IL4-10 fusion protein: a novel immunoregulatory drug combining activities of interleukin 4 and interleukin 10.

The objective of this study was to test the capacity of a newly developed fusion protein of interleukin 4 (IL-4) and IL-10 [IL4-10 fusion protein (FP)] to shift multiple pro-inflammatory pathways towards immune regulation, and to inhibit pro-inflammatory activity in arthritis models. The effects of IL4-10 FP in comparison with IL-4, IL-10 and IL-4 plus IL-10 on pro- and anti-inflammatory mediators, T cells and immunoglobulin (Ig) receptors in favour of immunoregulatory activity were studied. In addition, the capacity of IL4-10 FP to inhibit pro-inflammatory activity in ex-vivo and in-vivo arthritis models was investigated. IL4-10 FP robustly inhibited pro-inflammatory cytokine [IL-1ß, tumour necrosis factor (TNF)-α, IL-6 and IL-8] production in whole blood cultures, mediated by both the IL-10 and the IL-4 moiety. IL4-10 fusion protein induced IL-1 receptor antagonist (IL-1RA) production and preserved soluble TNF receptor (sTNFR) levels, strongly increasing IL-1RA/IL-1ß and sTNFR/TNF-α ratios. In addition, IL4-10 FP strongly inhibited T helper (Th) type 1 and 17 cytokine secretion, while maintaining FoxP3 expression and up-regulating Th2 activity. In addition, while largely leaving expression of activating Fc gamma receptor (FcγR)I, III and Fc epsilon receptor (FcεR) unaffected, it significantly shifted the FcγRIIa/FcγRIIb ratio in favour of the inhibitory FcγRIIb. Moreover, IL4-10 FP robustly inhibited secretion of pro-inflammatory cytokines by rheumatoid arthritis synovial tissue and suppressed experimental arthritis in mice, without inducing B cell hyperactivity. IL4-10 fusion protein is a novel drug, signalling cells to induce immunoregulatory activity that overcomes limitations of IL-4 and IL-10 stand-alone therapy, and therefore has therapeutic potential for inflammatory diseases such as rheumatoid arthritis.

A fusion protein of interleukin-4 and interleukin-10 protects against blood-induced cartilage damage in vitro and in vivo.

Essentials Targeted treatment for hemophilic arthropathy, still causing significant morbidity, is lacking. This study evaluates the efficacy of a fusion of protein of interleukin(IL)-4 and IL-10. In vitro the fusion protein prevents blood-induced cartilage damage in a dose-dependent manner. In hemophilic mice, the IL4-10 fusion protein ameliorates cartilage damage upon joint bleeding. SUMMARY: Background Joint damage still causes significant morbidity in hemophilia. It results from synovial inflammation and direct cartilage-degenerating properties of blood components. Interleukin (IL)-4 and IL-10 have been shown to protect cartilage from blood-induced damage. Recently an IL4-10 fusion protein has been developed to combine the function of IL-4 and IL-10 and increase their bioavailability. Objectives In this study we evaluate whether this IL4-10 fusion protein protects against blood-induced joint damage. Methods In vitro, human cartilage explants were exposed to whole blood and simultaneously to a broad concentration range of the IL4-10 fusion protein. Effects on cartilage matrix turnover were compared with the individual cytokines. Moreover, the influence of the fusion protein and its individual components on IL-1ß and IL-6 production was investigated. In hemophilia A mice, the effect of intra-articular treatment on synovitis and cartilage damage resulting from joint bleeding was evaluated by histochemistry. Results In vitro, the fusion protein prevented blood-induced cartilage damage in a dose-dependent manner, with equal effectiveness to the combination of the separate cytokines. In whole blood cultures 10 ng mL-1 fusion protein completely blocked the production of IL-1ß and IL-6 by monocytes/macrophages. In hemophilic mice, intra-articular injection of IL-4 and IL-10 did not influence synovitis or cartilage degeneration. In contrast, equimolar amounts of the fusion protein attenuated cartilage damage upon repeated joint bleeding, although synovial inflammation was hardly affected. Conclusions Overall, this study shows that the IL4-10 fusion protein prevents blood-induced cartilage damage in vitro and ameliorates cartilage degeneration upon joint bleeding in hemophilic mice.

Targeting CD1c-expressing classical dendritic cells to prevent thymus and activation-regulated chemokine (TARC)-mediated T-cell chemotaxis in rheumatoid arthritis.

