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BackgroundIn a subset of patients, anti tumour necrosis factor (TNF) therapeutic antibodies are immunogenic, resulting in the formation of antidrug antibodies (ADAs). Neutralising ADAs compete with TNF for its binding site and reduces the effective serum concentration, causing clinical non-response. It is however unknown to which extent ADAs are neutralising.ObjectivesTo study which proportion of antibodies to human(ised) anti-TNF (adalimumab, golimumab, certolizumab) as well as chimeric anti-TNF (infliximab) is neutralising.MethodsNeutralising capacity of ADAs was assessed using a TNF competition assay in ADA-positive sera of patients treated with adalimumab (n=21), golimumab (n=4), certolizumab (n=9) or infliximab (n=34) sent in to our diagnostic department.ResultsIn 34 sera with ADAs to adalimumab, golimumab or certolizumab, >97% of the antibodies were neutralising. In 34 sera with ADAs to infliximab >90% of the antibodies were neutralising. Further characterisation of the broader antibody response to infliximab revealed that non-neutralising antibodies to infliximab do not target murine domains, but may bind infliximab-unique domains not involved in TNF binding (located outside the paratope).ConclusionsOur study shows that ADAs to human(ised) as well as chimeric anti-TNF therapeutic antibodies are largely neutralising. This highly restricted ADA response suggests an immunodominant role for the paratope of anti-TNF therapeutics.

Clinical remission is today the treatment goal for rheumatoid arthritis (RA), which requires fast and assertive therapeutic decisions for a tight control of disease activity. Few objective parameters are available to guide clinical decisions, particularly in switcher patients. We designed a preliminary algorithm introducing immunogenicity assessment in the current approach to patients with RA receiving tumour necrosis factor inhibitors (TNFi).To evaluate the concordance between the new algorithm and current clinical practice, comparing the effectiveness of 'immunogenicity-based' versus 'empirical-based' switches in a cohort of patients with established RA receiving biologics.EULAR therapeutic response was evaluated in 105 patients with RA (naive or switchers) over one year, through generalised estimation equation (GEE) analyses. Serum drug trough levels were assessed by ELISA and antidrug antibodies (ADAb) by Bridging ELISA.During follow-up, 48.6% of patients had therapeutic decisions concordant with the proposed algorithm (Group A), and 51.4% had discordant decisions (Group B). One year after the therapeutic decision, patients from Group A had a higher probability of achieving response (OR=7.91, p0.001, 95% CI 3.27 to 19.13) and low disease activity (OR=9.77, p0.001, 95% CI 4.69 to 20.37) than patients in Group B.Immunogenicity assessment might help to optimise therapeutic decisions, leading to a better control of disease activity with significantly better clinical outcomes in patients with RA receiving TNFi.

Therapeutic monoclonal antibodies are effective drugs for many different diseases. However, the formation of anti-drug antibodies (ADA) against a biological can result in reduced clinical response in some patients. Measurement of ADA in the presence of (high) drug levels is difficult due to drug interference in most assays, including the commonly used antigen binding test (ABT). We recently published a novel method which enables the measurement of complexed antibodies against adalimumab (an anti-TNF antibody) in the presence of drug. Here we use this pH-shift-anti-idiotype ABT (PIA) to measure anti-adalimumab antibodies (AAA) in 99 rheumatoid arthritis (RA) patients treated for up to 3 years with adalimumab. 53 out of 99 RA patients produced AAA. In 50 of these PIA positive patients, AAA could be detected within the first 28 weeks of treatment. Patients in which AAA could be detected in the PIA after 28 weeks of treatment were more prone to declining adalimumab levels (

Human immunoglobulin G4 (IgG4) is a poor trigger of effector functions and, therefore, is the preferred subclass for therapeutic monoclonal antibodies that merely aim to block their in vivo targets. An example is natalizumab, a recombinant IgG4 antibody directed against alpha 4-integrin and used for treatment of multiple sclerosis. Efficient treatment requires that the pharmacokinetics of therapeutic monoclonal antibodies can be accurately monitored. For natalizumab, this requires special precautions due to recently reported structural peculiarities of human IgG4. Here we describe the development of an assay to determine serum levels of natalizumab. Compared with other IgG subclasses, human IgG4 possesses unique structural properties that influence its interactions in both in vivo and in vitro settings. Thus. IgG4 undergoes Fab arm exchange in vivo, resulting in effectively monovalent antibodies. Furthermore, IgG4 is able to bind to other human and nonhuman IgG via Fc interactions. We demonstrate how these features can interfere with measurement of specific IgG4 and describe how we addressed these issues, resulting in an assay that is not sensitive to Fab arm exchange by natalizumab or to IgG4 Fc interactions. (C) 2011 Elsevier Inc. All rights reserved

Adjuvant therapy with interferon-alpha (IFN) only benefits a small subgroup of melanoma patients and a predictive marker selecting responders does not exist. IFN induces increased ferritin and decreased C-reactive protein (CRP) levels; however, an association with treatment effect was not studied. Serum was collected from patients participating in the European Organization for Research and Treatment of Cancer 18 952 trial comparing adjuvant treatment with IFN to observation. Serial ferritin and CRP levels were determined using enzyme-linked immusorbent assays, before treatment and up to 24 months. Ferritin levels are influenced by sex and age; therefore ratios of serial ferritin and CRP values with corresponding pretreatment values were calculated. Cox regression model and landmark method at end of induction and 6 months were used to evaluate the association between ferritin, CRP and distant metastasis-free survival (DMFS). Baseline ferritin levels were comparable in the two treatment groups (P = 0.92). However, ferritin ratios were significantly higher in IFN-treated patients (N = 96) compared with untreated patients (N = 21) at end of induction (mean: 2.88 vs. 0.75; P = 0.0003) and at 6 months (mean: 3.18 vs. 1.02; P = 0.009). In the IFN arm, higher ferritin ratios at end of induction and at 6 months were not associated with improved outcome (respectively, P = 0.66 and 0.86). Concerning CRP ratios, no differences between the treatment groups, neither an association with DMFS, were observed. Administration of IFN in melanoma patients induced increase in ferritin levels but not in CRP levels. Ferritin and CRP ratios have no prognostic value regarding DMFS. Melanoma Res 21:344-351 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Eur J Clin Invest 2010; 40 (10): 893–902 Abstract Background Clusterin (Apolipoprotein J), a plasma protein with cytoprotective and complement-inhibiting activities, localizes in the infarcted heart during myocardial infarction (MI). Recently, we have shown a protective effect of exogenous clusterin in vitro on ischaemically challenged cardiomyocytes independent of complement. We therefore hypothesized that intravenous clusterin administration would reduce myocardial infarction damage. Methods Wistar rats undergoing experimental MI, induced by 40 min ligation of a coronary vessel, were treated with either clusterin (n = 15) or vehicle (n = 13) intravenously, for 3 days post-MI. After 4 weeks, hearts were analysed. The putative role of megalin, a clusterin receptor, was also studied. Results Administration of human clusterin significantly reduced both infarct size (with 75 ± 5%) and death of animals (23% vehicle group vs. 0% clusterin group). Importantly, histochemical analysis showed no signs of impaired wound healing in the clusterin group. In addition, significantly increased numbers of macrophages were found in the clusterin group. We also found that the clusterin receptor megalin was present on cardiomyocytes in vitro which, however, was not influenced by ischaemia. Human clusterin co-localized with this receptor in vitro, but not in the human heart. In addition, using a megalin inhibitor, we found that clusterin did not exert its protective effect on cardiomyocytes through megalin. Conclusions Our results thus show that clusterin has a protective effect on cardiomyocytes after acute myocardial infarction in vivo, independent of its receptor megalin. This indicates that clusterin, or a clusterin derivate, is a potential therapeutic agent in the treatment of MI.

