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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.

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.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.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.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.

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.

Introduction The human monoclonal antibody adalimumab is known to induce an anti-globulin response in some adalimumab-treated patients. Antibodies against adalimumab (AAA) are associated with non-response to treatment. Immunoglobulins, such as adalimumab, carry allotypes which represent slight differences in the amino acid sequences of the constant chains of an IgG molecule. Immunoglobulins with particular IgG (Gm) allotypes are racially distributed and could be immunogenic for individuals who do not express these allotypes. Therefore, we investigated whether a mismatch in IgG allotypes between adalimumab and IgG in adalimumab-treated patients is associated with the development of AAA. Methods This cohort study consisted of 250 adalimumab-treated rheumatoid arthritis (RA) patients. IgG allotypes were determined for adalimumab and for all patients. Anti-idiotype antibodies against adalimumab were measured with a regular radio immunoassay (RIA), and a newly developed bridging enzyme linked immunosorbent assay (ELISA) was used to measure anti-allotype antibodies against adalimumab. The association between AAA and the G1m3 and the G1m17 allotypes was determined. For differences between groups we used the independent or paired samples t-test, Mann-Whitney test or Chi square/Fisher's exact test as appropriate. To investigate the influence of confounders on the presence or absence of AAA a multiple logistic regression-analysis was used. Results Adalimumab carries the G1m17 allotype. No anti-allotype antibodies against adalimumab were detected. Thirty-nine out of 249 patients had anti-idiotype antibodies against adalimumab (16%). IgG allotypes of RA patients were associated with the frequency of AAA: patients homozygous for G1m17 had the highest frequency of AAA (41%), patients homozygous for G1m3 the lowest frequency (10%), and heterozygous patients' AAA frequency was 14% (P = 0.0001). Conclusions An allotype mismatch between adalimumab and IgG in adalimumab-treated patients did not lead to a higher frequency of AAA. On the contrary, patients who carried the same IgG allotype as present on the adalimumab IgG molecule, had the highest frequency of anti-adalimumab antibodies compared to patients whose IgG allotype differed from adalimumab. This suggests that the allotype of adalimumab may not be highly immunogenic. Furthermore, patients carrying the G1m17-allotype might be more prone to antibody responses.

An allotypic form of the low affinity IgG Fc receptor Fc gamma RIIa (CD32), termed low responder (LR) because of its weak reactivity with mouse (m) IgG1, interacts efficiently with human (h) IgG2. Fc gamma RIIaLR is the first known human FcR that binds this IgG subclass. In this study, we analyzed the role of Fc gamma RIIa in binding of stable hIgG-subclass dimers, and in induction of T cell mitogenesis using chimeric anti-CD3 mAb. We demonstrate that the functional polymorphism to hIgG2 is expressed on the majority of Fc gamma R-bearing peripheral blood cells: monocytes, neutrophils, and platelets. We were able to assess Fc gamma RII-mediated IgG-binding without interference of other Fc gamma R-classes, by blockade of Fc gamma RI on monocytes, and by using neutrophils of an individual deficient for the Fc gamma RIIIB gene. This study indicates as subclass specificity: hIgG3 >hIgG1,hIgG2 >> hIgG4 for Fc gamma RIIaLR and hIgG3,hIgG1 >> hIgG2 > hIgG4 for Fc gamma RIIaHR. Comparing the serum hIgG levels of individuals homozygous for the two fc gamma RIIa allotypic forms, we observed significantly lower hIgG2 serum levels in individuals expressing the hIgG2-binding LR allotypic form. This observation may implicate that Fc gamma RIIa regulates hIgG subclass production or turnover in man.

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)

We have examined the regulation of interleukin (IL)-4 production by human peripheral blood T cells. Production of IL-4 was shown to be regulated differently from IL-2 and interferon(IFN)-gamma production. Stimulation of peripheral blood lymphocytes with anti-CD3, anti-CD2, anti-CD28, Phorbol 12-myristate 13-acetate (PMA) or IL-2 as a single stimulant did not induce IL-4 production. However, combinations of anti-CD2 with either anti-CD28 or IL-2 resulted in IL-4 production, peaking at days 3-4. Stimulation with anti-CD3 instead of anti-CD2 gave similar results, but was less potent. After days 3-4, IL-4 levels decreased, most likely due to consumption of IL-4. PMA profoundly affected cytokine production, it enhanced IL-2 production by at least tenfold, whereas, in the same cell population, IL-4 production was almost completely inhibited. This was observed at the protein as well as at the mRNA level. In contrast, agents that increase intracellular cAMP levels inhibited IL-2 production but left IL-4 production unaffected. IFN-gamma production behaved similar to IL-2 production but the effects were less outspoken.