OBJECTIVES: Thymus and activation-regulated chemokine (TARC) attracts cells that express the C-C chemokine receptor type 4 (CCR4), including CD4 T cells. As expression of CCR4 is increased on peripheral T cells and intra-articular interleukin (IL)-17-producing cells in patients with rheumatoid arthritis (RA), we investigated whether TARC plays a role in the attraction of T cells to the synovial compartment. In addition, we assessed the role of classical dendritic cells (cDCs) in the production of TARC in RA. METHOD: TARC was measured in synovial fluid (SF) samples from RA and osteoarthritis (OA) patients. Spontaneous and thymic stromal lymphopoietin (TSLP)-induced TARC production by mononuclear cells (MCs) and CD1c cDCs from peripheral blood (PB) and SF was assessed. The role of TARC in CD4 T-cell migration towards cDCs was assessed and the contribution of CD1c-expressing cells to TARC production was studied. RESULTS: TARC concentrations were higher in SF of RA patients compared to OA patients. MCs from SF produced TARC spontaneously and produced more TARC upon stimulation than paired PBMCs. Blocking TARC strongly inhibited CD4 T-cell chemotaxis by TSLP-stimulated cDCs, associated with decreased production of tumour necrosis factor (TNF)-α. Depletion of cDCs from SFMCs strongly reduced TARC production. CONCLUSIONS: TARC levels are increased in RA SF and our data indicate that this results from production by SFMCs and in particular CD1c cDCs. TARC attracts T cells and TARC secretion by MCs is crucially dependent on the presence of CD1c cDCs. Considering the potential of SF cDCs to activate T cells and induce pro-inflammatory cytokine secretion, targeting intra-articular cDCs constitutes a novel therapeutic approach in RA.

Anti-ETAR and anti-AT1R autoantibodies are elevated in patients with endstage cystic fibrosis.

Autoantibodies against endothelin-1 type A receptor (ETAR) are present in systemic sclerosis complicated by lung fibrosis and pulmonary hypertension. As increased serum levels and local overproduction of endothelin-1 in the airways are reported in cystic fibrosis (CF) patients, we reasoned that anti-ETAR antibodies could be prevalent in endstage CF patients prior to lung transplantation (LTx). Also, ETAR autoantibodies are frequently associated with autoantibodies against the angiotensin II type 1 receptor (AT1R). We analyzed the presence of anti-ETAR and anti-AT1R autoantibodies in 43 LTx patients (chronic obstructive pulmonary disease (COPD), n=20; CF, n=13; interstitial lung disease (ILD), n=1). We observed overall higher anti-ETAR and anti-AT1R autoantibody titers in sera taken prior to LTx in the CF patient group as compared to COPD. No difference was found in autoantibody levels between patients with CF versus ILD. In sera taken post-LTx we found the same difference in anti-ETAR and anti-AT1R autoantibody titers between patients with CF versus COPD. No difference was found in antibody titers between sera taken prior to or 6 months after LTx. There was no association between autoantibody levels and other relevant demographic parameters, and we found no association between autoantibody titers and the development of the bronchiolitis obliterans syndrome. Both autoantibody titers were strongly correlated. We hypothesize that due to prolonged exposure to bacterial infection, increased levels of AT1R and ETAR result in a deregulated immune response causing autoantibody formation. Further research is expedient to elucidate the occurrence of autoantibodies against ETAR and AT1R and their role in disease progression.

