Self-determined motivation and physical activity in patients with rheumatoid arthritis: a cross-sectional study.

OBJECTIVE: To identify factors associated with two self-reported measures of physical activity (PA) in patients with rheumatoid arthritis (RA). METHOD: Hospital outpatients with RA from central Norway filled in questionnaires about symptoms, psychological factors, and PA. Outcomes were two alternative self-reported measures of PA: (i) fulfilling the aerobic PA recommendations of ≥ 150 min/week at moderate intensity or ≥ 75 min/week at vigorous intensity; or (ii) being in the PA maintenance stage of the Stages of Exercise Behaviour Change framework. Logistic regression was applied to identify factors associated with PA. Step 1 included the independent variables sex, age, and smoking habits. Step 2a added self-reported function, joint pain during the past 6 months, and fatigue to Step 1. Step 2b added Exercise Self-Efficacy and the Relative Autonomy Index (RAI), calculated from the Behavioural Regulation in Exercise Questionnaire-2, to Step 1. Step 3 included all the mentioned independent variables. Steps 1-3 were analysed for each PA measure. RESULTS: In total, 227 patients participated. The RAI had a statistically significant positive association with being physically active according to both PA definitions. Joint pain had a significant negative association with meeting the aerobic PA recommendations but was not associated with being in the PA maintenance stage. CONCLUSION: The degree of self-determined motivation was the most consistent variable associated with self-reported PA behaviour. Joint pain was associated with one of the two PA measures. Motivation and joint pain may be useful targets for intervention in clinical practice to improve PA engagement among patients with RA.

Genetic risk score associations for myocardial infarction are comparable in persons with and without rheumatoid arthritis: the population-based HUNT study.

Persons with rheumatoid arthritis (RA) have increased risk of myocardial infarction (MI). Overlapping associations with MI of weighted genetic risk scores (wGRS) for coronary artery disease (CAD) and RA is unknown in a population-based setting. Data from the prospective Nord-Trøndelag Health Study (HUNT2: 1995-1997 and HUNT3: 2006-2008) were used. wGRS added each participant's carriage of all risk variants weighted by the coefficient from published association studies. Published wGRS for CAD and RA were analysed in Cox regression with MI as outcome, age as analysis time, and censoring at the first MI, death, or 31.12.2017. 2609 of 61,465 participants developed MI during follow-up (mean 17.7 years). The best-fitting wGRS for CAD and RA included 157 and 27 single-nucleotide polymorphisms, respectively. In multivariable analysis including traditional CAD risk factors, the CAD wGRS was associated with MI [hazard ratio = 1.23 (95% CI 1.18-1.27) for each SD increase, p < 0.0001] in RA patients (n = 433) and controls. The RA wGRS was not significant (p = 0.06). Independently from traditional risk factors, a CAD wGRS was significantly associated with the risk for MI in RA patients and controls, whereas an RA wGRS was not. The captured genetic risk for RA contributed little to the risk of MI.

Comparison of methods to construct a genetic risk score for prediction of rheumatoid arthritis in the population-based Nord-Trøndelag Health Study, Norway.

OBJECTIVES: To evaluate selection methods among published single-nucleotide polymorphisms (SNPs) associated with RA to construct predictive genetic risk scores (GRSs) in a population-based setting. METHODS: The Nord-Trøndelag Health (HUNT) Study is a prospective cohort study among the whole adult population of northern Trøndelag, Norway. Participants in HUNT2 (1995-1997) and HUNT3 (2006-2008) were included (489 RA cases, 61 584 controls). The initial SNP selection from relevant genome-wide studies included 269 SNPs from 30 studies. Following different selection criteria, SNPs were weighted by published odds ratios. The sum of each person's carriage of all weighted susceptibility variants was calculated for each GRS. RESULTS: The best-fitting risk score included 27 SNPs [weighted genetic risk score 27 (wGRS27)] and was identified using P-value selection criterion ≤5 × 10-8, the largest possible SNP selection without high linkage disequilibrium (r2 < 0.8), and lasso regression to select for positive coefficients. In a logistic regression model adjusted for gender, age and ever smoking, wGRS27 was associated with RA [odds ratio 1.86 (95% CI 1.71, 2.04) for each s.d. increase, P < 0.001]. The AUC was 0.76 (95% CI 0.74, 0.78). The positive and negative predictive values were 1.6% and 99.7%, respectively, and the positive predictive value was not improved in sensitivity analyses subselecting participants to illustrate settings with increased RA prevalences. Other schemes selected more SNPs but resulted in GRSs with lower predictive ability. CONCLUSION: Constructing a wGRS based on a smaller selection of informative SNPs improved predictive ability. Even with a relatively high AUC, the low PPV illustrates that there was a large overlap in risk variants among RA patients and controls, precluding clinical usefulness.

