpc. Neutrophil-depleted rats had reduced circulating neutrophils from 14.5 to 19.5 dpc compared to Control, as well as decreased neutrophils in lung and placenta on 19.5 dpc. MAP increased in RUPP Control vs Sham Control rats, and neutrophil depletion attenuated this increase in MAP in RUPP rats without any effect on Sham rats. The RUPP-induced increase in fetal resorptions and complement activation product C3a were not affected by neutrophil depletion. Thus, these data are the first to indicate that neutrophils play an important role in RUPP hypertension and that cells of the innate immune system may significantly contribute to pregnancy-induced hypertension.

Novel serum protein biomarker panel revealed by mass spectrometry and its prognostic value in breast cancer.

INTRODUCTION: Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed proteins in sera from BC and healthy volunteers (HV), with the goal of developing a new prognostic biomarker panel. METHODS: Training set serum samples from 99 BC and 51 HV subjects were applied to four adsorptive chip surfaces (anion-exchange, cation-exchange, hydrophobic, and metal affinity) and analyzed by time-of-flight MS. For validation, 100 independent BC serum samples and 70 HV samples were analyzed similarly. Cluster analysis of protein spectra was performed to identify protein patterns related to BC and HV groups. Univariate and multivariate statistical analyses were used to develop a protein panel to distinguish breast cancer sera from healthy sera, and its prognostic potential was evaluated. RESULTS: From 51 protein peaks that were significantly up- or downregulated in BC patients by univariate analysis, binary logistic regression yielded five protein peaks that together classified BC and HV with a receiver operating characteristic (ROC) area-under-the-curve value of 0.961. Validation on an independent patient cohort confirmed the five-protein parameter (ROC value 0.939). The five-protein parameter showed positive association with large tumor size (P = 0.018) and lymph node involvement (P = 0.016). By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, immunoprecipitation and western blotting the proteins were identified as a fragment of apolipoprotein H (ApoH), ApoCI, complement C3a, transthyretin, and ApoAI. Kaplan-Meier analysis on 181 subjects after median follow-up of >5 years demonstrated that the panel significantly predicted disease-free survival (P = 0.005), its efficacy apparently greater in women with estrogen receptor (ER)-negative tumors (n = 50, P = 0.003) compared to ER-positive (n = 131, P = 0.161), although the influence of ER status needs to be confirmed after longer follow-up. CONCLUSIONS: Protein mass profiling by MS has revealed five serum proteins which, in combination, can distinguish between serum from women with breast cancer and healthy control subjects with high sensitivity and specificity. The five-protein panel significantly predicts recurrence-free survival in women with ER-negative tumors and may have value in the management of these patients.

Biomarkers of coagulation, inflammation, and angiogenesis are independently associated with preeclampsia.

PROBLEM: Although preeclampsia has been associated with inflammation, coagulation, and angiogenesis, their correlation and relative contribution are unknown. METHOD OF STUDY: About 114 women with preeclampsia, 31 with early onset (EOP) and 83 with late onset preeclampsia (LOP), and 100 normal pregnant controls were included. A broad panel of 32 biomarkers reflecting coagulation, inflammation, and angiogenesis was analyzed. RESULTS: Preeclampsia was associated with decreased antithrombin, IL-4 and placental growth factor levels and with increased C3a, pentraxin-3, and sFlt-1 levels, with more marked differences in the EOP group. The Th1-associated chemokines CXCL10 and CXCL11 were significantly higher in the preeclampsia and EOP group than in controls, respectively. No correlations between the biomarkers were found in preeclampsia. Multivariate logistic regression tests confirmed the results. CONCLUSIONS: Cytokines, chemokines and complement activation seem to be part of a Th1-like inflammatory reaction in preeclampsia, most pronounced in EOP, where chemokines may be more useful than cytokines as biomarkers. Biomarkers were not correlated suggesting partly independent or in time separated mechanisms.

Identification of potential markers related to neoadjuvant chemotherapy sensitivity of breast cancer by SELDI-TOF MS.

