Rheumatoid arthritis is a chronic, systemic joint disease in which an autoimmune response translates into an inflammatory attack resulting in joint damage, disability and decreased quality of life. Despite recent introduction of therapeutic agents such as anti-TNFα, even the best current therapies fail to achieve disease remission in most arthritis patients. Therefore, research into the mechanisms governing the destructive inflammatory process in rheumatoid arthritis is of great importance and may reveal novel strategies for the therapeutic interventions. To gain deeper insight into its pathogensis, we have developed for the first time a three-dimensional synovium-on-a-chip system in order to monitor the onset and progression of inflammatory synovial tissue responses. In our study, patient-derived primary synovial organoids are cultivated on a single chip platform containing embedded organic-photodetector arrays for over a week in the absence and presence of tumor-necrosis-factor. Using a label-free and non-invasive optical light-scatter biosensing strategy inflammation-induced 3D tissue-level architectural changes were already detected after two days. We demonstrate that the integration of complex human synovial organ cultures in a lab-on-a-chip provides reproducible and reliable information on how systemic stress factors affect synovial tissue architectures.

Endogenous nucleic acids and their receptors may be involved in the initiation of systemic autoimmune diseases including rheumatoid arthritis (RA). As the role of the DNA sensing Toll-like receptor (TLR) 9 in RA is unclear, we aimed to investigate its involvement in the pathogenesis of autoimmune arthritis using three different experimental models of RA. The data obtained revealed involvement of TLR9 in the T cell-dependent phase of inflammatory arthritis. In rats with pristane-induced arthritis (PIA), TLR9 inhibition before disease onset reduced arthritis significantly and almost completely abolished bone erosion. Accordingly, serum levels of IL-6, α-1-acid-glycoprotein and rheumatoid factor were reduced. Moreover, in TLR9-/- mice, streptococcal cell wall (SCW)-induced arthritis was reduced in the T cell-dependent phase, whereas T cell-independent serum-transfer arthritis was not affected. Remarkably, while TLR7 expression did not change during in vitro osteoclastogenesis, TLR9 expression was higher in precursor cells than in mature osteoclasts and partial inhibition of osteoclastogenesis was achieved only by the TLR9 antagonist. These results demonstrate a pivotal role for TLR9 in the T cell-dependent phases of inflammatory arthritis and additionally suggest some role during osteoclastogenesis. Hence, endogenous DNA seems to be crucially involved in the pathophysiology of inflammatory autoimmune arthritis.

Jaundice and cholestatic hepatic dysfunction are frequent findings in critically ill patients associated with increased mortality. Cholestasis in critically ill patients is closely associated with stimulation of pro-inflammatory cytokines resulting in impaired bile secretion and subsequent accumulation of bile acids.

Objective: The antimicrobial agent flucloxacillin is a potential cause of drug-induced liver disease, but the underlying mechanisms for toxicity have not been fully elucidated. As in-vitro and in-vivo findings suggest that biotransformation products contribute to hepatotoxicity, the purpose of this study was to characterize formation and accumulation of its metabolites in patients with renal failure. Methods: Twelve intensive care patients undergoing continuous venovenous hemofiltration received 4.0 g flucloxacillin as single and repeated infusion. Blood and dialysate samples were collected and analyzed for flucloxacillin and its metabolites by HPLC. Results: The overall amounts of the flucloxacillin metabolites 5'-hydroxymethyl-flucloxacillin (5-OH-FX), 5'-hydroxymethylflucloxacillin-penicilloic acid (5-OH-PA), and flucloxacillin-penicilloic acid (FX-PA) produced varied considerably between patients, and accounted for 3.62 -35.9% of total flucloxacillin concentration (flucloxacillin + metabolites) in the plasma. Clearance rates and sieving coefficients for 5-OH-FX and FX-PA were comparable to that of the parent drug, although removal of 5-OH-PA was decreased. Using an isolated perfused rat liver model we demonstrated that 5-OH-FX reached concentrations in the bile (240.5 +/- 84.2 nmoles/mL) that were sufficient to exert cytotoxic effects, unlike either of the two penicilloic acids. Conclusions: Based on data from perfused rat livers, high biliary concentrations of 5-OHFX might also be observed in our patients explaining why LDH, bilirubin, and alkaline phosphatase were elevated in up to 8/ 12 patients after repeated infusion of flucloxacillin. Liver toxicity of flucloxacillin might therefore be observed in patients with renal impairment after continuously elevated 5-OH-FX levels.