Reduced Th22 cell proportion and prevention of atopic dermatitis in infants following maternal probiotic supplementation.

BACKGROUND: In the randomized, controlled study Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT), maternal probiotic supplementation reduced the incidence of atopic dermatitis (AD) in the offspring. In the current study, we hypothesized that the effect was mediated by a shift in the T helper (Th) cells in the children. OBJECTIVE: To examine whether Th cell proportions were affected by maternal probiotic supplementation and thus could mediate the preventive effect of probiotics on AD. METHODS: A total of 415 pregnant women were randomized to ingest a combination of Lactobacillus rhamnosus GG (LGG), Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12) and Lactobacillus acidophilus La-5 (La-5) or placebo, and their offspring were assessed for AD during the first 2 years of life. Peripheral blood collected at 3 months of age was analysed for regulatory T cells (n=140) and Th subsets (n=77) including Th1, Th2, Th9, Th17 and Th22. RESULTS: The proportion of Th22 cells was reduced in children in the probiotic group compared to the placebo group (median 0.038% vs 0.064%, P=.009). The difference between the probiotic and placebo groups was also observed in the children who did not develop AD during the 2-year follow-up. The proportion of Th22 cells was increased in children who developed AD compared to the children who did not develop AD (0.090% vs 0.044%, P<.001). Mediation analysis indicated that the preventive effect of probiotics was partially mediated through the reduction in Th22 cells. CONCLUSION: Perinatal maternal probiotic supplementation with a combination of LGG, Bb-12 and La-5 reduced the proportion of Th22 cells in 3-month-old children. This may partially explain the preventive effect of probiotics on AD.

Fine-mapping the MHC locus in juvenile idiopathic arthritis (JIA) reveals genetic heterogeneity corresponding to distinct adult inflammatory arthritic diseases.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases, comprising seven categories. Genetic data could potentially be used to help redefine JIA categories and improve the current classification system. The human leucocyte antigen (HLA) region is strongly associated with JIA. Fine-mapping of the region was performed to look for similarities and differences in HLA associations between the JIA categories and define correspondences with adult inflammatory arthritides. METHODS: Dense genotype data from the HLA region, from the Immunochip array for 5043 JIA cases and 14 390 controls, were used to impute single-nucleotide polymorphisms, HLA classical alleles and amino acids. Bivariate analysis was performed to investigate genetic correlation between the JIA categories. Conditional analysis was used to identify additional effects within the region. Comparison of the findings with those in adult inflammatory arthritic diseases was performed. RESULTS: We identified category-specific associations and have demonstrated for the first time that rheumatoid factor (RF)-negative polyarticular JIA and oligoarticular JIA are genetically similar in their HLA associations. We also observe that each JIA category potentially has an adult counterpart. The RF-positive polyarthritis association at HLA-DRB1 amino acid at position 13 mirrors the association in adult seropositive rheumatoid arthritis (RA). Interestingly, the combined oligoarthritis and RF-negative polyarthritis dataset shares the same association with adult seronegative RA. CONCLUSIONS: The findings suggest the value of using genetic data in helping to classify the categories of this heterogeneous disease. Mapping JIA categories to adult counterparts could enable shared knowledge of disease pathogenesis and aetiology and facilitate transition from paediatric to adult services.

Safety and retention of combination triple disease-modifying anti-rheumatic drugs in new-onset rheumatoid arthritis.