Production of anti drug antibodies (ADA) in adalimumab treated RA patients is associated with reduced serum adalimumab levels and less clinical response. However, most current assays to measure ADA are unable to detect ADA in complex with adalimumab. Thus, ADA is only measured if antibody production exceeds drug levels in the serum, meaning that ADA formation is underestimated. The aim of this study is to develop a method to detect ADA in the presence of drug. A pH-shift-anti-idiotype Antigen binding test (PIA) was used to enable ADA measurement in the presence of adalimumab. ADA-adalimumab complexes were dissociated by acid treatment and addition of excess rabbit anti-idiotype-F(ab) before neutralization. Rabbit anti-idiotype-F(ab) blocks reformation of ADA-drug complexes by competing with patient ADA for adalimumab binding. Released ADA are measured by an antigen binding test (ABT). The PIA enabled detection of ADA in the presence of large excess of adalimumab and was used to measure ADA in 30 adalimumab treated rheumatoid arthritis (RA) patients during the first 28 weeks of treatment. It revealed ADA in 21 out of 30 tested patients, while the ABT detected ADA in only 5 patients. Indicating that an immunogenic reaction towards adalimumab is present in the majority of adalimumab treated patients. (C) 2010 Elsevier B.V. All rights reserved

OBJECTIVE: To investigate how antibodies against anti-tumour necrosis factor (anti-TNF) agents influence response after switching from infliximab to adalimumab in rheumatoid arthritis (RA). METHODS: This cohort study consisted of 235 patients with RA, all treated with adalimumab. At baseline 52 patients (22%) had been previously treated with infliximab ('switchers'), and 183 (78%) were anti-TNF naive. Disease activity (using the 28-joint count Disease Activity Score (DAS28)) and presence of antibodies against infliximab and adalimumab were assessed. Clinical response to adalimumab was compared between switchers and anti-TNF naive patients and their anti-infliximab and anti-adalimumab antibody status. RESULTS: After 28 weeks of adalimumab treatment the decrease in DAS28 (Delta DAS28) for the 235 patients was 1.6+/-1.5 (mean+/-SD). Anti-adalimumab antibodies were detected in 46 patients (20%). Delta DAS28 was 1.8+/-1.4 in patients without anti-adalimumab and 0.6+/-1.3 in patients with anti-adalimumab (p

Objective Treatment of patients with infliximab, a chimeric monoclonal IgG1 antibody against tumor necrosis factor, may result in the formation of infliximab-specific IgG antibodies. This study evaluated the clinical significance of these antibodies in patients with rheumatoid arthritis (RA). Methods Antiinfliximab antibodies were measured using a newly developed radioimmunoassay in a cohort of 51 consecutive patients with RA treated with infliximab, with a followup of 1 year. In addition, serum infliximab levels were determined by enzyme-linked immunosorbent assay. The results were analyzed in relation to the clinical response to treatment according to the European League Against Rheumatism criteria. Results Antibodies against infliximab were detected in 22 patients (43%). Patients without detectable antiinfliximab antibodies (n = 29 [57%]) were significantly more often classified as responders (20 of 29 [69%]) compared with patients with detectable antiinfliximab antibodies (8 of 22 [36%]; P = 0.04). Three patients had an infusion-related allergic reaction, all of whom had detectable antiinfliximab antibodies. Conclusion In this study, nearly half of the RA patients treated with infliximab developed antiinfliximab antibodies within the first year of treatment. This seems to be clinically relevant, since development of antiinfliximab antibodies is associated with a reduced response to treatment.

Neonates are highly susceptible to diseases and display biased type 2 immune responses, although no skewing to type 2 cytokines has been reported. In view of the emerging importance of IL-13 in type 2 inflammatory responses and clinical allergy, we analyzed IL-13 production by neonatal T cells. We found that, mainly CD8 T cells produced high levels of IL-13, while producing low levels of IL-4, IL-10 and IFN-gamma, upon primary and secondary stimulation. Our results point towards a possible immunoregulatory role of CD8 T cells in neonate responses. Moreover, they suggest that the abundance of IL-13 in the neonate immune system might account for the type 2 bias in neonates, providing a basis for the high disease susceptibility of newborns, for instance to allergic diseases.

OBJECTIVE—Plasma concentrations of soluble CD95 (sCD95) are raised in patients with systemic lupus erythematosus (SLE) before clinical relapses become manifest. Increased sCD95 concentrations may therefore be a familial characteristic that is associated with susceptibility to severe disease. To test this, sCD95 concentrations were measured in healthy first degree relatives of patients with severe and non-severe SLE. METHODS—Seventy seven first degree relatives of 26 patients with severe, and 72 relatives of 25 patients with non-severe lupus were studied. Controls were 42 first degree relatives of 17 patients with chronic cutaneous lupus erythematosus (CCLE) and 63 partners of the patients with their first degree relatives. Severe lupus was defined as both multi-organ disease and cyclophosphamide treatment, non-severe lupus as neither. Organ damage was assessed with the SLICC-ACR index, disease activity with SLEDAI. RESULTS—Soluble CD95 concentrations in relatives of patients with severe SLE were similar to those in relatives of patients with non-severe SLE, relatives of patients with CCLE, and controls (median (interquartile range) sCD95 concentration 0.59 (0.52-0.66) v 0.57 (0.50-0.63), 0.56 (0.51-0.71), and 0.55 (0.49-0.61) ng/ml, p=0.25, p=0.94, and p=0.17, respectively). Increased concentrations of sCD95, however, were found in patients with severe SLE compared with those in patients with non-severe SLE, patients with CCLE, and control relatives (0.77 (0.70-0.97) v 0.60 (0.54-0.67), 0.57 (0.54-0.71), and 0.57 (0.52-0.63) ng/ml, respectively, p

Graft-versus-host disease (GVHD) and infections are two major complications of allogeneic bone marrow transplantation (BMT). In the course of GVHD, one of the pathways that activated cytotoxic T cells use to execute their killing mechanisms is the Fas/Fas ligand pathway. This killing mechanism might be accompanied by the release of soluble Fas (sFas) in the circulation. To examine the association of serum sFas levels and post-BMT complications, we have analyzed sFas levels in sera of bone marrow recipients with and without GVHD. Postallogeneic BMT sFas levels were significantly increased during clinically relevant acute GVHD (aGVHD; P = .002). However, during infections sFas levels tended to decrease (P = .088). Yet, the simultaneous occurrence of GVHD and infections resulted in extreme high sFas levels. These results suggested that sFas release may be correlated with the amount of tissue damage, because aGVHD induces more damage than infections. The presence of significantly increased sFas levels during aGVHD provides new insights into the GVHD pathogenesis.

Interleukin-6 (IL6) plays a major role in the pathogenesis of multiple myeloma. In patients with monoclonal gammopathy serum levels of sIL6R have been found to be increased. The role of IL6 in the regulation of soluble receptors is still unclear. In a phase I/II study we treated 12 myeloma patients with high-affinity chimeric anti-IL6 monoclonal antibodies. This treatment resulted in a total in vivo blockage of IL6 activity and as a result we had an unique opportunity to gain insight into the possible regulation effects of IL6 on these soluble IL6 receptors. Pre-treatment sIL6R levels were elevated in 9 of the 12 patients; pre-treatment sgp130 levels were significantly increased in all patients. Total blockage of IL6 activity by the high-affinity cMab did not influence sIL6R in 10 of these 12 patients and sgp130 levels remained stable in all patients. Of the 2 patients whose sIL6R levels increased during therapy, one had progressive disease and the other developed an acute infection. We conclude that in most end-stage myeloma patients sIL6R and sgp130 serum levels are elevated, but that there is no relation between IL6 activity and sIL6R or sgp130 levels.