IntroductionOxidative stress affects clinical outcome in critically ill patients. Although high-density lipoprotein (HDL) particles generally possess anti-oxidant capacities, deleterious properties of HDL have been described in acutely ill patients. The impact of anti-oxidant HDL capacities on clinical outcome in critically ill patients is unknown. We therefore analyzed the predictive value of anti-oxidant HDL function on mortality in an unselected cohort of critically ill patients.MethodWe prospectively enrolled 270 consecutive patients admitted to a university-affiliated intensive care unit (ICU) and determined anti-oxidant HDL function using the HDL oxidant index (HOI). Based on their HOI, the study population was stratified into patients with impaired anti-oxidant HDL function and the residual study population.ResultsDuring a median follow-up time of 9.8 years (IQR: 9.2 to 10.0), 69% of patients died. Cox regression analysis revealed a significant and independent association between impaired antioxidant HDL function and short-term mortality with an adjusted HR of 1.65 (95% CI 1.22-2.24; p = 0.001) as well as 10-year mortality with an adj. HR of 1.19 (95% CI 1.02-1.40; p = 0.032) when compared to the residual study population. Anti-oxidant HDL function correlated with the amount of oxidative stress as determined by Cu/Zn superoxide dismutase (r = 0.38; p < 0.001).ConclusionImpaired anti-oxidant HDL function represents a strong and independent predictor of 30-day mortality as well as long-term mortality in critically ill patients.

OBJECTIVES:Daptomycin is bactericidal against Gram-positive bacteria, with peak-dependent effect but trough-dependent toxicity. This study was performed to develop dosing recommendations in continuous venovenous haemodiafiltration (CVVHDF).PATIENTS AND METHODS:Nine critically ill patients in intensive care units of the Medical University Hospital of Vienna, requiring CVVHDF due to acute renal failure and antimicrobial treatment, were included. Blood and effluent samples were collected over 72 h to determine daptomycin concentrations by HPLC. Pharmacokinetic parameters were based on 10 sampling timepoints during the first 24 h, and peak and trough samples thereafter. An open two-compartment model was fitted to each subject's plasma concentration-time data. Simulations of serum concentration-time profiles after different doses and intervals were performed using ADAPT 5.RESULTS:Peak plasma concentrations with 6 mg/kg daptomycin were 62.2 ± 16.2, 66.1 ± 17.3 and 78.5 ± 22.1 mg/L on days 1, 2 and 3, respectively. The total clearance was 6.1 ± 4.9 mL/min, and the elimination half-life was 17.8 ± 9.7 h. Daptomycin was filtrated and could therefore be measured in the effluent. Protein binding was lower than that seen in healthy volunteers. The unbound fraction was 16 ± 4.5%. All subjects maintained trough serum concentrations above 4 mg/L, at which relevant pathogens are considered daptomycin-susceptible. Accumulation resulted when daptomycin was given every 24 h. Simulation of 8 mg/kg daptomycin given every 48 h resulted in adequate levels without accumulation.CONCLUSIONS:We recommend 8 mg/kg daptomycin every 48 h in patients on CVVHDF and therapeutic drug monitoring, if possible.

BACKGROUND:Clinical studies support a role for anidulafungin as first-line treatment of invasive candidiasis in critically ill patients and postulate no need for dose adjustments in mild to severe renal failure. Although intensive care patients requiring renal replacement therapy are at particular risk of invasive fungal infection, no pharmacokinetic data on anidulafungin during continuous venovenous haemofiltration (CVVHF) are available.PATIENTS AND METHODS:Ten patients with CVVHF due to acute renal failure were included. Anidulafungin was infused on 3 consecutive days starting with a loading dose of 200 mg on day 1, followed by doses of 100 mg on each of days 2 and 3. During the 72 h study phase of CVVHF, blood and ultrafiltrate samples were collected at corresponding times. Anidulafungin concentrations were determined by HPLC.RESULTS:Peak plasma concentrations were reached 3 h after the start of infusion and were 8.5±3.6 mg/L at the pre-filter port. The mean arterial area under the curve (AUC0-24) of the study population was 109.9±49.82 mg·h/L, the total clearance was 1.08±0.41 L/h, the volume of distribution was 41.97±22.64 L and the elimination half-life was 28.78±10.40 h. Anidulafungin was not filtered, but CVVHF resulted in a substance loss of ∼20%, due to adherence to synthetic surfaces.CONCLUSIONS:Pharmacokinetics of anidulafungin during CVVHF resembled findings in healthy adults and adults with fungal infections. Therefore we recommend a loading dose of 200 mg intravenous anidulafungin on the first day and 100 mg on consecutive treatment days in patients during CVVHF.