BACKGROUND: While efficacy of combination treatment with methotrexate (MTX), sulfasalazine (SSZ) and hydroxychloroquine (HCQ) ('triple therapy') has been shown in clinical trials, few studies have examined its longevity in a real-life setting. AIM: Our aim was to assess the tolerability, longevity and efficacy of a triple disease-modifying anti-rheumatic drug (DMARD) regimen initiated in new-onset rheumatoid arthritis (RA) patients. METHODS: Patients who met 1987 American College of Rheumatology criteria for RA with disease duration less than 2 years were offered triple therapy upon diagnosis. Treatment was intensified according to a response-driven step-up algorithm, which included progression to leflunomide (LEF) or a biologic agent. RESULTS: Of 181 new-onset RA patients, 119 commenced triple therapy. Median duration of triple therapy was 39 weeks, and 23.5% remained on it at last follow up, with median follow up 104 weeks. Continuous therapy with any three-DMARD combination (including LEF) occurred in 32% at last follow up, with median duration of 70 weeks. Cessation of at least one of MTX, SSZ or HCQ occurred because of an adverse event in 38%, remission in 7% and incomplete response in 28% of patients. SSZ accounted for 49% of initial drug withdrawals for an adverse event. Continuation of three-drug therapy did not significantly influence the proportion of patients achieving remission or low disease activity (LDA). CONCLUSIONS: Triple therapy in new-onset RA was reasonably well tolerated, persisting for median 39 weeks. SSZ intolerance commonly reduces longevity of triple therapy. Treating to the target of remission or LDA is more important than the number of DMARD continued.

Genetic variation influences the risk of bleeding after cardiac surgery: novel associations and validation of previous findings.

BACKGROUND: Severe post-operative bleeding in cardiac surgery is associated with increased morbidity and mortality. We hypothesized that variation in genetic susceptibility contributes to post-operative bleeding in addition to clinical factors. METHODS: We included 1036 adults undergoing cardiac surgery with cardiopulmonary bypass. Two different endpoints for excessive post-operative bleeding were used, either defined as blood loss exceeding 2 ml/kg/h the first 4 h post-operatively or a composite including bleeding, transfusions, and reoperations. Twenty-two single nucleotide polymorphisms (SNPs) central in the coagulation and fibrinolysis systems or in platelet membrane receptors were genotyped, focusing on replication of earlier non-replicated findings and exploration of potential novel associations. Using logistic regression, significant SNPs were added to a model with only clinical variables to evaluate whether the genetic variables provided additional information. RESULTS: Univariate tests identified rs1799809 (located in the promoter region of the PROC gene), rs27646 and rs1062535 (in the ITGA2 gene), rs630014 (in the ABO gene), and rs6048 (in the F9 gene) as significantly associated with excessive post-operative bleeding (P < 0.05, P-values confirmed by permutation). The SNPs were significant also after adjustment with clinical variables, showing almost unchanged odds ratios except for rs1799809 (P = 0.06). Addition of the genetic covariates to a logistic regression model with clinical variables significantly improved the model (P < 0.01). CONCLUSION: We identified five SNPs associated with post-operative bleeding after cardiac surgery, of which two validated previously published associations. Addition of genetic information to models with only clinical variables improved the models. Our results indicate that common genetic variations significantly influence post-operative bleeding after cardiac surgery.

Genetic and clinical risk factors for fluid overload following open-heart surgery.

BACKGROUND: Post-operative fluid overload following cardiac surgery is associated with increased morbidity and mortality. We hypothesised that genetic variations and pre-operative clinical factors predispose some patients to post-operative fluid overload. METHODS: Perioperative variables were collected prospectively for 1026 consecutive adults undergoing open-heart surgery at St. Olavs University Hospital, Norway from 2008-2010. Post-operative fluid overload was defined as a post-operative fluid balance/kg ≥ the 90th percentile of the study population. Genotyping was performed for 31 single-nucleotide polymorphisms related to inflammatory/vascular responses or previously associated with complications following open-heart surgery. Data were analysed using logistic regression modelling, and the findings were internally validated by bootstrapping (n = 100). RESULTS: Homozygous carriers of the common G allele of rs12917707 in the UMOD gene had a 2.2 times greater risk of post-operative fluid overload (P = 0.005) after adjustment for significant clinical variables (age, duration of cardiopulmonary bypass, and intraoperative red cell transfusion). A genetic risk score including 14 single-nucleotide polymorphisms was independently associated with post-operative fluid overload (P = 0.001). The number of risk alleles was linearly associated with the frequency of fluid overload (odds ratio per risk allele 1.153, 95 % confidence interval 1.056-1.258). Nagelkerke's R(2) increased with 7.5% to a total of 25% for the combined clinical and genetic model. Hemofiltration did not reduce the risk. CONCLUSION: A common variation in the UMOD gene previously shown to be related to renal function was associated with increased risk of post-operative fluid overload following cardiac surgery. Our findings support a genetic susceptibility to disturbed fluid handling following cardiac surgery.

Pre-operative and intraoperative determinants for prolonged ventilation following adult cardiac surgery.