Objective To determine a concentration-effect curve of adalimumab in rheumatoid arthritis (RA) patients taking into account the effect of methotrexate (MTX) on concentration and effect and to identify a therapeutic range for adalimumab concentrations. Methods In a prospective observational cohort study, 221 consecutive patients with RA were treated with 40 mg adalimumab subcutaneously every other week. The relationship between adalimumab trough level and clinical efficacy after 28 weeks of follow-up was determined in a concentration-effect curve. A receiver-operator characteristics (ROC) curve established a therapeutic cut-off concentration. The effect of MTX on adalimumab trough levels was shown by dividing patients that are and are not concomitantly using MTX in the concentration-effect curve and a concentration table. Results Clinical efficacy improved with increasing adalimumab concentration and reached a maximum (mean disease activity score in 28 joints improvement of 2) with levels between 5-8 mu g/mL. Levels exceeding 8 mu g/mL were illustrated to have no additional beneficial effect on disease activity. The ROC curve showed an area under the curve of 0.695 (95% CI 0.626 to 0.764) for European League Against Rheumatism response and adalimumab levels: good responders versus non-responders and moderate responders. A cut-off of 5 mu g/mL had a sensitivity of 91% and a specificity of 43%. Adalimumab levels are influenced by concomitant MTX use: patients on adalimumab monotherapy had a median adalimumab level of 4.1 mu g/mL (IQR 1.3-7.7), whereas patients concomitantly taking MTX had a median level of 7.4 mu g/mL (IQR 5.3-10.6, p

Objectives Millions of patients worldwide are treated with therapeutic monoclonal antibodies. These biological therapeutics can be immunogenic, resulting in anti-drug antibody formation which leads to loss of response. Fully human biological agents, such as the anti-tumour necrosis factor α (anti-TNFα) antibody adalimumab, are considered to be weakly immunogenic, but anti-adalimumab antibodies (AAA) were recently detected in more than half of treated patients with rheumatoid arthritis (RA) within 28 weeks of treatment. A study was undertaken to determine the mechanism by which AAA lead to loss of response. Methods The specificity of the repertoire of AAA was investigated in a cohort of 50 AAA-positive RA patients. Inhibition experiments using TNFα and patient-derived anti-adalimumab monoclonal antibodies were performed. Results The antibody response against adalimumab is highly restricted: Fab fragments of a single monoclonal antibody specific for the idiotype of adalimumab inhibited 98.65% (25th–75th percentiles: 98.25–99.90) of the total anti-adalimumab reactivity in serum from 50 AAA-positive patients. The anti-adalimumab response was confined to the TNFα binding region of adalimumab, thereby neutralising its therapeutic efficacy. In line with this restricted specificity, small immune complexes were found in the circulation of AAA-forming patients. Conclusions The humoral immune response against adalimumab is highly restricted and limited to the idiotype of the therapeutic antibody. All antibodies result in functional neutralisation of the drug, thereby providing a mechanism by which AAA formation leads to clinical non-response.

A substantial part of rheumatoid arthritis (RA) patients is chronically treated with adalimumab. Some of these patients produce antibodies against adalimumab, which correlate with lower serum drug levels and reduced clinical response. Long term exposure to antigens may result in antigen specific IgG4 production as was demonstrated in studies on prolonged exposure to antigens such as different allergens, Factor VIII and IFN-beta. Here, we investigate whether long term treatment of RA patients with the therapeutic monoclonal antibody adalimumab leads to the production of specific IgG4 antibodies. We developed radio immunoassays to detect total IgG or IgG4 against adalimumab and applied these in a cohort of 271 consecutive RA patients during 3 years of adalimumab treatment. In 32 % of the 271 patients antibodies against adalimumab were detectable. IgG4 antibodies were detected in 29 % of the patients. The proportion IgG4 of total IgG against adalimumab varies widely between patients, and IgG4 was found to contribute significantly to the anti drug antibody (ADA) response in some patients. In the immune response against adalimumab in adalimumab-treated RA patients a considerable part of the ADA is IgG4. Although IgG4 is often considered to be harmless due to its lack of effector function, neutralization of adalimumab by IgG4 antibodies will lead to a reduced clinical response

Objective: To investigate the effects of various therapeutic modalities used to treat inflammatory bowel disease on the acute phase response in experimental colitis. Design and method: In an acetic acid model of acute experimental colitis in rats, histological assessment was carried out to determine the degree of inflammation in all study groups. Rats were pre-treated with either 5-aminosalicylic acid (5-ASA), betamethasone, zileuton (a 5-lipoxygenase inhibitor), or saline. Serum levels of endotoxin, interleukin-6 and alpha-2-macroglobulin were measured at various time points. Results: A 1 ml 4% acetic acid enema induced moderate to severe colitis in all rats. Plasma endotoxin could not be detected

Context Short-term data on the immunogenicity of monoclonal antibodies showed associations between the development of antidrug antibodies and diminished serum drug levels, and a diminished treatment response. Little is known about the clinical relevance of antidrug antibodies against these drugs during long-term follow-up. Objective To examine the course of antidrug antibody formation against fully human monoclonal antibody adalimumab and its clinical relevance during long-term (3- year) follow-up of patients with rheumatoid arthritis (RA). Design, Setting, and Patients Prospective cohort study February 2004- September 2008; end of follow-up was September 2010. All 272 patients were diagnosed with RA and started treatment with adalimumab in an outpatient clinic. Main Outcome Measures Disease activity was monitored and trough serum samples were obtained at baseline and 8 time points to 156 weeks. Serum adalimumab concentrations and antiadalimumab antibody titers were determined after follow-up. Treatment discontinuation, minimal disease activity, and clinical remission were compared for patients with and without antiadalimumab antibodies. Results After 3 years, 76 of 272 patients (28%) developed antiadalimumab antibodies- 51 of these (67%) during the first 28 weeks of treatment. Patients without antiadalimumab antibodies had much higher adalimumab concentrations (median, 12 mg/L; IQR, 9-16 mg/L) compared with patients with antibody titers from 13 to 100 AU/mL (median, 5 mg/L; IQR, 3-9 mg/L; regression coefficient, -4.5; 95% CI, -6.0 to -2.9; P

The presence of anti-drug antibodies (ADA) in adalimumab-treated patients is associated with reduced serum adalimumab levels and a lower clinical response. Currently, there is no standard for measurement of anti-drug antibodies and many factors influence the results. Consequently, the incidence of ADA as reported in different studies varies considerably. Here we investigated the differential effect of drug interference in two common types of assays used to measure anti-adalimumab: an antigen binding test (ABT) and a more often-used bridging elisa. We measured ADA to adalimumab in a cohort of 216 rheumatoid arthritis patients treated with adalimumab for 28 weeks. Only 15 samples (7%) were positive in the bridging elisa, compared to 29 (13%) in the ABT, despite the fact that the bridging elisa was the most sensitive assay. Furthermore, in an ABT specific for IgG4, 48 samples (22%) were found positive. The bridging elisa was found to detect only the bivalent form of (drug-specific) IgG4, resulting in an underestimation of ADA levels. However, the predominant reason for the different outcomes of these assays was a differential susceptibility to drug interference. In particular, the bridging elisa only detected ADA in the absence of detectable amounts of circulating adalimumab and is therefore not suited for measurement of ADA in complex with the drug. In summary, we showed that a bridging elisa is susceptible to drug interference and typically measures ADA only in absence of detectable drug levels. (C) 2011 Elsevier B.V. All rights reserved

IL-8 was measured in knee joint synovial fluid of 60 patients with rheumatoid arthritis, 8 with gout, 6 with osteoarthritis and 4 with meniscus lesions. IL-8 could be demonstrated in most SF samples. The highest levels were observed in rheumatoid joint effusions, yet mean levels were not significantly different between the different subgroups (mean +/- SE; RA 1537 +/- 3049 pg/ml, gout 570 +/- 952 pg/ml, OA/ML 178 +/- 188 pg/ml). In RA patients, IL-8 levels could not be related to various serological, clinical or radiological parameters. However, a correlation was observed between SF levels of IL-8 with those of lactate, LDH, beta 2-microglobulin and glucose. These observations suggest that next to the laboratory parameters IL-8 will be a parameter of the activity of the local inflammatory process. The results also demonstrate that IL-8 is not a disease-specific marker of joint inflammation.