Neoadjuvant chemotherapy (NACT) is known to be beneficial for patients with locally advanced breast cancer. However, there is still no unified standard on the evaluation of NACT. To identify the potential markers related to NACT sensitivity of breast cancer, in the present study, we examined the protein spectrum of breast cancer tissues before and after NACT using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Totally, 87 protein samples were extracted from tissues of breast cancer, with 30 from patients before NACT, 30 from patients after NACT, and 27 from patients without any treatment. To eliminate confounding factors a couple of tissue samples from the same patient were mixed. SELDI-TOF MS analysis demonstrated that the intensities of eight different protein peaks, i.e., 26,055.46, 17,898.94, 8,949.50, 11,652.02, 11,053.48, 38,546.56, 5,825.89, and 22,250.63 Da, were higher in samples after NACT than those before NACT. Although further experiments are needed to prove the reliability of the proteins identified in this study, our results will help the establishment of protein model based on drug resistance-related protein peaks to screen whether a patient is suitable for adopting NACT and to improve cancer treatment.

Serum proteomic profiling reveals that pretreatment complement protein levels are predictive of esophageal cancer patient response to neoadjuvant chemoradiation.

OBJECTIVE: To identify serum-based biomarkers predicting response to neoadjuvant chemoradiotherapy (neo-CRT) in esophageal cancer. PURPOSE: Increasingly, the standard of care for esophageal cancer involves neo-CRT followed by surgery. The identification of biomarkers predicting response to therapy may represent a major advance, enabling clinical trials and improved outcomes. BACKGROUND DATA: Patients with esophageal cancer (n = 31) received a standard neo-CRT regimen. Histopathologic response to therapy was assessed by using the Mandard tumor regression grade (TRG) classification. Serum was collected pretreatment and at 24-hour and 48-hour time points into treatment. Serum samples were analyzed by using Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and enzyme-linked immunosorbent assay. A leave-one-out cross-validation predictive algorithm assessed the ability of validated biomarkers to correctly predict therapeutic outcome. RESULTS: Fifty-one percent (16) of patients were poor responders (TRG 3-5), whereas 49% (15) responded well (TRG 1-2). On CM10 biochips, serum expression of 9 protein peaks was significantly different between the response groups. Two differential spectrum peaks were identified as complement C4a and C3a and were subsequently analyzed by enzyme-linked immunosorbent assay. Pretreatment serum C4a and C3a levels were significantly higher in poor responders versus good responders. Subdivision of the response groups by TRG indicated an inverse correlation between levels of C4a and C3a and pathological response to neo-CRT. The leave-one-out cross-validation analysis revealed that these serum proteins could predict response to neo-CRT with a sensitivity and specificity of 78.6% and 83.3%, respectively. CONCLUSIONS: This translational application of proteomics technology identifies pretreatment serum levels of C4a and C3a as predictive biomarkers of response. Large validation studies in an independent cohort are merited.

Immunomodulation of serum complement (C3) and macrophages by synthetic pyrethroid fenvalerate: in vitro study.

Fenvalerate, a type II synthetic pyrethroid, has emerged as one of the most potent indoor toxicants. Despite its widespread usage, the adverse effect of this insecticide on immune defense mechanism has not been comprehensively investigated. In this in vitro study we report the effect of fenvalerate on two pivotal components of the immune network, namely the complement system and macrophages. Fenvalerate treated human sera showed serum complement activation as evident by significant (p<0.05) increase in C3b, C3d and C3a levels and a significant (p<0.05) decline in CH50 levels. Further detailed study demonstrates that the activation of complement system is through alternative pathway. This is possibly responsible for various allergic manifestations often reported in subjects exposed to fenvalerate. In addition, fenvalerate induce cellular apoptosis and cytotoxicity, as demonstrated by cytoplasmic vacuolization, heterochromatin condensation, hypodiploid nuclei and DNA fragmentation in macrophages. Considerable deleterious effects on macrophages in conjunction with uncontrolled serum complement activation are probably one of the major mechanisms contributing for the immunosuppressive effects of fenvalerate.

Complement factor C5a in acute exacerbation of Chronic Obstructive Pulmonary Disease.

The complement component C5a is a potent inflammatory peptide, which may be involved in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). We analysed the induced sputum and plasma of 28 patients with stable COPD, 12 healthy smokers and 7 non-smokers. In 13 of the patients with COPD, we also observed paired samples during acute exacerbation. The concentrations of C5a/C5a desArg and C3a/C3a desArg were measured using cytometric bead array. Both C5a and C3a concentrations in induced sputum of stable patients with COPD were significantly increased compared to the control groups of healthy smokers and non-smokers. In addition, there was a significant elevation in C5a values in exacerbation of COPD that was independent from the airway C3a levels. Airway C5a levels were negatively correlated with forced expiratory volume in first second (FEV1)% predicted and diffusing capacity of the lung (TLCO). Plasma C5a concentrations in patients with COPD were significantly higher than in healthy smokers, but no further significant systemic C5a elevation was detected with acute exacerbation of COPD. There was no important difference in local or systemic C5a concentrations between healthy smokers and non-smokers. These in vivo results clearly show that local and systemic C5a concentrations in COPD are elevated, and that the local, in contrast to systemic, C5a concentrations additionally increase in the acute exacerbation of COPD. It seems that the cigarette smoke is not related to C5a increase. The elevated local and systemic C5a levels, and additional individual local C5a increase during the exacerbation support the importance of C5a in COPD.