Immunoglobulin M, C-reactive protein and complement activation in rat hepatic ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (I/R) models have shown that C-reactive protein (CRP) and immunoglobulin M (IgM) are involved in complement activation. Binding of CRP and IgM to damaged cell membranes initiates complement activation and aggravates I/R injury in various organs. However, the time course of CRP- and IgM-mediated complement activation and the relation to hepatocellular injury and inflammation in liver I/R are unknown. AIM: To evaluate the time course of IgM- and CRP-related complement activation and the relation to hepatocellular injury and inflammation in a hepatic I/R rat model. METHODS: Male Wistar rats were allocated to (1) five groups of animals exposed to 60 min of partial ischemia (70%) induced via clamping of the left segmental portal triad, followed by 0, 3, 6, 12 or 24 h of reperfusion (n = 6 in each group); (2) five groups of sham-operated animals with corresponding reperfusion times (n = 5), and (3) a control group sacrificed before ischemia (n = 5). Hepatocellular injury, inflammatory response, rat plasma CRP and IgM levels and immunohistochemical depositions of CRP, IgM and C3 were assessed for each group. RESULTS: Histopathological injury scores of hematoxylin and eosin sections of ischemic liver lobes demonstrated increasing values throughout the reperfusion time with a peak at 12 h. Plasma aminotransferases (alanine aminotransferase and aspartate aminotransferase) significantly increased after 3 h of reperfusion, peaking at 6 h (3,100 ± 800 U/l; p < 0.05). Hepatic neutrophil influx significantly increased from 3 to 6 h of reperfusion (p < 0.05) and demonstrated the highest value at 12 h (1.1 ± 0.2 U/mg of protein). Plasma IL-6 levels in the ischemia groups showed peak values after 6 h of reperfusion, decreasing significantly thereafter (p < 0.05). Plasma CRP values reached highest levels after 3 h of reperfusion (mean 91 ± 5% of control pool), decreasing significantly thereafter. Rat IgM concentrations in plasma did not significantly change throughout the reperfusion time. Immunohistochemical depositions of IgM, CRP and C3 in ischemic lobes demonstrated a similar pattern in time, reaching maximum values at 12 h of reperfusion. The percentages of depositions of CRP and IgM were significantly correlated [r(S) = 0.569; p < 0.001; Spearman test]. The time course of C3 and CRP depositions throughout reperfusion and C3 and IgM staining were significantly similar [r(S) = 0.797 and r(S) = 0.656, respectively; p < 0.0001; ANOVA]. CONCLUSIONS: CRP and IgM depositions demonstrate a parallel time course throughout the reperfusion to hepatocellular damage, inflammatory response and activated complement deposition in this rat hepatic I/R model. Furthermore, the time course of CRP and IgM depositions was significantly similar to that of activated complement depositions.

Anti-BPIFA1/SPLUNC1: a new autoantibody prevalent in patients with endstage cystic fibrosis.

BACKGROUND: Bactericidal/permeability increasing protein fold containing family A (BPIFA) 1, is a secreted protein of the upper airways that shares structural homology with BPI and exhibits comparable antimicrobial capacities. We hypothesized that CF patients have circulating IgG or IgA anti-BPIFA1 autoantibodies, similarly as reported for BPI autoantibodies. METHODS: We analyzed pre- and post-transplantation sera from 67 endstage lung disease patients who underwent lung transplantation (LTx) because of COPD (n=27), CF (n=25), and ILD (n=15). RESULTS: Anti-BPIFA1 (48%) and anti-BPI (92%) were elevated in CF patients compared to healthy controls, with anti-BPIFA1 IgG isotype being most prevalent, whereas anti-BPI is of the IgA isotype. Levels of anti-BPI autoantibodies significantly declined post-LTx, whereas anti-BPIFA1 did not. No relation was found between autoantibodies against BPIFA1 and BPI. CONCLUSION: Our results indicate that BPIFA1 is a novel target for autoantibodies in CF. The function of these autoantibodies needed to be investigated in future studies.

Complement activation on the surface of cell-derived microparticles during cardiac surgery with cardiopulmonary bypass - is retransfusion of pericardial blood harmful?

OBJECTIVES: To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. METHODS: Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. RESULTS: Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. CONCLUSIONS: In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.

Secretory type II phospholipase A2 in culprit coronary lesions is associated with myocardial infarction.

BACKGROUND: Secretory type-II phospholipase A(2) (sPLA(2)-II) is a cardiovascular risk marker since higher levels of this acute phase protein imply an increased risk for coronary artery disease. Moreover, it is hypothesized that local activity of sPLA(2)-II in the atherosclerotic plaque facilitates an inflammatory response to induce plaque instability or rupture. We have studied the presence of sPLA(2)-II in culprit lesions in the coronary arteries of patients with acute myocardial infarction (AMI) or angina pectoris. MATERIALS AND METHODS: We performed a histological examination of culprit lesions in 41 patients with stable (SAP) or unstable angina pectoris (UAP), or AMI using directed coronary atherectomy (DCA). Frozen slides were analysed immuno-histochemically for the presence of sPLA(2)-II, macrophages and smooth muscle cells. Immunopositive areas were calculated as a percentage of the total tissue area using image analysis software. RESULTS: Intracellular sPLA(2)-II was found in atherosclerotic lesions in the macrophages of the intima as well as in vascular smooth muscle cells. Next to this, extracellular sPLA(2)-II depositions were also found. These depositions were significantly more extensive in patients with AMI, i.e. 26%(median)[6%(25th(percentile))-44%(75th(percentile))] of the intima area, than in patients with SAP 0%(median) (0%(25th)-10%(75th); P = 0.013) or UAP 0%(median) (0%(25th)-0%(75th); P = 0.04). CONCLUSIONS: Extracellular sPLA(2)-II is more abundantly present in atherosclerotic culprit lesions that have led to myocardial infarction. This suggests a role for extracellular sPLA(2)-II in the development of complications of atherosclerotic lesions in coronary arteries.