BACKGROUND: Prolonged ventilation is a serious complication after cardiac surgery, but few risk prediction models exist. Our objectives were to develop a specific risk prediction model based on pre-operative variables, to identify whether selected intraoperative variables could improve prediction, and to compare our model with the EuroSCORE. METHODS: Data from 5027 patients undergoing open-heart surgery in 2000-2007 were used for logistic regression model development. Internal validation was performed by bootstrapping. Discrimination and calibration were assessed with areas under the receiver operating characteristic curve (AUC) and the Hosmer-Lemeshow test. Our pre-operative model was compared with predictions based on the additive and logistic EuroSCORE. RESULTS: Age, previous cardiac surgery, peripheral arterial disease, left ventricular hypertrophy, chronic pulmonary disease, renal insufficiency, pre-operative hemoglobin concentration, urgent or emergency operation, and operation other than isolated coronary artery bypass grafting were identified as pre-operative predictors for prolonged ventilation (model I). Discrimination and accuracy were excellent (AUC: 0.848 and shrinkage factor: 94%). Calibration was good (Hosmer-Lemeshow test: P = 0.43). Inclusion of a few intraoperative variables somewhat improved the model, increasing shrinkage factors (96%) and discrimination ability (AUC model II = 0.870 and model III = 0.875 for two alternative such models). Our pre-operative model showed better performance than the logistic or additive EuroSCORE. CONCLUSIONS: The pre-operative risk prediction model for prolonged ventilation with easily obtainable variables in routine clinical work performed well and was only slightly improved by inclusion of intraoperative variables. Performance was better than with the EuroSCORE.

Mortality risk prediction in cardiac surgery: comparing a novel model with the EuroSCORE.

BACKGROUND: Several models for prediction of early mortality after open-heart surgery have been developed. Our objectives were to develop a local mortality risk prediction model, compare it with the European System for Cardiac Operative Risk Evaluation (EuroSCORE), and investigate whether the addition of intra-operative variables could enhance the accuracy of risk prediction. METHODS: All 5029 patients undergoing open-heart surgery in 2000-2007 were included in the study. Logistic regression with bootstrap methods was used to develop a pre-operative risk prediction model for in-hospital mortality. Next, several intra-operative variables were added to the pre-operative model. Calibration and discrimination were assessed, and the model was internally validated for prediction in future datasets. We thereafter compared the pre-operative model with the additive and logistic EuroSCOREs. RESULTS: Our pre-operative model included eight risk factors that are routinely registered in our department: age, gender, degree of urgency, operation type, previous cardiac surgery, and renal, cardiac, and pulmonary dysfunction. The model estimated mortality accurately throughout the dataset except in the 1% of patients at extremely high risk, in which mortality was somewhat overestimated. The estimated shrinkage factor was 0.930. The areas under the receiver operating characteristic curve for our pre-operative model and the logistic EuroSCORE were 0.857(0.823-0.891) and 0.821(0.785-0.857) (P=0.02). There was no significant difference in performance between the pre-operative and the intra-operative model (P>0.10). CONCLUSION: Our pre-operative model was simple and easy to use, and showed good predictive ability in our population. Internal validation indicated that it would accurately predict mortality in a future dataset.

Biocompatibility and pathways of initial complement pathway activation with Phisio- and PMEA-coated cardiopulmonary bypass circuits during open-heart surgery.

A randomized open-heart surgery study comprising 30 patients was undertaken to compare the biocompatibility of Phisio-(phosphorylcholine) and PMEA-(poly-2-methoxyethyl acrylate) coated cardiopulmonary bypass (CPB) circuits and to assess the initial complement pathway activation during open-heart surgery. Blood samples were obtained at five time points, from the start of surgery to 24 hours postoperatively. The following analyses were performed: haemoglobin, lactate dehydrogenase, leukocyte and platelet counts, myeloperoxidase and neutrophil-activating peptide-2, thrombin-anti-thrombin complexes, syndecan-1 and the complement activation products C1rs-C1-inhibitor complexes, C4bc, C3bc, C3bBbP and the terminal complement complex (TCC). No significant inter-group difference was found in any parameters, except for the concentration of TCC which was moderately lower in the PMEA group at termination of CPB. Complement activation during open-heart surgery was mainly mediated through the alternative pathway. In conclusion, PMEA- and Phisio-coated circuits displayed similar biocompatibility with respect to inflammatory and haemostatic responses during and after open-heart surgery.