Autoimmune responses to heterogeneous nuclear ribonucleproteins (hnRNP) occur in many systemic autoimmune diseases, particularly in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus. In RA, humoral and/or cellular autoimmunity to hnRNP-A2/B1 is the most prominent anti-nuclear reactivity, being detectable in more than 50% of patients. However, its pathogenic role has not been fully elucidated yet. Here, we report that splenocytes from rats with pristane-induced arthritis transfer disease after in vitro restimulation with hnRNP-A/B antigens. Remarkably, disease transfer can be blocked by nuclease treatment of hnRNPs and is also achieved with splenocytes stimulated with hnRNP-A/B associated DNA or RNA oligonucleotides (ON) alone. Induction of proinflammatory cytokines in splenocytes stimulated with hnRNP-A/Bs or ONs involves Toll-like receptors (TLR) 7 and 9 but not TLR3. Furthermore, although T cells are the main mediators of disease transfer they require restimulation with TLR-activated antigen-presenting cells such as macrophages in order to become arthritogenic. Thus, the autoantigenic properties of hnRNPs appear to be mediated by their associated nucleic acids binding to TLR7 and 9. Our data explain the specific selection of hnRNP-A2/B1 as autoantigen in RA and reveal the requirement of interaction between innate and adaptive immunity to initiate and drive inflammation in autoimmune arthritis.

BACKGROUND:Pulmonary endarterectomy (PEA) provides a potential cure for patients with chronic thromboembolic pulmonary hypertension (CTEPH). However, successfully operated patients can continue to suffer from a limitation of exercise capacity, despite normalization of pulmonary vascular resistance (PVR). The purpose of the present study was to explore the cardiopulmonary exercise test (CPET) profile and the pulmonary hemodynamic response to exercise in these patients. METHODS:Thirteen successfully operated patients with CTEPH and persistent dyspnea and control subjects underwent a CPET and a right-sided heart catheterization at rest and during exercise. RESULTS:The CPET profile of the patients was characterized by mild hyperventilation and decreased peak oxygen uptake (VO2). While there were no differences in resting hemodynamics between patients and control subjects, PVR was higher in the patients after 10 min of exercise (111 ± 46 dynes/s/cm(5) vs 71 ± 42 dynes/s/cm(5), P = .04), and pulmonary arterial compliance (Ca) was lower (5.5 ± 2.3 mL/mm Hg vs 8.1 ± 3.5 mL/mm Hg, P = .048). Ca under exercise correlated with peak VO2 in the patients (R(2) = 0.825, P = .022). CONCLUSIONS:After successful PEA, patients with persistent exertional dyspnea display an abnormal pulmonary hemodynamic response to exercise, characterized by increased PVR and decreased Ca. Decreased Ca under exercise is a strong predictor of limited exercise capacity in these patients.

The heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) has been described as an important autoantigen in rheumatoid arthritis (RA) since it is targeted by autoantibodies, autoreactive T cells, and is aberrantly expressed in synovial cells in patients. To identify hnRNP-A2-specific T-cell epitopes possibly associated with pathogenicity, we used an innovative approach. We first scanned 280 overlapping hnRNP-A2 peptides for binding to the RA-associated class II molecules HLA-DR4 and HLA-DR1, leading to a comprehensive selection of binders. The selected peptides were tested in IFN-gamma-specific ELISPOT assay: PBMC from 18% of RA patients showed a significant IFN-gamma response to hnRNP-A2 peptides, 15% to the overlapping sequences 117-133 and/or 120-133, whereas PBMC from healthy individuals tested negative. We measured proliferative responses to these two peptides in another cohort of patients with RA or osteoarthritis: positive responses were found in 28% of RA, but also in 11% of osteoarthritis patients and these responses could be blocked by anti-MHC class II Ab. Remarkably, the presence of 117/120-133-specific T cells was significantly associated with active disease in RA patients, and bone erosion appeared to be more common in T-cell positive patients. These data suggest involvement of hnRNP-A2 specific cellular autoimmune responses in RA pathogenesis.