PURPOSE OF REVIEW: In the last decade, biologicals revolutionized rheumatology. An increasing number of patients benefit from biotherapeuticals. However, some patients do not respond to treatment and others lose their response after a certain time. Immunogenicity is one of the factors linked to secondary nonresponse but its clinical significance has remained controversial. RECENT FINDINGS: In recent years, knowledge of how to assess immunogenicity of biologicals has improved. Various reports show an inverse relationship between drug levels and antibody formation against the drug. Studies associated immunogenicity of therapeutic antibodies with clinically significant nonresponse in a subgroup of patients. Clinically relevant immunogenicity is influenced by several factors including dosing and concomitant medication. It has been shown that immunogenicity against biologicals can be persistent or transient. SUMMARY: Immunogenicity affects a significant number of patients treated with biologicals. Monitoring of drug levels as well as of antibodies against therapeutic antibodies may lead to more rational treatment strategies

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.

A comparative study was performed to examine the lethal effects of several cytokines injected into mice sensitized with actinomycin D (Act-D). Consistent with published data, human tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) (0.2-5 micrograms) caused the death of the animals within 8-12 hr after injection. Human interleukin-6 (IL-6) and interleukin-8 (IL-8) (0.6-6 micrograms) known to be induced by TNF-alpha did not show any lethal effects, indicating that TNF-alpha-associated lethality is not mediated by IL-6 or IL-8. Human tumor necrosis factor-beta (TNF-beta) (also called lymphotoxin), which shares structural and functional properties with TNF-alpha, was as potent as TNF-alpha in its lethal effects. Murine interferon-gamma (IFN-gamma) (0.04-5 micrograms) was also tested and showed no lethal effects in this model. In addition, a synthetic peptide corresponding to amino acid residues 163-171 of IL-1 beta, and which has been shown to lack the inflammatory effects of IL-1 beta, also caused no lethality among Act-D sensitized mice. The pretreatment of mice with IL-6, IL-8, or IFN-gamma had no protective effects on TNF-alpha or IL-1 beta-induced lethality in contrast to the protection observed by a pretreatment with TNF-alpha/IL-1 beta themselves or with endotoxin. Histopathologic data showed that severe tissue injury in vital organs is associated with the rapid lethality among sensitized mice.

Production of interleukin-2 (IL-2) by human T-lymphocytes can be augmented by costimulation via CD28. It has been reported that signaling via CD28 acts by stabilization of lymphokine mRNAs (Lindsten, T., June, C. H., Ledbetter, J. A., Stella, G., and Thompson, C. B. (1989) Science 244, 339-343). Here we demonstrate that costimulation via CD28 also provides a signal which activates transcription of the IL-2 gene A CD28-responsive element (CD28RE) in the IL-2 enhancer at position -162 to -152 was identified. This so far unidentified element shows sequence similarity to the kB enhancer motif. In vitro binding studies have demonstrated that the via CD28-induced signal synergizes with either phorbol myristate acetate or anti-CD3 for the induction of a nuclear factor that binds CD28RE and the human immunodeficiency virus (HIV-1) NF-kB motif. The significance of the sequence similarity of CD28RE with the kB enhancer motif was demonstrated by cross-competition studies using unlabeled CD28RE, HIV-1 NF-kB binding site, and a mutated version of the NF-kB motif. In addition, we found that NF-kB-dependent reporter gene expression was induced by costimulation via CD28. These results indicate that besides an effect on lymphokine mRNA stabilization, stimulation via CD28 acts at the level of transcription via coinduction of an NF-kB-like activity.