Objective The aim of this study was to test the hypothesis that the reason for non-response (caused by immunogenicity or not) to a fi rst tumour necrosis factor (TNF) inhibitor defi nes whether a second TNF inhibitor will be effective. Methods This cohort study consisted of 292 consecutive patients with rheumatoid arthritis (RA), all treated with etanercept. A total of 89 patients (30%) were treated previously with infl iximab or adalimumab (‘switchers’), and the remaining 203 (70%) were anti-TNF naive. All switchers were divided into two groups: with and without antibodies against the previous biological. Differences in clinical response to etanercept between switchers with and without antibodies and patients who were anti-TNF naive were assessed after 28 weeks of treatment using changes in Disease Activity Score in 28 joints (DAS28). Results After 28 weeks of treatment, response to etanercept did not differ between patients who were anti-TNF naive and switchers with anti-drug antibodies (ΔDAS28=2.1±1.3 vs ΔDAS28=2.0±1.3; p=0.743). In contrast, switchers without anti-drug antibodies had a diminished response to etanercept treatment compared to patients who were TNF naive (ΔDAS28=1.2±1.3 vs ΔDAS28=2.1±1.3; p=0.001) and switchers with antibodies (ΔDAS28=1.2±1.3 vs ΔDAS28=2.0±1.3; p=0.017). Conclusion Patients with RA with an immunogenic response against a fi rst TNF-blocking agent had a better clinical response to a subsequent TNF blocker compared to patients with RA without anti-drug antibodies. Hence, determining immunogenicity can be helpful in deciding in which patient switching could be benefi cial and can be part of a personalised treatment regimen.

BACKGROUND: In this study, we investigated the pharmacokinetics of rituximab in patients with CD20 positive non-Hodgkin lymphoma, to get more insight into the factors that influence the pharmacokinetics of rituximab. This may aid to understand variability of treatment outcome, in patients with a CD20 positive malignancy treated with rituximab. METHODS: In this study, patients with a CD20 positive B-cell malignancy who were treated with rituximab containing regimens were included. Induction treatment schedules consisted of a combination of rituximab with chemotherapy for 4-8 cycles. Maintenance treatment consisted of a 2 or 3-monthly dose of 375 mg/m2 rituximab intravenously for 2 years. On the day of the treatment with rituximab, preinfusion blood samples were taken. Also, after the end of treatment, selected blood samples were taken. Rituximab levels were measured with a validated enzyme-linked immunosorbent assay (ELISA). An antigen binding assay was applied for determination of human-antibodies against chimeric-antibodies (HACAs). RESULTS: Eight patients were on induction therapy. Rituximab levels of one patient on induction therapy remained very low after the first course. This patient had a chronic lymphoid leukemia with circulating tumor cells and a high tumor burden. Apart from one patient with mantle cell lymphoma, all patients on induction therapy had a complete response. Five patients were on maintenance therapy. Trough levels of 4 patients on three-monthly schedule maintenance therapy remained constant, with a median concentration of 6 mu g/mL (range 0.5-11.7 microg/mL). One patient had a relapse during his maintenance treatment. The elimination half-life at steady state of rituximab in all patients was estimated to be 19.2 (+/- 15.2%) days with a between-subject variability of 54%, indicating wide variability. Possible pharmacokinetic-pharmacodynamic relationship was observed as rituximab levels of the non-responders remained low compared to the rituximab levels of the responders. For all patients, concentration of HACAs remained below the quantification limit. SUMMARY/CONCLUSION: Considerable inter-individual variability of rituximab levels was observed. Although the patient population was small, the results support the need for more research into the pharmacokinetics and factors that might influence the pharmacokinetic-pharmacodynamic relationships of rituximab in patients with non-Hodgkin lymphoma.

To study the local immunological effects of intravesical bacillus Calmette-Guérin (BCG) therapy in superficial bladder cancer patients, the production of interleukin-1 (IL-1), IL-2, IL-6, tumour necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma) was investigated in the urine. Urine specimens were collected during the six weekly BCG instillations, before instillation, and 2, 4, 6, 8, and 24 h thereafter. Results were standardized to urine creatinine. In general, the concentration of IL-1 increased markedly during the first three BCG instillations, reaching a plateau from instillations 3 to 6. IL-2 was not detected after the first BCG instillation, but from the second instillation onwards the mean IL-2 concentration increased rapidly. With respect to IL-6, patients had relatively high levels in the urine after the first BCG instillation. A relatively moderate increase of the IL-6 concentration was observed during the following weeks. Like IL-2, TNF alpha was only detected after repeated BCG instillations. Generally the highest TNF levels were found after BCG instillation 5. The presence of IFN gamma could not be demonstrated. With respect to the occurrence of the cytokines during the first 24 h after the BCG instillation, TNF, IL-2, and IL-6 were detectable 2 h after the instillation. In contrast, IL-1 seemed to appear later, i.e. from 4 h onwards. TNF decreased most rapidly; it was nearly absent in 6-h samples. Generally IL-2 was not detectable in the 8-h samples, whereas IL-1 and IL-6 were present up to 8 h after instillation of BCG. The presence of TNF was found less frequently than the presence of IL-1, IL-2, and IL-6. Neutralization experiments indicated that most of the IL-1 present in the urine after BCG treatment was IL-1 alpha. In conclusion, activation of BCG-specific T cells was indicated by the detection of IL-2. The presence of IL-1, IL-6, and TNF alpha might suggest activation of macrophages by intravesically administered BCG, although production by other cell types cannot be excluded. It is suggested that these cytokines, in combination with the leucocytes that are known to be recruited to the bladder in reaction to the BCG treatment, may play an important role in the antitumour activity of BCG against bladder cancer. For monitoring purposes, collection of urine might be performed during the first 6 h after BCG instillations 4-6.(ABSTRACT TRUNCATED AT 400 WORDS)

Objective:To investigate the dynamics of IgG1 and IgG4 anti-citrullinated protein antibody (ACPA) subclasses during anti-tumour necrosis factor (TNF) treatment in patients with rheumatoid arthritis (RA).Methods:IgG, IgG1 and IgG4 ACPA levels were determined by ELISA on anti-citrullinated fibrinogen (ACF) and IgG1 : IgG4 ACPA ratios were calculated. A pilot study was performed in 28 ACF-positive patients treated with infliximab for one year. Confirmation of the results was obtained using a cohort of 180 consecutive patients treated with adalimumab for 28 weeks.Results:The median reduction in ACF levels was 31% for total IgG, 29% for IgG1, 40% for IgG4 and 22% for the IgG4 : IgG1 ACF ratio in the infliximab cohort. In adalimumab-treated patients, ACF levels declined 14% for total IgG and IgG1, and 36% for IgG4 ACF; the IgG4 : IgG1 ratio was reduced by 24% (all percentage values pConclusion:ACPA subclass distribution is modulated by effective anti-inflammatory treatment. The preferential decline of IgG4 ACPA, reflected by the decreased IgG4 : IgG1 ratio, suggests a beneficial effect of anti-TNF treatment on chronic antigenic stimulation by citrullinated proteins. This effect may be directly anti-TNF mediated or the result of effective dampening of the inflammation in the rheumatoid joint.

Stress reactions exist in many conditions in which plasma interleukin 6 (IL-6) is elevated. Examples are burns and sepsis. In these situations fever is often present. These stress situations are always accompanied with high levels of adrenalin and corticosteroids. These hormones, especially when given together, elicit a definite response of acute phase proteins in normal rats. In two stress models, (i) laparotomy and (ii) fever induced by administration of PGE2 in the lateral intracerebral ventricle, we observed a rise of adrenalin and corticosteron followed by an elevated level of plasma IL-6. Therefore, we studied the effect of adrenalin and corticosteron on the plasma level of IL-6. Adrenalin evokes high levels of IL-6, and this effect can be blocked by propranolol. When IL-6 release is blocked in this way, the response of alpha 2 macroglobulin and the cysteine protease inhibitor, both fast-reacting acute phase proteins in rat, is strongly depressed. Isoprenalin, an adreno beta 2 agonist, also causes very high levels of IL-6, indicating that the release of IL-6 can be mediated by an adreno beta 2 receptor whose presence has been demonstrated in monocytic cells. The results suggest a relation between stress situations and IL-6 and may be another factor besides the presence of endotoxins, virus, etc. explaining the high levels of IL-6 observed in many serious clinical situations.