Inflammatory markers in late-life depression: results from a population-based study.

BACKGROUND: Previous studies have reported conflicting results concerning the association between several inflammatory markers and depression. The association between inflammation and depression may depend on the presence of specific chronic diseases or be relevant in specific sub-groups of depressed patients only. OBJECTIVE: To assess associations between inflammatory markers and depression in older people, taking account of confounding and effect-modifying factors. METHOD: Population-based study of 1285 participants of the Longitudinal Aging Study Amsterdam, aged 65 and over. Plasma concentrations of Interleukin-6 (IL-6) and C-reactive protein (CRP) were measured. Major depression (first- or recurrent episode) and sub-threshold depression were assessed. Associations were adjusted for confounding variables. Associations with inflammatory markers were further studied with regard to severity and duration of depression, and with regard to specific depressive symptoms. RESULTS: High levels of IL-6 (above 5 pg/mL) were associated with major depression (odds ratio 2.49 (1.07-5.80), both in recurrent and first episodes. No significant effect of either one of the markers on specific symptom dimensions of depression was found. Mildly elevated plasma levels of CRP (above 3.2 mg/L) were associated with higher CES-D scores, but not after correction for the confounding effect of age and chronic diseases. LIMITATIONS: The cross-sectional design limits conclusions regarding causality. CONCLUSIONS: A high plasma level of IL-6, but not CRP, is associated with an increased prevalence of major depression in older people, independent of age, chronic diseases, cognitive functioning and anti-depressants. Present results suggest new directions for clinical research into the prevention of physical consequences of depression.

The plasma kallikrein-kinin system and risk of cardiovascular disease in men.

BACKGROUND: The plasma kallikrein-kinin system (PKKS) has been implicated in cardiovascular disease, but activation of the PKKS has not been directly probed in individuals at risk of coronary heart disease (CHD) or stroke. OBJECTIVE: To determine the involvement of the PKKS, including factor XI, in cardiovascular disease occurring in a nested case-control study from the Second Northwick Park Heart Study (NPHS-II). METHODS AND RESULTS: After a median follow-up of 10.7 years, 287 cases of CHD and stroke had been recorded and 542 age-matched controls were selected. When FXIIa-C1 esterase inhibitor (C1-inhibitor) concentrations were divided into tertiles (lowest tertile as reference), the odds ratios (ORs) at 95% CIs for CHD were 0.52 (0.34-0.80) in the middle tertile and 0.73 (0.49-1.09) in the highest tertile (P = 0.01 for the overall difference; P = 0.01 for CHD and stroke combined). For kallikrein-C1-inhibitor complexes, the ORs for stroke were 0.29 (0.12-0.72) and 0.67 (0.30-1.52) in the middle and high tertiles, respectively (P = 0.02). FXIIa-C1-inhibitor and kallikrein-C1-inhibitor complexes were negatively related to smoking and fibrinogen (P < 0.005). FXIa-inhibitor complexes correlated strongly with FXIIa-inhibitor complexes. CONCLUSIONS: Lower levels of inhibitory complexes of the PKKS enzymes and particularly of FXIIa contribute to the risk of CHD and stroke in middle-aged men. This observation supports the involvement of the PKKS in atherothrombosis.

Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis.

Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand- and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a Glu --> Ala mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases.

Tissue factor serum levels and the risk of future coronary artery disease in apparently healthy men and women: the EPIC-Norfolk prospective population study.