Comparable biocompatibility of Phisio- and Bioline-coated cardiopulmonary bypass circuits indicated by the inflammatory response.

BACKGROUND: The biocompatibility of cardiopulmonary bypass surfaces has been improved by heparin and polymer surface modifications. The present study compared the effect of two such coatings on the inflammatory reactions after open heart surgery. METHODS: Thirty patients undergoing elective heart surgery were randomly assigned to receive one of two types of coated circuits: Bioline (n=15) or phosphorylcholine (Phisio, n=15). The platelet and leukocyte counts, neutrophil activation (myeloperoxidase), complement activation (C3a and TCC), concentrations of lactate dehydrogenase, 27 cytokines (including interleukins, chemokines and growth factors), thrombin-antithrombin complexes, and the endothelial cell marker syndecan-1 were analyzed at five predetermined time points until 24 hrs post operatively. RESULTS: Most measurements were comparable in both groups. However, myeloperoxidase was significantly higher in the Bioline group (p < 0.001). Postoperative lactate dehydrogenase concentrations were significantly higher in the Phisio group (p<0.01) and the maximal concentration of thrombin-antithrombin complexes 2 hours postoperatively tended to be higher in the Phisio group (p=0.08), consistent with a longer aortic cross-clamp and cardiopulmonary bypass time. CONCLUSIONS: The two circuits exhibited a comparable degree of in vivo biocompatibility.

The additive contribution from inflammatory genetic markers on the severity of cardiovascular disease.

Inflammation plays a key role in the development of atherosclerosis. Genetic differences in molecules related to inflammation have therefore been linked to the susceptibility for and severity of atherosclerosis. We hypothesized that the additive contribution from different genes of importance for inflammation would enhance the severity of cardiovascular disease. Blood samples were collected from 230 adults admitted for elective coronary angiography. A total of 130 patients had significant (>50%) stenosis in at least one main coronary artery branch and 100 had not. Six polymorphisms in five different genes were analysed: myeloperoxidase (MPO) -129G/A and -463G/A, toll-like receptor 4 (TLR4) Asp299Gly, interleukin-6 (IL6) -174G/C, surfactant protein D (SFTPD) Met11Thr and regulated upon normal T-cell expressed and secreted (CCL5) -403G/A. The IL6 polymorphism was significantly associated (P = 0.017) to angiographic significant coronary artery disease, and this relation remained after adjustment for age, gender, smoking and hypercholesterolaemia (P = 0.007). The TLR4 (P = 0.050) and SFTPD (P = 0.058) polymorphisms were also associated with the presence of coronary stenosis in univariate but not in multivariate analyses. For MPO and CCL5 no associations were found. There was a significant linear association between the number of high-risk gene variants (IL6-174CC, SFTPD 11CC and TLR4 299AA) and the proportion of patients with coronary artery disease (P < 0.0005). Inherited factors related to inflammation may increase susceptibility for severe coronary artery disease. Furthermore, the additive contribution from different inflammatory genetic markers strongly enhances the individual severity of cardiovascular disease.

Soluble ICAM-1 and VCAM-1 as markers of endothelial activation.

Activated endothelium releases the soluble adhesion molecules vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1). Measurement of fluid-phase adhesion molecules is therefore used to quantify endothelial activation, but it is unclear which is the better marker. The aims of the study were to compare the relationships between mRNA, surface and total expression and released VCAM-1 and ICAM-1 in endothelial cell cultures during activation, and to compare human umbilical vein endothelial cells (HUVEC) with the microvascular cell line HMEC-1. sVCAM-1 better represented mRNA and surface expression changes in HUVEC undergoing endotoxin stimulation than did sICAM-1. Very little VCAM-1 was released from endotoxin-stimulated HMEC-1, and sICAM-1 seemed a better activation marker for these cells. During incubation of HUVEC in media with glucose concentrations of 5.6, 10.6 or 20.6 mM, VCAM-1 was released to the media in a dose-dependent way without changes in surface expression. ICAM-1 was not influenced by the glucose concentration. There are situations when VCAM-1 concentrations in the media do not mirror the surface expression on HUVEC in culture, indicating that measurements of soluble adhesion molecules may not necessarily be representative of the conditions on the cell surface. Endothelium from different locations showed varying responses with respect to VCAM-1 and ICAM-1 liberation upon endotoxin stimulation. Thus, both sVCAM-1 and sICAM-1 should be quantified in clinical studies of endothelial activation until their characteristics are better clarified.