BACKGROUND: A substantial proportion of patients with rheumatoid arthritis (RA) do not respond, or lose initial response, to adalimumab treatment. One explanation for non-response is that patients develop anti-adalimumab antibodies.OBJECTIVES: To evaluate the incidence of formation of antibody against adalimumab and the association with serum adalimumab concentrations and clinical response.METHODS: In a cohort of 121 consecutive patients with RA treated with adalimumab, serum adalimumab concentrations and antibodies against adalimumab were measured together with clinical response variables before and up to 28 weeks after the start of treatment.RESULTS: Anti-adalimumab antibodies were detected in 21 patients (17%) during 28 weeks of treatment. EULAR non-responders had antibodies significantly more often than good responders (34% vs 5%; p = 0.032). Patients with antibodies showed less improvement in disease activity (mean (SD) delta DAS28 0.65 (1.35)) than patients without antibodies (mean delta DAS28 1.70 (1.35)) (p = 0.001). Patients with antibodies during follow-up had lower serum adalimumab concentrations at 28 weeks than patients without antibodies (median 1.2 mg/l, range 0.0-5.6 vs median 11.0 mg/l, range 2.0-33.0, respectively; pCONCLUSIONS: Serum antibodies against adalimumab are associated with lower serum adalimumab concentrations and non-response to adalimumab treatment.

OBJECTIVES: Correlation of serum trough infliximab levels and antibodies to infliximab (anti-infliximab) with clinical response in ankylosing spondylitis. METHODS: In accordance with the international ASsessment in Ankylosing Spondylitis (ASAS) consensus statement, patients were treated with infliximab (5 mg/kg) every 6 weeks after a starting regimen. Preinfusion sera were collected at baseline, 24 and 54 weeks. At every visit, the 20% improvement response (ASAS-20) was assessed and laboratory tests performed. RESULTS: 24 of the 38 (63%) patients fulfilled ASAS-20 response criteria after 24 weeks of treatment and 21 (53%) after 54 weeks. After 54 weeks, 11 (29%) patients showed undetectable serum trough infliximab levels and detectable anti-infliximab; six of these patients developed an infusion reaction. Anti-infliximab was found significantly more often (p = 0.04) in ASAS-20 non-responders compared with responders at week 54. Serum trough infliximab levels were significantly (p

OBJECTIVE—The measurement of cytokine production of activated lymphocytes and monocytes in the whole blood cell (WBC) culture system may provide a sensitive tool for evaluating the actual ongoing immune response of patients with rheumatoid arthritis (RA). METHODS—Lipopolysaccharide (LPS) up to 250 pg/ml was used for the stimulation of monocytes for measuring the production of tumour necrosis factor α (TNFα), interleukin 6 (IL6) and IL12, while the anti-CD3 (1 µg/ml) and anti-CD28 (5 µg/ml) combination was used for T cell stimulation with the measuring of IL4 and interferon gamma (INFγ) production. Twenty seven patients with RA and 23 healthy controls were studied. RESULTS—The results showed a decreased IL6 (LPS stimulus 4-6 pg/ml) and IL12 (LPS stimulus 16-62 pg/ml) production in the RA patients. The maximal production of both cytokines was comparable with the normal controls. T cell stimulation showed a significant decreased INFγ production in the RA patients. CONCLUSIONS—These findings obtained in the WBC culture system are highly suggestive for a decreased TH-1 derived cytokine production by a diminished IL12 production in RA patients. Another possibility is that both IL12 and INFγ production in WBCs are inhibited by eventual circulating serum factors.

We examined the relationship between clusterin and activated complement in human heart infarction and evaluated the effect of this protein on ischemic rat neonatal cardiomyoblasts (H9c2) and isolated adult ventricular rat cardiomyocytes as in vitro models of acute myocardial infarction. Clusterin protects cells by inhibiting complement and colocalizes with complement on jeopardized human cardiomyocytes after infarction. The distribution of clusterin and complement factor C3d was evaluated in the infarcted human heart. We also analyzed the protein expression of clusterin in ischemic H9c2 cells. The binding of endogenous and purified human clusterin on H9c2 cells was analyzed by flow cytometry. Furthermore, the effect of clusterin on the viability of ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes was analyzed. In human myocardial infarcts, clusterin was found on scattered, morphologically viable cardiomyocytes within the infarcted area that were negative for complement. In H9c2 cells, clusterin was rapidly expressed after ischemia. Its expression was reduced after reperfusion. Clusterin bound to single annexin V-positive or annexin V and propidium iodide-positive H9c2 cells. Clusterin inhibited ischemia-induced death in H9c2 cells as well as in isolated adult ventricular rat cardiomyocytes in the absence of complement. We conclude that ischemia induces the upregulation of clusterin in ischemically challenged, but viable, cardiomyocytes. Our data suggest that clusterin protects cardiomyocytes against ischemic cell death via a complement-independent pathway.

The complement system is a humoral effector in the innate immune system. Three activation pathways exist in the complement system, known as the classical pathway, the lectin pathway and the alternative pathway. Dysfunction of lectin pathway activation is caused by MBL deficiency. MBL deficiency in a cohort of healthy Caucasian blood bank donors was investigated with MBL genotyping and MBL plasma concentration. Recognition of the yeast-derived zymosan by MBL was investigated with Western blot. The involvement of the alternative pathway amplification loop in enhancing MBL-mediated opsonization of zymosan was investigated in a novel opsonophagocytosis assay for flowcytometry. Sera deficient for MBL, factor D or properdin were tested, and purified MBL, factor D or properdin were used to recover opsonization. The optimal receiver–operator characteristic (ROC) cut-off value for dividing the Caucasian cohort in MBL-sufficient and MBL-deficient was calculated at 0.7 μg/ml. Thirty-eight percent of the group had concentrations below 0.7 μg/ml. Zymosan eluates opsonized with MBL-sufficient sera contain high oligomers of MBL, while eluates from MBL-deficient donors contained hardly any MBL. The MBL-, factor D- and properdin-deficient sera showed reduced opsonophagocytosis by human control neutrophils, as compared to normal MBL-sufficient sera. This reduction in opsonization was restored to normal levels by addition of purified MBL, factor D and properdin. The absence of opsonization in the factor D- and properdin-deficient sera, but presence in normal serum after blocking with anti-C1q-F(ab) 2 and anti-MBL-F(ab) 2 , demonstrates the involvement of the amplification loop in MBL-initiated zymosan opsonization, even at very low serum concentrations (up to 3%, v/v). In conclusion, our data demonstrate that the MBL-mediated route of complement activation depends on the alternative pathway amplification loop for optimal opsonization of zymosan.

Methotrexate (MTX) and mycophenolic acid (MPA) are used in the clinic for their immunosuppressive properties. MTX is widely used for the treatment of rheumatoid arthritis (RA). MPA is used to prevent graft rejection and is now experimentally used in systemic lupus erythematosis and RA. It is known that both drugs interfere with DNA synthesis. However, the precise mechanism of action is still debated. We have analysed the effect of the drugs on cytokine production in whole blood during short cultures. The production of T-cell cytokines was inhibited by both drugs. MTX inhibits cytokine production because MTX induces apoptosis in activated T-cells. MPA inhibits cytokine production by preventing T-cells to progress to the S-phase of the cell cycle. Cytokine production by monocytes was slightly decreased by the drugs. The reason for this inhibition is not clear. These results indicate that T-cells are the main target cells of the immunosuppressive drugs MPA and MTX.