The effects of normoxic versus hyperoxic cardiopulmonary bypass on oxidative stress and inflammatory response in cyanotic pediatric patients undergoing open cardiac surgery: a randomized controlled trial.

OBJECTIVES: This study investigates the effects of controlled reoxygenation cardiopulmonary bypass on oxidative stress, inflammatory response, and organ function in children undergoing repair of cyanotic congenital heart defects. METHODS: Sixty-seven cyanotic patients (median age 15 months, interquartile range 6-49 months) undergoing corrective cardiac surgery were randomized to receive either controlled normoxic (50-0 mm Hg; n = 35) or hyperoxic (150-180 mm Hg; n = 32) cardiopulmonary bypass. Troponin I and 8-isoprostane, C3a, interleukins 6, 8, and 10, cortisol, protein S100, and alpha-glutamate transferase were measured preoperatively and 10 and 30 minutes after starting bypass, on removal of the aortic crossclamp, and 12 and 24 hours thereafter. RESULTS: Overall, troponin I and 8-isoprostane levels were lower in the controlled normoxic group (-29%, 95% CI -48% to -3%, P = .03, and -26%, 95% CI -44% to -2%, P = .03, respectively). Protein S100 release was also lower in the normoxic group 10 minutes after starting bypass (-26%, 95% CI -40% to -9%, P = .005) and 10 minutes after aortic crossclamp removal (-23%, 95% CI -38% to -3%, P = .02, respectively), but similar at other time points in the two groups (P >or= .17). The alpha-glutamate transferase release was significantly lower in the normoxic group 10 minutes after aortic crossclamp removal (-28%, 95% CI -44% to -9%, P = .006, respectively) but was similar at other times (P >or= .11). Release of C3a, interleukins 6, 8, and 10, and cortisol was similar in the two groups throughout (P >or= .15). CONCLUSION: Controlled reoxygenation on starting cardiopulmonary bypass is associated with reduced myocardial damage, oxidative stress, and cerebral and hepatic injury compared with hyperoxic bypass and similar whole body inflammatory and stress response in cyanotic children undergoing open cardiac surgery.

Serum protein profiling for diagnosis of breast cancer using SELDI-TOF MS.

In search for novel markers for breast cancer, we aimed to identify and validate novel serum protein profiles specific for breast cancer, and assess the influence of clinical (subjects age) and pre-analytical (sample storage duration) variables on the constructed classifiers. To this end, sera of breast cancer patients (n=152) and healthy controls (n=129), randomly divided into a training and test set, were analysed by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS). In the training set, 14 peak clusters were found to differ significantly in expression between cases and controls. None of the peak clusters were influenced by subjects age and sample storage duration. Ten peak clusters were also found significantly discriminative in the test set. Peak clusters were structurally identified as C3a des-arginine anaphylatoxin, (tentative) inter-alpha-trypsin inhibitor heavy chain 4 fragments and a fibrinogen fragment. Logistic regression analyses on the training set yielded a classification model with a moderate performance on the test set, corresponding to those reported in previously performed validation studies. Most likely originating from the highly heterogeneous nature of breast cancer, selection of breast cancer subgroups for comparison with healthy controls is expected to improve results of future diagnostic SELDI-TOF MS studies.

Complement activation in patients with primary antiphospholipid syndrome.