Complement activation by neutrophil granulocytes.

Complement plays a vital role in the body's defence systems. Cardiopulmonary bypass induces a detrimental inflammatory reaction in which the complement system is known to participate through direct effects as well as through activation of neutrophils, platelets and endothelial cells. On the other hand, it has been suggested that in the setting of cardiopulmonary bypass, complement may be activated by neutrophils, perhaps due to fragmentation caused by the heart-lung machine. We therefore investigated whether intact or fragmented neutrophils were able to activate the complement system, and whether neutrophil-platelet interaction could influence such complement activation. Lepirudin-anticoagulated plasma was incubated at 37 degrees C with resting or activated intact neutrophils or neutrophils combined with platelets, or increasing amounts of fragmented neutrophils. Complement activation was evaluated by measurement of C1rs-C1 inhibitor complexes, C4bc, C3bBbP, C3bc, C5a and sC5b-9. We found significant activation of complement only by unphysiological doses of fragmented neutrophils or supernatant from fragmented neutrophils, consistent with a limited clinical significance related to neutrophil destruction during cardiopulmonary bypass. Unstimulated neutrophils induced C3bPBb formation but little formation of other activation products, indicating an increased C3 hydrolysis which was kept under control by regulatory mechanisms. Neutrophils and platelets combined increased classical activation and decreased alternative activation, similar to the findings with platelets alone. Our data confirm that in the setting of acute neutrophil fragmentation or activation, complement activation is much more important in the inflammatory network as an event upstream to neutrophil activation than vice versa.

The inflammatory response to stent grafting of the thoracic aorta.

AIM: The aim of this study was to evaluate the inflammatory reactions in patients with thoracic aortic aneurysms before, during and after stent graft treatment and to relate markers of leukocyte activation to the use of radiographic contrast media. METHODS: Blood samples were drawn from 7 patients undergoing elective stent graft treatment for thoracic aneurysms. The samples were analyzed for leukocyte and platelet counts and the concentrations of iohexol (radiographic contrast medium), myeloperoxidase, lactoferrin, neutrophil activating peptide-2 (NAP-2), soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule-1 (sVCAM-1), the complement activation products C3bc and the terminal complement complex (TCC). The preoperative results were compared with 10 healthy blood donors of similar age. RESULTS: Preoperatively, the aneurysm patients had significantly elevated concentrations of myeloperoxidase, neopterin and complement activation products compared to controls. Myeloperoxidase and lactoferrin increased after the first contrast dose and peaked at 8 h postoperatively. Platelet counts decreased, while NAP-2, sVCAM-1 and TCC increased from 8 h postoperatively. CONCLUSION: We conclude that patients with thoracic aneurysms have a low-grade inflammation prior to intervention. Stent graft treatment induces further activation, and markers of endothelial, platelet, and complement activation were increased for several days after the procedure. Radiographic contrast media could be an important contributor to the activation of neutrophil leukocytes.

Changes in neutrophil surface-receptor expression after stimulation with FMLP, endotoxin, interleukin-8 and activated complement compared to degranulation.

Neutrophil activation induces changes in the expression of surface receptors and may lead to degranulation. Surface expression of beta2-integrins, l-selectin, complement receptor 1 (CR-1), decay-accelerating factor (DAF), C5a receptor, intercellular adhesion molecule-1 (ICAM-1) and ICAM-3 was compared by flow cytometry on isolated neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (FMLP), endotoxin or interleukin-8 and on neutrophils in whole blood anti-coagulated with the thrombin inhibitor lepirudin and stimulated with cobra venom factor to induce complement activation. Myeloperoxidase and lactoferrin in the supernatants were quantified in enzyme immunoassays. With high enough doses, all stimulants induced significant upregulation of beta2-integrins, CR-1 and DAF and downregulation of l-selectin. ICAM-3 was either unchanged or somewhat downregulated. Only FMLP and PMA induced significant upregulation of ICAM-1. Combined measurement of beta2-integrins and l-selectin permitted graded evaluation of early neutrophil activation. Measurement of degranulation showed no differences compared to unstimulated controls due to substantial spontaneous degranulation of isolated neutrophils by rewarming from 4 degrees C and incubation at 37 degrees C. Spontaneous activation was less in ethylenediaminetetraacetic acid-anti-coagulated blood, but calcium chelation may also inhibit the stimulated responses. There was large activation of unstimulated neutrophils in lepirudin-anti-coagulated blood at 37 degrees C, obscuring changes induced by stimulation, which may render this anti-coagulant unsuitable for studies of neutrophils.