INTRODUCTION: Tissue factor (TF) has been implicated in coronary artery disease (CAD). High levels of circulating TF are found in patients with acute atherothrombotic events. Whether high serum TF levels predict risk of future CAD independent of known risk factors remains unknown. METHODS: We conducted a prospective case-control study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk population study. Cases (n=1037) were apparently healthy men and women, aged 45-79 years, who developed fatal or non-fatal CAD during follow-up. Controls (n=2005) were matched by age, sex, and enrolment time. Serum TF levels were measured using high-affinity antibodies. RESULTS: In men, median TF levels were not significant higher in cases than in controls (59.0 pg mL-1, range: 16.7-370.4 vs. 54.9 pg mL-1, range: 16.2-452.4). In women, median TF levels were not significant higher in controls than in cases (73.4 pg mL-1, range: 16.7-492.3 vs. 50.5 pg mL-1, range: 16.5-376.7). The incidence of smoking was about double in the lowest compared with the highest TF quartile. Correcting for sex, age, body mass index, smoking, diabetes, systolic blood pressure, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol and C-reactive protein levels, the risk of future CAD was 1.05 (95% CI: 0.81-1.36) for people in the highest TF quartile, compared with those in the lowest (P-value for linearity=0.8). CONCLUSION: High levels of serum TF were not independently associated with an increased risk of future CAD in apparently healthy individuals.

Ischemia induces nuclear NOX2 expression in cardiomyocytes and subsequently activates apoptosis.

In previous work we have demonstrated increased expression of NOX2 in cardiomyocytes of infarcted human hearts. In the present manuscript we investigated the functional role of NOX2 in ischemically challenged H9c2 cells, a rat cardiomyoblast cell line, and adult rat cardiomyocytes. Expression of NOX2 in H9c2 cells was confirmed by RT-PCR. In Western-blot experiments, increased NOX2 expression was detected during ischemia, which was inhibited by transcription and translation inhibitors. Surprisingly, under ischemia, in addition to an increased cytosolic expression, NOX2 was localized mainly in the nucleus of apoptotic cardiomyocytes, where it colocalized with nitrotyrosine residues and activated caspase 3. Inhibition of reactive-oxygen-species generation with the flavoenzyme inhibitor diphenylene iodonium (DPI) and the NADPH-oxidase inhibitor apocynin led to a significantly decreased induction of apoptosis as assessed by quantification of caspase-3 activity and by TUNEL analysis. These results demonstrate that NOX2 is expressed in the nucleus of cardiomyocytes during apoptosis and that it likely participates in proapoptotic signaling. To the best of our knowledge, this is the first demonstration of nuclear NOX2 expression and its involvement in cardiomyocyte apoptosis.

Estrogen replacement raises rat CRP without evidence of complement activation.

Given current controversies regarding anti- and pro-inflammatory effects of estrogen, there is a need to explore relationships between gonadal hormones and inflammation using appropriate animal models. It has been proposed that rats are not appropriate for such research since, contrary to the effect of estrogen in humans, earlier animal studies had reported that estrogen downregulates serum C-reactive protein (rCRP) levels in the rat. With these considerations in mind, we re-examined the effects of estrogen withdrawal and replacement on CRP expression and complement activation in the rat. F-344 rats underwent bilateral ovariectomy or sham surgery at 9-10 months of age. Four months later, ovariectomized rats were treated with traditional high-dose 17beta-estradiol (Hi-E2) capsules, lower-dose (Lo-E2) 17beta-estradiol capsules, or placebo capsules for 7 days prior to sacrifice. Levels of plasma rat C-reactive protein (rCRP) were significantly lower in ovariectomized vs. sham-operated animals (415.5 +/- 10.6 vs. 626.6 +/- 23.0 mg/L, p < 0.001). Estrogen replacement significantly raised rCRP levels in ovariectomized animals (690.0 +/- 28.0 mg/L in Lo-E2 and 735.5 +/- 35.8 mg/L in Hi-E2, respectively, p < 0.001). Plasma rCRP levels correlated significantly with both hepatic rCRP (r = 0.79, p < 0.001) and serum estradiol (r = 0.70, p < 0.001) levels. However, no significant differences were observed in indices of complement activation (C4b/c) or CRP-complement complex generation (rCRP-C3 complex). In the mature female rat, ovariectomy reduces and estrogen replacement raises rCRP. Effects of estrogen on plasma rCRP induction are mediated, at least in part, through hepatic mechanisms and do not appear to require or be associated with complement activation.

Sustained activation of neutrophils in the course of Kawasaki disease: an association with matrix metalloproteinases.