Ligation of the T cell membrane antigen CD28 strongly enhances cytokine secretion in human T lymphocytes that are activated via T cell receptor (TcR)/CD3 or CD2 molecules. This study was undertaken to investigate whether, as has been indicated for activation via TcR/CD3, stimulation via CD28 is dependent on the activation of protein kinase C (PKC). Two inhibitors of PKC, 1-alkyl 2-methyl-glycerol and staurosporine, caused a dose-dependent inhibition of T cell proliferation induced by anti-CD3 monoclonal antibodies (mAb). The induction of interleukin (IL) 2 secretion was found to be more sensitive to the effects of the PKC inhibitors than the up-regulation of IL 2 receptor expression. In marked contrast, the anti-CD28 mAb-mediated enhancement of T cell proliferation and IL 2 secretion were insensitive to the action of either compound. We conclude that two independent signaling pathways may be operational in human T cells. The first used by TcR/CD3 depends on the activation of PKC, whereas the second is employed by CD28 and functions independently of PKC.

A monoclonal antibody and an affinity purified polyclonal antibody, both raised against recombinant human IL-6, have been employed in an ELISA procedure to quantitate human IL-6. Both antibodies were very potent in neutralizing the biological activity of recombinant as well as natural human IL-6. The monoclonal antibody was used as the capture antibody whilst the polyclonal antibody, in biotinylated form, was used as the detecting antibody in combination with a streptavidin horseradish peroxidase conjugate and a signal amplification system. The detection limit for natural as well as recombinant IL-6 was 1 pg/ml. A good correlation was found between the ELISA and the B9 biological assay when IL-6 was measured in crude culture supernatants, in synovial fluids of rheumatoid arthritis patients and in the sera of patients with diverse diseases. Immunoprecipitation of IL-6, produced by different cell types, such as monocytes, endothelial cells and smooth muscle cells or derived from biological fluids, such as the serum of a patient with septic shock or the synovial fluid of a rheumatoid arthritis patient, revealed in every case only molecules in the molecular weight range of 21,000-26,000.

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1-alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF-alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1-mu-M dexamethasone. Monoclonal antibodies to rTNF-alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.

It has been reported recently that freshly isolated human myeloma cell cultures proliferate in response to added interleukin-6 (IL-6). Endogenous levels of IL-6 found in the same cultures suggested that an autocrine growth loop may contribute to cell growth. However, the lack of homogenous cell populations in primary myeloma cultures has made it difficult to distinguish between paracrine and autocrine growth mechanisms. To precisely address the autocrine growth issue we have evaluated the growth of the human myeloma cell line, U266. We have found that a neutralizing anti-IL-6 monoclonal antibody can inhibit U266 proliferation. Furthermore, the addition of IL-6 antisense oligonucleotides also inhibits U266 proliferation. These effects are reversed by adding IL-6, suggesting the presence of an autocrine loop. Using bioassays with two different IL-6-dependent cell lines, we were able to detect IL-6 in concentrated U266 supernatants. IL-6 mRNA was detected by polymerase chain reaction amplification of cDNA. Cell cycle parameter analysis shows that IL-6 acts to release a block in G1. Taken together these results present conclusive evidence for IL-6-mediated autocrine growth in the U266 human myeloma cell line.

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

We observed that interaction of secondary necrotic (sn) cells with human serum or plasma leads to loss of DNA staining. The decrease turned out to be a result of nucleosome release and was specific for apoptotic cells as necrotic cells did not show this phenomenon. We named this activity in plasma nucleosome releasing factor (NRF). NRF activity was completely inhibited by trypsin inhibitors suggesting that a serine protease is involved. Upon testing a number of plasma candidate serine proteases we found that plasmin did have NRF activity. However, plasminogen-deficient plasma still had NRF activity indicating that NRF is not plasmin. We conclude that a yet unidentified plasma serine protease is involved in removal of nucleosomes from sn cells. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved

Background: During sepsis, ischemia-reperfusion injury and sterile inflammation, levels of circulating histones and nucleosomes have been found to correlate with disease severity. Although the pathophysiological effects of circulating nucleosomes are not fully understood, free histones have been described to mediate cytotoxicity and contribute to disease severity. Several plasma proteins have been identified that modulate these cytotoxic effects by neutralizing or degrading histones. We have previously described that the plasma serine protease Factor VII-activating protease (FSAP) is activated upon contact with late apoptotic or necrotic cells and induces release of nucleosomes from these cells. During this release, histone H1 is cleaved. In addition, it has been described that FSAP is (auto)activated upon contact with histones and subsequently degrades histone H3. Aims: To investigate the regulation of histone-induced cytotoxicity by FSAP. Moreover, we aimed to compare differences in the cytotoxicity of histones and nucleosomes (histone+DNA) and potential differences in their proteolysis by FSAP. Methods: To determine FSAP activation and proteolysis of histones in serum we incubated normal donor or FSAP-deficient serum with 100ug/ml calf thymus histones. Upon its activation FSAP is rapidly bound by plasma inhibitors and we measured complexes of FSAP and its plasma inhibitor alpha 2-antiplasmin as an indirect readout for FSAP activation. To detect FSAP mediated proteolysis of histones we performed immunoblotting for histone H3. Results were corroborated using plasma-purified or recombinant FSAP. To determine histone-mediated cytotoxicity, we measured LDH levels after overnight incubation of HEK293 or ECRF cells with 100ug/ml calf thymus histone that had undergone a pre-incubation step in the presence or absence of healthy donor serum, FSAP-deficient serum or FSAP-depleted serum. Moreover, we tested the cytotoxicity of purified nucleosomes that were pre-incubated in the presence or absence of benzonase to degrade DNA. Results: EndogenousFSAP in serum was activated upon contact with histones, and histone proteolysis was observed. Similarly, histones were proteolyzed upon incubation using active plasma-purified or pre-activated recombinant FSAP, whereas no histone proteolysis was observed using recombinant inactive FSAP. These results indicate that FSAP is responsible for the observed histone degradation. FSAP-mediated histone degradation was able to protect HEK293 and ECRF cells against histone-induced cytotoxicity, showing that the resultant histone breakdown products had lost their cytotoxic effects. Recombinant inactive FSAP did not affect histone-induced cytotoxicity, indicating that the catalytic activity of FSAP is required for the observed protective effect of FSAP on cytotoxicity. When FSAP-deficient or FSAP-depleted serum was used, this led to greatly diminished histone proteolysis and reduced histone-induced cytotoxicity. Using our assay, we found that nucleosomes did not show cytotoxic effects unless they had been pre-incubated with benzonase to release histones. Notably, whereas intact nucleosomes were resistant against degradation by FSAP, FSAP protected against the cytotoxicity induced by histones released from these nucleosomes. Conclusion: FSAP in serum is activated by histones and subsequently proteolyses histones ,resulting in protection of cells from histone-induced cytotoxicity. Histones in the form of nucleosomes did not induce cytotoxicity, however after DNA removal the histones regained cytotoxicity. FSAP efficiently protected against the cytotoxicity of the unbound histones. We predict that the form in which histones are released into the blood is highly relevant in the context of human health and disease. We speculate that when endogenous DNases in serum degrade the DNA in nucleosomes, or alternatively, when highly cytotoxic histones are inadvertently released from dead cells in their free form rather than in the form of nucleosomes, FSAP mediates their efficient degradation to protect against the damaging effects of free histones. Neutralization of histones by FSAP may be an effective therapeutic approach to treat systemic inflammation. Disclosures Zeerleder: ViroPharma: Other: Receives an unrestricted grant from Viropharma.

Assessment of clinically relevant immunogenicity is a much-debated topic. With respect to the neutralising capacity of antidrug antibodies (ADAs) to tumour necrosis factor (TNF) blockers, we recently demonstrated that the vast majority of antibodies in fact compete with TNF binding.1 Therefore, we are pleased to find this notion further supported by the letter of Rinaudo-Gaujous et al .2 The aim of our study was to quantify to which extent ADAs were neutralising. Using the TNF competition assay, we concluded that …

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.

Tissue factor, the main initiator of blood coagulation, is shed into plasma by blood cells and endothelium. While studying such circulating plasma tissue factor with a commercially available immunoassay, we found unsatisfactory results and therefore developed a new and highly sensitive enzyme-linked immunosorbent assay (ELISA). High-affinity monoclonal antibodies raised against recombinant soluble tissue factor were used and the new assay had a detection limit of 40fmol/L, approximately six-fold lower than existing assays. Normal ranges in 20 healthy donors were established in serum and in citrated EDTA and heparinized plasma. Tissue factor was also measured in three successive plasma samples from 43 patients with type 2 diabetes mellitus. In citrated plasma from healthy donors, tissue factor concentrations were 2.5 (1.0–9.3) pmol/L (median with range) and were not significantly different in diabetics. With a commercially available immunoassay, seven plasma samples were below the detection limit. Use of the new assay reduced intra-individual variation in diabetics from 49% to 14% and we conclude that high-affinity antibodies may markedly improve immunoassay performance.