Multiple organ dysfunction syndrome (MODS) is a frequent complication of severe sepsis and septic shock. Although massive inFlammatory reaction is considered to be one of the main triggers for the development of MODS, outcome is not necessarily improved by blocking the action of these mediators. Therefore, the downstream effects of these inFlammatory mediators, such as the induction of apoptosis, might be pivotal in the pathogenesis of MODS. However, only indirect markers for apoptosis, such as soluble FAS, can be measured in plasma. During the later stages of apoptosis, nucleosomes are released from cell’s chromatin by internucleosomal cleavage by endonucleases. We investigated whether circulating nucleosomes constitute a marker for apoptosis in sepsis.

Methotrexate (MTX) and mycophenolic acid (MPA) are used in the clinic for their immunosuppressive properties. MTX is widely used for the treatment of rheumatoid arthritis (RA). MPA is used to prevent graft rejection and is now experimentally used in systemic lupus erythematosis and RA. It is known that both drugs interfere with DNA synthesis. However, the precise mechanism of action is still debated. We have analysed the effect of the drugs on cytokine production in whole blood during short cultures. The production of T-cell cytokines was inhibited by both drugs. MTX inhibits cytokine production because MTX induces apoptosis in activated T-cells. MPA inhibits cytokine production by preventing T-cells to progress to the S-phase of the cell cycle. Cytokine production by monocytes was slightly decreased by the drugs. The reason for this inhibition is not clear. These results indicate that T-cells are the main target cells of the immunosuppressive drugs MPA and MTX.

Objective: Multiple organ dysfunction syndrome is a frequent complication of severe sepsis and septic shock and has a high mortality. We hypothesized that extensive apoptosis of cells might constitute the cellular basis for this complication. Design: Retrospective study. Setting: Medical and surgical wards or intensive care units of two university hospitals. Patients: Fourteen patients with fever, 15 with systemic inflammatory response syndrome, 32 with severe sepsis, and eight with septic shock. Interventions: None. Measurements and Main Results: We assessed circulating levels of nucleosomes, specific markers released by cells during the later stages of apoptosis, with a previously described enzyme-linked immunosorbent assay in these 69 patients with fever, systemic Inflammatory response syndrome, severe sepsis, or septic shock. Severity of multiple organ dysfunction syndrome was assessed with sepsis scores, and clinical and laboratory variables. Elevated nucleosome levels were found in 64%, 60%, 94%, and 100% of patients with fever, systemic inflammatory response syndrome, severe sepsis, or septic shock, respectively. These levels were significantly higher in patients with septic shock as compared with patients with severe sepsis, systemic inflammatory response syndrome, or fever, and in nonsurvivors as compared with survivors. In patients with advanced multiple organ dysfunction syndrome, nucleosome levels correlated with cytokine plasma levels as well as with variables predictive for outcome. Conclusions: Patients with severe sepsis and septic shock have elevated plasma levels of nucleosomes. We suggest that apoptosis, probably resulting from exposure of cells to excessive amounts of inflammatory mediators, might by involved in the pathogenesis of multiple organ dysfunction syndrome.

Objective: To investigate the kinetics of nucleosome leakage from apoptotic cells in an in vitro system and extrapolate the results to autoimmune disease, in particular systemic lupus erythematosus. Methods: A sensitive nucleosome enzyme linked immunosorbent assay (ELISA) was developed, using a monoclonal antibody (mAb) against histone 3 and an mAb against nucleosomes. Nucleosome release during apoptotic cell death was studied in Jurkat cells. AnnexinV binding (early apoptosis) and propidium iodide positivity (late apoptosis) of the cells were compared with nucleosome release at different times after apoptosis induction. Results: Nucleosomes appeared in culture supernatant of Jurkat cells 24 to 48 hours after apoptosis induction, when the cells had been late apoptotic for more than 12 hours. Conclusion: Nucleosomes are released from late apoptotic Jurkat cells, with a 12 hour delay from the appearance of AnnexinV binding cells. This result suggests that in vivo scavenger mechanisms have 12 hours to remove apoptotic material from the circulation.

The 5q31-33 region contains a number of genes, including IL-13, that could be involved in the aetiology of allergic asthma. Other genes of interest in this region are the Th2 cytokines IL-4 and IL-5; the p40 chain of the Th1 cytokine inducer IL-12; IL-3; IL-9; CD14; the s2-adrenergic receptor; the corticosteroid receptor and the transcription factors interferon regulatory factor 1 (IRF1) and transcription factor 7 (TCF7). Other diseases such as schistosomiasis may also be (at least partly) controlled by this region. In humans, the severity of infection caused bySchistosomamansoniis linked to a marker on chromosome 5g31 [1]. In mice, resistance to this disease is regulated by Th2 cytokines; in particular, neutralisation of IL-13 leads to reduced pulmonary granuloma formation and total serum IgE levels, while Th2 cytokine production (IL-4, IL-5, and IL-13) remains intact [2]. The important role of Th2 cytokines in human asthma is underscored by the fact that T cells, acquired from bronchial biopsies, display an increased capacity to produce IL-4, IL-5, and IL-13 [3-5]. Interestingly, pulmonary expression of IL-13 is observed in both allergic and non-allergic asthma [6], while IL-4 expression may be more restricted to allergic asthma [7]. Because of their biological effects, IL-4 and IL-13, located at close proximity on 5q31, are very likely candidates to be involved in the inheritance of asthma. Firstly, for an antibody isotype switch to IgE, B cells require stimulation by either IL-4 or IL-13 [8, 9], and secondly these cytokines induce VCAM-1 expression on endothelial cells and pulmonary fibroblasts [10, 11], which may cause the accumulation of eosinophils at the site of the allergic reaction [12]. Recently, other effector functions of IL-4 and IL-13 have been described, independent of IgE and eosinophils.

Item does not contain fulltext The high-affinity receptor for IgG, FcgammaRI, shares its capacity to bind IgG2a immune complexes (IgG2a-IC) with the low-affinity receptor FcgammaRIII and complement factors, hampering the definition of its biological role. Moreover, in vivo, FcgammaRI is occupied by monomeric IgG2a, reducing its accessibility to newly formed IgG2a-IC. By using a variety of FcgammaR(-/-) mice, we demonstrate that in the absence of FcgammaRI, the IgG2a-IC-induced cellular processes of phagocytosis, cytokine release, cellular cytotoxicity, and antigen presentation are impaired. FcgammaRI(-/-) mice showed impaired hypersensitivity responses, strongly reduced cartilage destruction in an arthritis model, and impaired protection from a bacterial infection. We conclude that FcgammaRI contributes substantially to a variety of IgG2a-IC-dependent immune functions and immunopathological responses.

Treatment with anti-tumour necrosis factor (TNF) is very effective in most patients with ankylosing spondylitis (AS), but inefficacy occurs in about 40% of cases.1 Antibody formation against TNF blocking agents is an increasingly recognised problem;2 however, no data have yet been reported on antibody formation against adalimumab (anti-adalimumab) in AS. Lack of response can be explained in two ways: (1) TNF might not be important for disease activity in certain patients; and (2) TNF inhibition might be insufficient. The latter could be caused by excessive production of TNF, low compliance of the patient, insufficient dosing or an enhanced clearance of adalimumab due to antibody formation. Adalimumab is a fully human monoclonal antibody against TNF but, despite this fact, an immune response …

IgE antibodies play a crucial role in allergic type I reactions. Only IL-4 and IL-13 are able to induce an immunoglobulin isotype switch to IgE in B cells. A major question is to what extent these cytokines contribute to the production of IgE in allergic patients. To address this question we used an in vitro culture system in which the production of IgE is dependent on endogenously produced IL-4 and IL-13. In cultures of purified T and B cells from allergic asthma patients and non-atopic controls, T cells were polyclonally stimulated to obtain IL-4, IL-13 and subsequently IgE secretion. The absolute amount of IgE produced was not significantly different between patients and controls. When neutralizing IL-4 antibodies were included during culture, the production of IgE was dramatically inhibited in both patients and controls (production of IgE was reduced to 12%). However, neutralization of IL-13 led to a significantly stronger inhibition of IgE production in the patient group: production of IgE was reduced to 23 ± 3% versus 50 ± 10% in the control group. Corresponding with these results, we also observed a higher production of IL-13 by the patients, while the production of IL-4 was not significantly different. A more detailed analysis of the production of IL-13 revealed that patients' T cells were less sensitive to a negative signal controlling IL-13 production. Our results indicate that, at least in vitro, IgE production in allergic asthma patients is more dependent on IL-13 than in non-atopics, due to enhanced IL-13 production and to enhanced IgE production in response to IL-13.