Kawasaki disease (KD) is an acute febrile syndrome of childhood, characterized by vasculitis of the medium-sized arteries. White blood cell counts and the inflammatory parameter C-reactive protein (CRP) are known to be elevated in the acute phase of the disease. In this study we investigated the course of inflammatory cell type-specific parameters in KD over a longer period of time. Plasma levels of human neutrophil elastase (HNE), matrix metalloproteinases-2 and -9 (MMP2, MMP9), and neutrophil gelatinase-associated lipocalin (NGAL), macrophage neopterin and CRP were measured. Plasma samples were collected in the acute, subacute and early convalescent stage, and three months after the onset of disease. Median CRP and neopterin normalized within two weeks. In contrast, six weeks and three months after onset of disease, levels of HNE were still elevated, with median values of 163 ng/ml and 156 ng/ml, respectively (control children median < 50 ng/ml; for all time-points P < 0.0001). Values of NGAL correlated with the levels of HNE (r = 0.39, P = 0.013). These results demonstrate a longer state of neutrophil activation in KD than was previously assumed. The potential relationship between this prolonged neutrophil activation, coronary artery lesion formation and their persistence, as well as the risk of premature atherosclerosis warrants further evaluation.

Infliximab treatment reduces complement activation in patients with rheumatoid arthritis.

BACKGROUND: Tumour necrosis factor (TNF) blocking agents decrease C reactive protein (CRP) levels in rheumatoid arthritis (RA). It has been shown that CRP may contribute to complement activation in RA. OBJECTIVE: To assess the effect of intravenous infliximab treatment on complement activation, especially that mediated by CRP, in RA. METHODS: 35 patients with active RA (28 joint count Disease Activity Score (DAS28) >4.4) were treated with intravenous injections of infliximab (3 mg/kg, at weeks 0, 2, 6, 14, and 22). Clinical response and plasma levels of complement activation products, of CRP and of CRP-complement complexes, which are specific markers for CRP mediated complement activation, were assessed at the indicated time points up to 22 weeks. The relationship between CRP and CRP-complement complexes was analysed by paired t test between two time points and by generalised estimated equation, to test differences of variables over time. RESULTS: At 2 weeks after the first dose, infliximab significantly reduced overall C3 and C4 activation and plasma levels of CRP and CRP-complement complexes were also significantly reduced at this time point. The effects of infliximab on CRP and complement continued throughout the observation period and were more pronounced in patients with a good response to infliximab treatment. CONCLUSION: Treatment with infliximab decreases plasma levels of CRP and CRP dependent complement activation products and concomitantly may reduce complement activation in RA. Complement activation may be among the effector mechanisms of TNF in RA.

Serum inflammatory proteins and cognitive decline in older persons.

OBJECTIVE: To assess whether serum levels of the inflammatory proteins alpha(1)-antichymotrypsin (ACT), C-reactive protein (CRP), interleukin-6 (IL-6), and albumin are associated with cognitive decline in older persons. METHODS: The study sample consisted of 1,284 participants in the Longitudinal Aging Study Amsterdam, aged 62 to 85 years. Cognition was assessed on general cognition (Mini-Mental State Examination [MMSE]), memory (Auditory Verbal Learning Test), fluid intelligence (Raven's Colored Progressive Matrices), and information-processing speed (Coding Task) at baseline and at 3-year follow-up. RESULTS: The highest tertile of ACT was associated with an increased risk of decline on the MMSE (age-, sex-, education-adjusted odds ratio [OR] 1.60; 95% CI: 1.05 to 2.43) but not on any other cognitive test score. CRP, IL-6, and albumin were not associated with cognitive decline on any cognitive test in our study. CONCLUSIONS: This population-based study showed that the serum inflammatory protein alpha1-antichymotrypsin is associated with cognitive decline in older persons, whereas C-reactive protein, interleukin-6, and albumin are not.

Pro- and non-coagulant forms of non-cell-bound tissue factor in vivo.