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.

4-Hydroxy-oxyphenbutazone (4OH-OPB), is currently in phase II trials for its immunosuppressive effect in patients with rheumatoid arthritis. 4OH-OPB and other compounds related to phenylbutazone were tested for their effect on in vitro cytokine production by monocytes and lymphocytes present in peripheral mononuclear cells (PBMC) or whole blood (WB) cultures, and compared against phenylbutazone and oxyphenbutazone, two known anti-inflammatory drugs. In PBMC cultures, 4OH-OPB was by far the most potent inhibitor, and both monokines and Th1 and Th2 lymphokines were efficiently inhibited at low concentrations. In WB cultures, 4OH-OPB was less effective than in PBMC cultures, but was still the best inhibitor of lymphokine production and, furthermore, was the only inhibitor of monokine production. The increase in 4OH-OPB concentration needed to induce the same inhibition of cytokine production in WB as in PBMC culture could be mimicked by the addition of erythrocytes to the PBMC cultures. Experiments with radioactively-labeled 4OH-OPB suggest that 4OH-OPB is taken up very rapidly into erythrocytes and is secreted by the erythrocytes with much slower kinetics via a multidrug-resistance-associated protein. The secreted compound is most likely structurally different from 4OH-OPB, as in PBMC and WB cultures, the inhibition of cytokine production seems to be caused by a different mechanism. In PBMC cultures, the inhibition of cytokine production is accompanied by a loss of cell viability, while this is not the case when 4OH-OPB inhibits cytokine production in WB. Our data suggest that 4OH-OPB may be useful as an immunosuppressive drug for patients with inflammatory diseases.

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.

Apoptotic cells activate complement via various molecular mechanisms. It is not known which of these mechanisms predominate in a physiological environment. Using Jurkat cells as a model, we investigated complement deposition on vital, early and late apoptotic (secondary necrotic) cells in a physiological medium, human plasma, and established the main molecular mechanism involved in this activation. Upon incubation with recalcified plasma, binding of C3 and C4 to early apoptotic cells was similar to background binding on vital cells. In contrast, late apoptotic (secondary necrotic) cells consistently displayed substantial binding of C4 and C3 and low, but detectable, binding of C1q. Binding of C3 and C4 to the apoptotic cells was abolished by EDTA or Mg-EGTA, and also by C1-inhibitor or a monoclonal antibody that inhibits C1q binding, indicating that complement fixation by the apoptotic cells was mainly dependent on the classical pathway. Late apoptotic cells also consistently bound IgM, in which binding significantly correlated with that of C4 and C3. Depletion of plasma for IgM abolished most of the complement fixation by apoptotic cells, which was restored by supplementation with purified IgM. We conclude that complement binding by apoptotic cells in normal human plasma occurs mainly to late apoptotic, secondary necrotic cells, and that the dominant mechanism involves classical pathway activation by IgM.

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.

Mycophenolic acid (MPA) and methotrexate (MTX) are immunosuppressive drugs used for the treatment of various immunological disorders. MPA is an inhibitor of inosine monophosphate dehydrogenase and MTX is a folate antagonist that inhibits tetrahydrofolate reductase. Production of T cell cytokines in whole blood cultures, as well as in PBMC cultures, is inhibited by a low concentration of both drugs. Inhibition of cytokine production after monocyte stimulation was less evident. The mechanism by which inhibition is achieved is different for both drugs. Inhibition of T cell cytokine production by MPA was more profound and started earlier compared to the inhibition by MTX. MTX induced apoptosis in T cells that became activated, whereas MPA prevented activation of T cells by arresting the cell cycle in the G0/G1 phase. Addition of guanosine and adenosine can overcome this cell cycle arrest, even after several days. Furthermore MPA inhibited the expression of activation markers HLA-DR and CD71 on T cells. The observation that MTX cannot prevent T cell activation but induces apoptosis in activated T cells, and that MPA reversibly prevents activation of T cells could explain the immunosuppressive effects of both these drugs.

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.