Chemokines and leukocyte activation in the fetal circulation during preeclampsia.

Preeclampsia is a potentially life-threatening disease for both mother and fetus. Endothelial dysfunction is pivotal in the pathogenesis of this disorder, possibly reflecting a state of persistent inflammation. In the present study, we examined whether signs of inflammation with production of chemokines and leukocyte activation were present in the fetal circulation during preeclampsia. Venous cord blood was sampled during cesarean sections from 36 neonates born after uncomplicated pregnancies and from 35 born after severe preeclamptic pregnancies with premature newborns. The expression of adhesion molecules on neutrophils and monocytes was analyzed by flow cytometry, and plasma levels of chemokines and soluble adhesion molecules were analyzed by enzyme immunoassay. Newborns of preeclamptic mothers had increased expression of CD15s (P=0.003), CD49d/CD29 (P=0.01/0.005), and CD31 (P=0.007) on neutrophils and CD15s (P<0.001), CD11c (P=0.009), and CD54 (P=0.001) on monocytes. This activation of neutrophils and monocytes was accompanied by raised plasma levels of the CXC chemokines interleukin-8 (P=0.007) and growth-related oncogene-alpha (P=0.01) and decreased plasma levels of soluble E-selectin (P=0.001) and L-selectin (P=0.002). Although raised levels of adhesion molecules on leukocytes or decreased levels of soluble adhesion molecules in plasma were not related to prematurity or the degree of preeclampsia, raised interleukin-8 levels were found only in neonates of preeclamptic mothers with the highest blood pressures. Our findings suggest the activation of neutrophils and monocytes in the fetus during preeclampsia involving enhanced chemokine activation, possibly contributing to the fetal morbidity of this disorder.

Veno-venous bypass in liver transplantation: heparin-coated perfusion circuits reduce the activation of humoral defense systems in an in vitro model.

We studied the effects of bypass circuit surface heparinization on kallikrein-kinin, coagulation, fibrinolytic and complement activation in a closed model system for simulating veno-venous bypass (WBP) in orthotopic liver transplantation (OLT). The circuits were identical to those in routine use during clinical OLT in our institution. Fresh whole human blood diluted 1:2 with Ringer's acetate was circulated at a non-pulsatile flow (2 l/min) and at a constant temperature (37.5 degrees C) for 12 h. In 10 experiments, the entire inner surface of the circuits was coated with end-point attached heparin (HC). In the remaining 10, non-treated PVC tubing was used (NC). Components of the plasma kallikrein-kinin, coagulation, fibrinolytic and complement systems were analyzed using functional techniques (chromogenic peptide substrate assays) and enzyme immunoassays at baseline, 3 and 12 h. Significant activation of the initial (C3bc) and terminal (TCC) components of the complement system were found in both the NC and HC groups after 3 and 12 h: C3bc: NC: baseline = 4 (3.5-7.7), 3 h = 17.3* (12.5-27), 12h = 31* (17.7-63.6), HC: baseline = 4.9 (3.2-6.8), 3h = 9* (6-14.4), 12h = 13.7* (7.4-18.1). TCC: NC: baseline = 0.4 (0.2-0.6), 3h = 5*(0.8-11.9), 12 h: 13.1* (4.2-25.7). HC: baseline = 0.5 (0.1-0.6), 3 h = 0.6* (0.1-0.8), 12 h = 1.2* (0.3-2) AU/ml; median and range (*: p < 0.05). The C3bc and TCC concentrations were significantly higher in the NC group at 3 and 12 h, compared to the HC group: C3bc (NC vs. HC group): 3 h, p < 0.001; 12 h, p < 0.001. TCC (NC vs. HC group): 3h, p < 0.001; 12 h, p < 0.001. Significant increases in the values of thrombin-antithrombin complexes (p = 0.003), prothrombin fragment 1 + 2 (p = 0.006) and plasmin-alpha2-antiplasmin complexes (p = 0.016) were found in the non-coated group, but not in the heparin-coated group during the observation period, showing that the coagulation and fibrinolytic systems were activated in the non-coated circuits. We conclude that heparin-coating of the internal surface of the extracorporeal perfusion circuit used for WBP reduces activation of the plasma cascade systems in a closed venous system in vitro.