OBJECTIVE: To investigate the significance of complement activation in patients with primary antiphospholipid syndrome (APS). METHODS: Thirty-six patients with primary APS, 42 control patients with non-systemic lupus erythematosus (SLE) connective tissue diseases, and 36 healthy volunteers were analysed retrospectively. Serum complement levels (C3, C4, CH(50)) and anaphylatoxins (C3a, C4a, C5a) were examined in all subjects, and serum complement regulatory factors (factor H and factor I) were measured in patients with primary APS. Plasma anticoagulant activity was determined in a mixing test using the activated partial thromboplastin time. RESULTS: Serum complement levels were significantly lower in patients with primary APS than in patients with non-SLE connective tissue diseases (mean (SD) C3: 81.07 (17.86) vs 109.80 (22.76) mg/dl, p<0.001; C4: 13.04 (8.49) vs 21.70 (6.96) mg/dl, p<0.001; CH(50): 31.32 (8.76) vs 41.40 (7.70) U/ml, p<0.001) or healthy volunteers. Only two healthy subjects with low serum C4 levels showed hypocomplementaemia, whereas most patients with primary APS showed raised serum C3a and C4a. No subjects showed raised C5a. Patients with primary APS with low serum C3 or C4 had significantly higher levels of C3a or C4a than healthy controls. No patients had low serum complement regulatory factors. Among patients with primary APS, hypocomplementaemia was significantly more common in those with high anticoagulant activity than in those with low or normal activity. CONCLUSION: Hypocomplementaemia is common in patients with primary APS, reflecting complement activation and consumption, and was correlated with anticoagulant activity, suggesting that antiphospholipid antibodies may activate monocytes and macrophages via anaphylatoxins produced in complement activation.

Is cell salvage safe in liver resection? A pilot study.

STUDY OBJECTIVE: To investigate the quality of cell salvaged (CS) blood in patients undergoing hemihepatectomy (study group) and compare it with CS-blood from aortic surgery (control group). DESIGN: Observational study. SETTING: Operating room in a university hospital. MEASUREMENTS: 6 patients undergoing hemihepatectomy or aortobifemoral bypass with intraoperative blood loss of more than 800 mL. Samples were drawn from the central venous catheter, from the reservoir of a CS recovery system, and from the processed blood in each patient to determine interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor (TNF), complement C3a, and the terminal complement complex C5b-9. Microbiological analysis included colony count after cultivation in aerobic and anaerobic medium as well as enrichment culture for 6 days. MAIN RESULTS: In the hemihepatectomy group, levels of IL-6, C3a, and C5b-9 were significantly higher in the reservoir than in samples obtained from the central venous catheter. After the washing procedure, levels of IL-6, C3a, and C5b-9 were lower in the liver resection group than in each patient's own plasma levels. In all patients undergoing aortobifemoral bypass and in 5 patients undergoing hemihepatectomy, blood samples were sterile or showed growth of commensal skin microflora in low numbers (coagulase-negative staphylococci or propionibacteria). In one patient in the liver resection group, we could not exclude contamination with intestinal flora. CONCLUSION: Cell salvaged blood in liver resection seems to be safe for retransfusion with respect to cytokine release and complement activation, but requires further investigation in regard to bacterial contamination.

The use of ultrafiltration for inflammatory mediators removal during cardiopulmonary bypass in coronary artery bypass graf surgery.

OBJECTIVE: To investigate the effectiveness of ultrafiltration in removing inflammatory mediators released by cardiopulmonary bypass and to correlate ultrafiltration with alterations in organic function according to the Sequential Organ Failure Assessment Score. METHODS: Forty patients were included and randomized into two groups: "no ultrafiltration" (n=20; Group I) and "ultrafiltration" (n=20; Group II). Activated complement 3 and 4, interleukins 1beta, 6, 8 and tumor necrosis factor alfa were measured prior to anesthesia induction (Time 1), 5 minutes before cardiopulmonary bypass (Time 2), in the ultrafiltrated fluid (Time 3), 30 minutes (Time 4), and 6 (Time 5), 12 (Time 6), 24 (Time 7), 36 (Time 8) and 48 (Time 9) hours following cardiopulmonary bypass. Sequential Organ Failure Assessment Score was evaluated at Time 1, 6 and 9. Statistical significance was established at p < 0.05. RESULTS: In the ultrafiltrated fluid, only tumor necrosis factor alfa levels were detected. Levels of activated complement 3 at Times 5 and 7 and activated complement 4 at Times 5 and 6 were significantly higher in the unfiltered Group, and levels of interleukin 6 were higher in the filtered Group at Times 7 and 8. Interleukins 1beta, 8, tumor necrosis factor alfa, and the Sequential Organ Failure Assessment score were not significantly different between the groups. CONCLUSIONS: Ultrafiltration significantly filtered tumor necrosis factor alfa but did not influences serum levels of this cytokine. Ultrafiltration with the type of filter used in this study had no effect in organic dysfunction and should be used only for volemic control in patients undergo cardiopulmonary bypass.