Summary We hypothesized that restenosis after coronary stenting is predicted by elevated levels of markers of thrombus formation and inflammation. Plasma levels of representative markers of inflammation, the thrombin and plasmin activation systems and adhesion molecules were measured in 59 patients with stable angina pectoris before, immediately after and 6 hours (h), 12 h, 24 h, one month and six months after elective stent implantation (radioactive phosphorus-32 stents /RSs/ n=16,bare-metal stents /BMSs/ n=43). All patients underwent clinical and angiographic follow-up (FUP) six months after stenting. RSs had significantly higher angiographic severity of restenosis than BMSs (47.1 ± 20.1% vs. 27.6 ± 22.0%, p=0.003). Repeated measures ANOVA revealed significant differences between the BMS and RS groups as regards the increases in plasma levels of vascular cell adhesion molecule-1 (VCAM-1, p=0.022), plasminogen activator inhibitor-1 (PAI-1, p=0.047), tissue-type plasminogen activator (tPA, p=0.047) and CD40 ligand (CD40L, p=0.038). tPA levels tended to increase immediately after stenting in both groups, whereas the PAI-1 level one month after stenting was elevated significantly only in the RS group. In the RS group, the plasma levels of CD40L were increased at 24 h and six months after stenting, and the VCAM-1 level rose immediately after stenting and remained high during the FUP. Multivariate analysis on pooled laboratory data of both groups revealed elevated levels of VCAM-1 at 12 h and at six months as significant predictors of the severity of stent restenosis. In conclusion, the process of inflammation and thrombosis occurring after coronary interventions seems to be prolonged and enhanced in patients with highgrade restenosis at the follow up.

OBJECTIVES:Natriuretic peptides emerged during recent years as potent prognostic markers in patients with heart failure and acute myocardial infarction. In addition, natriuretic peptides show strong predictive value in patients with pulmonary embolism, sepsis, renal failure, and shock. The present study tests the prognostic information of N-terminal pro-B-type natriuretic peptide (NT-pro-BNP) in an unselected cohort of critically ill patients. DESIGN:Prospective, observational study. SETTING:A tertiary intensive care unit in a university hospital. PATIENTS:A total of 289 consecutive patients admitted to the intensive care unit during a 16-month period with the following data: age 64 +/- 14 yrs, male n = 191, Simplified Acute Physiology Score II of 52 +/- 24, mechanical ventilation n = 180 (62%), vasopressors n = 179 (62%), renal failure n = 24 (8%). INTERVENTIONS:None. MEASUREMENTS AND MAIN RESULTS:Plasma NT-pro-BNP samples (Roche Diagnostics) were obtained on intensive care unit admission. Data are given as median [range]. Intensive care unit survivors had significantly lower NT-pro-BNP values compared with intensive care unit nonsurvivors (3394 [24-35,000] vs. 6776 [303-35,000] pg/mL, survivors vs. nonsurvivors, respectively, p = .001). Hospital survivors were characterized by significantly lower NT-pro-BNP values (2656 [24-35,000] vs. 8390 [303-35,000] pg/mL, survivors vs. nonsurvivors, respectively, p = .001). NT-pro-BNP levels were not significantly different in patients with primary cardiac diagnosis compared with those with a noncardiac admission diagnosis (4794 [26-35,000], n = 202 vs. 3349 [24-35,000], n = 87, cardiac vs. noncardiac, respectively, p = .28). In a logistic regression model, Simplified Acute Physiology Score II and NT-pro-BNP were independently associated with hospital survival (chi = 35.6, p = .0001 and chi = 11.3, p = .0008, Simplified Acute Physiology Score II and NT-pro-BNP, respectively). Areas under the receiver operating characteristic curves of NT-pro-BNP and Simplified Acute Physiology Score II were not statistically significant different regarding the prediction of outcome. CONCLUSIONS:NT-pro-BNP on admission is an independent prognostic marker of outcome in an unselected cohort of critically ill patients. A single measurement of NT-pro-BNP might facilitate triage of emergency and intensive care unit patients.