Publisher Summary This chapter reviews the role of cytokines in the pathogenesis of sepsis. The various models for sepsis are also discussed, including the human models and animal models. The human model for sepsis most frequently studied is the experimental human endotoxemia. Cytokines usually have an auto- or paracrine-mode of action, although in some situations, in particular in sepsis, they exert effects at a distance from their site of production. Cytokines may be grouped based on their main biological effects: (1) those with main effects on the activation and proliferation of lymphocytes, (2) those with effects on hematopoiesis, (3) those with pro- and anti-inflammatory properties, and (4) those that orchestrate cell growth and differentiation during tissue repair. The notion that cytokines are involved in the pathogenesis of sepsis is based on several lines of evidence: (1) the intravenous administration of cytokines [TNF, IL1, and IL2] induces a septic shock-like syndrome in animals or humans, (2) the inhibition of the effects of some cytokines—by administration of neutralizing antibodies, soluble cytokine receptors, or receptor antagonists—attenuates sepsis in animal models, and (3) the administration of anti-inflammatory cytokines, such as IL10, mitigates severe sepsis in animals.

Background Over the last years, an increasing body of evidence has highlighted the clinical significance of drug immunogenicity[1][1]. Anti-drug antibodies (ADA), by forming immune complexes with the drug, promote its faster clearance from circulation, reducing therapeutic effectiveness[2][2]. However, ADA have also been associated with increased incidence of drug-related adverse events (AE), particularly infusion-related AE during infliximab therapy, despite severe thromboembolic phenomena associated with antibodies against adalimumab have also been reported in the literature[3][3],[4][4],[5][5]. Objectives We assessed the association between ADA and infusion-related AE in patients with Rheumatoid Arthritis (RA), Ankylosing Spondylitis (AS), Psoriatic Arthritis (PsA) and Inflammatory Bowel Disease (IBD), receiving infliximab therapy. Methods We conducted a prospective cohort study over 2-years evolving 84 consecutive patients (22 AR, 33 AS, 9 PsA and 30 IBD) receiving infliximab at 3-5 mg/Kg every 6 or 8 wks, at day care unit of Hospital Garcia de Orta, Almada. Therapeutic response and low disease activity were defined according to the EULAR guidelines (RA and RA-like PsA), ASAS group guidelines (AS and AS-like PsA) and by an expert clinician (IBD patients). ADA were detected by an optimized Bridging ELISA, just prior to next infliximab infusion. Clinicians were blind for the tests results. Results Seventy-six percent of patients were female, with mean (SD) age of 48 (10.2) years, disease duration of 8 (6.4) years, receiving biologic therapy by 2.9 (2.0) years. All RA patients and 89% of PsA were receiving concomitant MTX. IBD patients were receiving concomitant azathioprine and also hydrocortisone plus anti-histaminic prior to each infliximab infusion. During the follow-up period, a total of 25 patients (30%) had detectable ADA (41% of RA, 33% of PsA, 18% of AS and 23% of IBD patients). Of those, 44% developed an infusion-related AE (4 RA patients, 2 PsA, 2 AS and 4 IBD patients). In all of those cases, ADA detection occurred prior to the AE. All patients with RA, PsA and AS who developed infusion-related AE were unable to maintain therapeutic response over time, while 2 out of 4 patients with IBD were still considered responders during the follow-up period. All the reactions were mild-moderate requiring hydrocortisone and anti-histaminic administration. Conclusions A significant porpotion of ADA-positives patients develop an infusion-related AE and cannot sustain therapeutic response. The maintenance of therapy in such cases may have serious deleterious effects with no additional therapeutic benefits. Further robust studies are warranted to better evaluate safety aspects related with immunogenicity. References 1. Garces, S et al. Ann Rheum Dis. 2012 Dec 6. [Epub ahead of print] 2. van Schouwenburg, PA et al. Ann Rheum Dis. 2013 Jan;72(1):104-9 3. Wolbink, GJ et al. Arthritis Rheum. 2006 Mar;54(3):711-5 4. Maggi, E et al. Expert Rev Clin Immunol. 2011 Jan;7(1):55-63 5. Korswagen, LA et al. Arthritis Rheum. 2011 Apr;63(4):877-83 Disclosure of Interest None Declared [1]: #p-6 [2]: #p-7 [3]: #p-8 [4]: #p-9 [5]: #p-10

Background Clinical remission is today the treatment goal for Rheumatoid Arthritis (RA), which requires fast and assertive therapeutic decisions for a tight control of disease activity. Few objective parameters are available to guide clinical decisions, namely in switcher patients. We designed a preliminary algorithm introducing immunogenicity assessment in the current approach to RA patients receiving biotechnologic therapies. Objectives To evaluate the concordance between the new algorithm and current clinical practice, comparing the effectiveness of “immunogenicity-based” versus “empirical-based” switches in a cohort of patients with established RA receiving biologics. Methods: EULAR therapeutic response was evaluated in 105 RA patients (naive or switchers) over one year, through GEE analysis. Serum drug trough levels were assessed by ELISA and anti-drug antibodies (ADAb) by Bridging ELISA. Results During follow-up, 48.6% of patients (Group A) had concordant therapeutic decisions. One year after the therapeutic decision, patients from Group A had a higher probability of achieving response (OR = 7.91, p Conclusions Immunogenicity assessment might help to optimise therapeutic decisions, leading to a better control of disease activity with significant better clinical outcomes in RA patients receiving biotechnologic therapies.

Background The clinical and scientific relevance of monitoring immunogenicity in clinical practice have been recognized and recommended by the European Agency of Medicine (EMA) 1 . However, its assessment is technically challenging and no consensus exists about the way immunogenicity can be monitored. Newly developed fluid-phase radio-immuno assays (RIA) have proven quite effective in detecting anti-drug antibodies and as been presented as a “gold standard” method to assess immunogenicity 2,3 . However, RIA requires high doses of radioactivity and special conditions, preventing its use as a routine assay. By contrast, enzyme-linked immunoassay (ELISA) it’s a simple and cheap method, ideal candidate to be used as a high-throughput screening assay. Bridging ELISA is a particular type of ELISA with increased specificity over the conventional ELISAs methods. Several optimizations of this method were made so that it can become a good screeningassay to monitor drug immunogenicity in routine clinical practice. Objectives We tested, in a same cohort of patients receiving infliximab, this newly optimized bridging ELISA in comparison with a fluid-phase antigen-binding RIA to quantify anti-infliximab antibodies. Methods A total of 82 consecutive patients were evaluated (38 rheumatoid arthritis patients, 27 ankylosing spondylitis, 9 psoriatic arthritis and 18 patients with inflammatory bowel disease), 61 females, with a mean age of 41 (4.2) years, that were receiving Infliximab in a dosage of 3-5mg/kg every 6 or 8 weeks for a mean period of 3.5 (2.0) years. Blood samples were collected immediately before the next infliximab infusion. Anti-infliximab antibodies were quantified by 1) Bridging ELISA where the antibodies bind to the infliximab coated in a solid phase and revealed by the addition of biotinylated infliximab and by 2) fluid-phase RIA-ABA that uses a sepharose-immobilized protein A, IgG total and IgG4-specific, to bind IgGs in the patient’s serum. Anti-infliximab specific IgGs are revealed by theaddition of 125 I-labeled infliximab F(ab’)2. A simple ELISA method was used to quantify serum infliximab levels,based on the principle that infliximab are captured through their ability to bind immobilized TNFα on a solid phase. The binding assessment is revealed by incubation with biotinylated rabbit IgG directed to infliximab idiotype. Therapeutic response was assessed according to validated criteria established for each disease. Results A total of 22 (27%) were tested positive for the presence of anti-infliximab abs using RIA, coinciding with the samples that were also positives in Bridging ELISA. Bridging ELISA cannot detect monovalent IgG4. No samples testing exclusively IgG4 specific anti-infliximab were detected. All patients (100%) with detectable anti-infliximab antibodies had undetectable serum trough drug levels and were not able to sustain the therapeutic response. Conclusions By its simplicity, cost and robustness, Bridging ELISA is a suitable test to be implemented in routine clinical practice as a screening assay to monitor drug immunogenicity. References E.M.A. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003946.pdf L et al. Curr Opin Immunol, 2008; 20: 431-5. Svenson M et al. Rheumatology (Oxford), 2007; 46: 1828-34. Disclosure of Interest None Declared