BACKGROUND: Concentrations of non-cell-bound (NCB; soluble) tissue factor (TF) are elevated in blood collecting in the pericardial cavity of patients during cardiopulmonary bypass (CPB). Previously, we reported microparticles supporting thrombin generation in such blood samples. In this study we investigated the extent of microparticle association of the NCB form of TF in pericardial and systemic blood, and whether this microparticle-associated form is active in thrombin generation compared with non-microparticle-bound, (fluid-phase) TF. METHODS: Systemic and pericardial blood samples were collected before and during CPB from six patients undergoing cardiac surgery. Microparticles were isolated by differential centrifugation and their thrombin-generating capacity measured in a chromogenic assay. Microparticle-associated and fluid-phase forms of NCB TF were measured by ELISA. Microparticle-associated TF was visualized by flow cytometry. RESULTS: In pericardial samples, 45-77% of NCB TF was microparticle-associated, and triggered factor VII (FVII)-mediated thrombin generation in vitro. Microparticles from systemic samples triggered thrombin generation independently of FVII, except at the end of bypass (P = 0.003). The fluid-phase form of TF did not initiate thrombin generation. Both forms of NCB TF were, at least in part, antigenically cryptic. CONCLUSIONS: We demonstrate the occurrence of two forms of NCB TF. One form, which is microparticle-associated, supports thrombin generation via FVII. The other form, which is fluid-phase, does not stimulate thrombin formation. We hypothesize that the microparticle-associated form of NCB TF may be actively involved in postoperative thromboembolic processes when pericardial blood is returned into the patients.

Circulating inflammatory mediators predict shock and mortality in febrile patients with microbial infection.

The host response to microbial infection is associated with the release of inflammatory mediators. We hypothesized that the type and degree of the systemic response as reflected by levels of circulating mediators predict morbidity and mortality, according to the invasiveness of microbial infection. We prospectively studied 133 medical patients with fever and culture-proven microbial infection. For 3 days after inclusion, the circulating levels of activated complement C3a, interleukin (IL)-6, and secretory phospholipase A(2) (sPLA(2)) were determined daily. Based on results of microbiological studies performed for up to 7 days, patients were classified as having local infections (Group 1, n = 80 positive local cultures or specific stains for fungal or tuberculous infections) or bacteremia (Group 2, n = 52 plus 1 patient with malaria parasitemia). Outcome was assessed as the development of septic shock and as mortality up to 28 days after inclusion. Fifteen patients (11%) developed septic shock and overall mortality was 18% (n = 24). Bacteremia was associated with shock and shock predisposed to death. Circulating mediator levels were generally higher in Group 2 than in Group 1. Circulating levels of IL-6 and sPLA(2) were higher in patients developing septic shock and in nonsurvivors, particularly in Group 1. High C3a was particularly associated with nonsurvival in Group 2. In Group 1, the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for the peak sPLA(2) for shock development was 0.79 (P < 0.05). The AUC of the ROC curve of the peak IL-6 and sPLA(2) for mortality was 0.69 and 0.68 (P < 0.05), respectively. In Group 2, the AUC of the ROC for peak C3a predicting mortality was 0.73 (P < 0.05). In conclusion, in medical patients with fever and microbial infection, the systemic inflammatory host response predicts shock and death, at an early stage, dependent on the invasiveness of microbial infection. The results suggest a differential pathogenetic role of complement activation on the one hand and release of cytokine and lipid mediators on the other in bacteremic and local microbial infections, respectively. They may partly explain the failure of strategies blocking proinflammatory cytokines or sPLA(2) in human sepsis and may extend the basis for attempts to inhibit complement activation at an early stage in patients at risk of dying from invasive microbial infections.

Increased Nox2 expression in human cardiomyocytes after acute myocardial infarction.

BACKGROUND/AIMS: Recent studies indicate the presence of reactive oxygen species (ROS) producing homologues of the enzymatic subunit (Nox2) of phagocytic NADPH oxidase in non-phagocytic cells. Interestingly, in these cells, ROS produced by the Nox2 homologue(s) was shown to play a role in various regulatory processes, including cell death, proliferation, and aging. The purpose of this study was to investigate whether human cardiomyocytes express Nox2. METHODS: The expression of Nox2 was studied in human cardiomyocytes using western blot and immunohistochemical analysis. To analyse the putative expression of Nox2 in human heart disease, cardiac samples from patients who had died subsequent to acute myocardial infarction (AMI) were studied. RESULTS: Both in western blot and immunohistochemical studies, Nox2 expression was found in normal human cardiomyocytes. In patients with AMI, a significant increase in Nox2 expression was found both in viable and in jeopardised cardiomyocytes in the infarcted area. In addition, in the "remote from infarction" area, Nox2 expression was present in cardiomyocytes, but was not increased. CONCLUSIONS: Nox2 or its homologue(s) is expressed in normal and jeopardised human cardiomyocytes. This expression is increased in patients with AMI, suggesting a role for this ROS producing Nox2 homologue(s) in the human heart after AMI.