Whole blood cell culture has great advantage over isolated peripheral blood mononuclear cell culture, because it needs only small amounts of blood and is fast to perform. The current report focuses on the measurement of IL-6 and TNF alpha produced by peripheral blood monocytes of patients with systemic lupus erythematosus (SLE) in the whole blood cell culture system. After an initial triggering with lipopolysaccharide (LPS), a specific stimulus for monocytes, a decreased production of IL-6 relative to the controls was observed. Dividing our SLE patients according to treatment with corticosteroids, overall the IL-6 production was decreased in the patients treated with corticosteroids. TNF alpha production was comparable with normals, with the exception of an increased spontaneous production and using LPS stimulus of 4 pg/ml. In the patients treated with corticosteroids a decreased TNF production was observed, in contrast to the non-treated patients in which an increased TNF production was found compared with the controls using LPS doses higher than 62 pg/ml. The impaired acute phase reaction (APR) that has been described in the literature, might be explained by our observation of a decreased production of mainly IL-6. However, also this study showed that treatment has a strong impact on ex vivo IL-6 and TNF production.

In vitro as well as in vivo observations have shown that IL6 plays a key role in the pathogenesis of multiple myeloma. Therefore we started a phase I/II dose escalating study with chimeric monoclonal anti-IL6 antibodies (cMab) in multiple myeloma (MM) patients resistant to second-line chemotherapy. Here we describe the pharmacological data as well as a new method for calculating the endogenous IL6 production. The cMab (CLB IL6/8; Kd: 6.25 x 10(-12) M) was given in two cycles of 14 daily infusions, starting on day 1 and day 28. Daily dose: 5 mg in patients 1-3, 10 mg in patients 4-6, and 20 mg in patients 7-9 (total dose 140, 280, and 560 mg of anti-IL6, respectively). Using the pharmacokinetic data of free IL6 and the binding characteristics of the cMab, the endogenous IL6 production could be calculated from day to day using a one-compartment open model. The median half-life time of this antibody was 17.6 d. No human antichimeric antibodies were induced. Pre-treatment median endogenous IL6 production in the MM patients was 60 micrograms/d (range 13.8-230; normal controls < 7 micrograms/d). During treatment with anti-IL6 cMabs, the endogenous IL6 production immediately decreased in all patients to below 3 micrograms/d and never reached the pre-treatment value during the treatment period, except in two patients who developed an active infection, resulting in an IL6 production of 128 and 1,208 micrograms/d, respectively. We concluded that in MM patients endogenous IL6 production is 2-30 times higher than in healthy individuals. The anti-IL6 cMab strongly suppress this endogenous IL6 production, probably by blocking a positive feed-back loop, but this cMab does not prevent infection-induced IL6 production. The chimeric anti-IL6 Mabs have a long half-life time, a low immunogenicity, and are able to block IL6-dependent processes in vivo.

Rheumatoid arthritis is a T cell-mediated autoimmune disease. The lack of knowledge of the involved target antigens severely hampers research on relevant T cells in patients. Here we describe the functional analysis of freshly isolated T cells from the peripheral blood and the site of the lesion (synovial fluid or synovial membrane) of patients with rheumatoid arthritis. Healthy donors and osteoarthritis patients served as controls. Using various polyclonal stimuli, we analyzed CD4+ T cells with respect to proliferation and their ability to produce lymphokines. Our data show that lesion-derived CD4+ T cells of patients with rheumatoid arthritis are severely defective in proliferation and lymphokine (interleukin-2, interleukin-4, tumor necrosis factor-alpha, interferon-gamma) production. This activation defect was most pronounced at lower cell densities and was present in both synovial fluid derived and synovial membrane derived CD4+ T cells of all patients tested. No difference was found between responses of synovial fluid derived CD4+ T cells from osteoarthritis patients and those observed with peripheral blood derived T cells from all groups. The observed defect in lesion-derived CD4+ T cells from rheumatoid arthritis patients was not due to the effect of inflammatory factors in the synovial fluid because preincubation with synovial fluid could not induce a similar defect in control T cells. Together, our data show a rheumatoid arthritis specific, general defect in the activation of lesion-derived CD4+ T cells.

Introduction: Cell death is a central event in the pathogenesis of sepsis and is reflected by circulating nucleosomes. Circulating nucleosomes were suggested to play an important role in inflammation and were demonstrated to correlate with severity and outcome in sepsis patients. We recently showed that plasma can release nucleosomes from late apoptotic cells. Factor VII-activating protease (FSAP) was identified to be the plasma serine protease responsible for nucleosome release. The aim of this study was to investigate FSAP activation in patients suffering from various inflammatory diseases of increasing severity. Methods: We developed ELISAs to measure FSAP-C1-inhibitor and FSAP-a2-antiplasmin complexes in plasma. FSAP-inhibitor complexes were measured in the plasma of 20 adult patients undergoing transhiatal esophagectomy, 32 adult patients suffering from severe sepsis and 8 from septic shock and 38 children suffering from meningococcal sepsis. Results: We demonstrate plasma FSAP to be activated upon contact with apoptotic and necrotic cells by an assay detecting complexes between FSAP and its target serpins a2-antiplasmin and C1-inhibitor, respectively. By means of that assay we demonstrate FSAP activation in post-surgery patients, patients suffering from severe sepsis, septic shock and meningococcal sepsis. Levels of FSAP-inhibitor complexes correlate with nucleosome levels and correlate with severity and mortality in these patients. Conclusions: These results suggest FSAP activation to be a sensor for cell death in the circulation and that FSAP activation in sepsis might be involved in nucleosome release, thereby contributing to lethality.

Because of its neutrophil-activating properties, interleukin-8 (IL-8) may play an important role in the pathophysiology of sepsis. We measured circulating IL-8 levels in 47 patients with clinical sepsis. Levels on admission were elevated in 42 of the 47 patients (89%) and were comparable in patients with gram-positive or gram-negative infections. Patients with shock had significantly higher IL-8 levels than normotensive patients (P = 0.0014, Wilcoxon-Mann-Whitney test), whereas no differences in IL-8 levels were found between patients with or without adult respiratory distress syndrome. Patients who died had higher IL-8 levels on admission than the patients who survived. The largest differences in IL-8 levels between survivors and nonsurvivors was found when only patients with positive cultures were considered (P = 0.0342). IL-8 levels appeared to correlate significantly with lactate levels and inversely with leukocyte and platelet numbers and mean arterial pressure. In addition, the IL-8 level in the sepsis patients was found to correlate significantly with levels of IL-6, elastase-alpha 1-antitrypsin, and C3a. Serial observations revealed that in most patients IL-8 levels decreased, irrespective of the outcome. Thus, our results demonstrate that IL-8 levels are increased in most patients with sepsis and correlate with some important clinical, biochemical, and inflammatory parameters. These findings suggest a role for IL-8 in the pathophysiology of sepsis.