Human serum amyloid P component (SAP) is a glycoprotein structurally belonging to the pentraxin family of proteins, which has a characteristic pentameric organization. Mice with a targeted deletion of the SAP gene develop antinuclear Abs, which was interpreted as evidence for a role of SAP in controlling the degradation of chromatin. However, in vitro SAP also can bind to phosphatidylethanolamine, a phospholipid which in normal cells is located mainly in the inner leaflet of the cell membrane, to be translocated to the outer leaflet of the cell membrane during a membrane flip-flop. We hypothesized that SAP, because of its specificity for phosphatidylethanolamine, may bind to apoptotic cells independent of its nuclear binding. Calcium-dependent binding of SAP to early, nonpermeable apoptotic Jurkat, SKW, and Raji cells was indeed observed. Experiments with flip-flopped erythrocytes confirmed that SAP bound to early apoptotic cells via exposed phosphatidylethanolamine. Binding of SAP was stronger to late, permeable apoptotic cells. Experiments with enucleated neutrophils, with DNase/RNase treatment of late apoptotic Jurkat cells, and competition experiments with histones suggested that binding of SAP to late apoptotic cells was largely independent of chromatin. Confocal laser microscopic studies indeed suggested that SAP bound to these apoptotic cells mainly via the blebs. Thus, this study shows that SAP binds to apoptotic cells already at an early stage, which raises the possibility that SAP is involved in dealing with apoptotic cells in vivo.

IL-13 plays a crucial role in the development of allergic asthma by several mechanisms, including induction of IgE antibodies, airway eosinophilia and hyper-reactivity. We previously established a deregulated production of IL-13 by T cells from allergic asthma patients. In this report we describe the identification of a novel IL-13 promoter polymorphism (C to T exchange) at position -1055. The IL-13 -1055 TT genotype is associated with allergic asthma (P = 0.002), altered regulation of IL-13 production (P < 0.002), and increased binding of nuclear proteins to this region. We postulate that the presence of this polymorphism predisposes to the development of allergic asthma.

We examined the influence of donor T lymphocytes on human peripheral blood leukocytes (PBL) engraftment into severe combined immune deficient (SCID) mice. Mice were injected with unfractionated or subset-depleted human PBL, and treated at various times with OKT3, a cytotoxic monoclonal antibody against human CD3 + T lymphocytes. PBL engraftment, high levels of human Ig, and high incidence of lymphoproliferative disease (lpd) were found in mice transplanted with unfractionated PBL and CD8- or CD14-depleted PBL, and in mice treated with OKT3 at distance from PBL transfer. Animals xenografted with CD3- or CD4-depleted PBL, or treated at transplantation time with OKT3, had very low levels of human Ig and did not develop lpd. PBL engraftment was minimal or absent in these animals, as determined by immunohistochemistry, dot-blot, and RT-PCR analyses. These results demonstrate that the presence of donor CD4 + T lymphocytes at transplantation time is necessary for observing human PBL engraftment into SCID mice, an essential condition for human Ig production and lpd development.

T lymphocytes can be induced to produce interleukin (IL)-2 and proliferate upon T cell receptor (TcR) occupancy together with a CD28-induced co-stimulatory signal. The T cell surface molecule CD28 is believed to function as a regulator in T cell activation at both the level of lymphokine mRNA stabilization and gene transcription. Activation of IL-2 gene transcription via CD28 has been shown to be mediated through a kappa B-like sequence, called CD28RE. DNA binding analysis revealed that the CD28-induced signal is involved in the induction of CD28RE binding activity. Here, we demonstrate that the induction of CD28RE binding activity is not specific for the CD28-induced signal. Our data indicate that distinct mitogenic T cell activation signals converge on the induction of CD28RE binding activity, and suggest a crucial role for this activity in the IL-2 enhancer responsiveness to different modes of T cell activation.

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 …

Activation of T cells through the antigen-specific T-cell receptor in combination with a costimulatory signal results in efficient cytokine gene transcription. The CD28-induced signal represents a major costimulatory signal for T cells. A CD28 response element, named CD28RE, was first identified in the interleukin-2 (IL-2) promoter region. Here we demonstrate that the NF-kappaB sequence in the IL-6 promoter functions as a CD28 response element. Mutations in this sequence rendered the IL-6 promoter unresponsive to CD28 costimulation. Moreover, this element could replace the IL-2 CD28RE in conferring CD28 responsiveness to the IL-2 promoter. In analogy to the known CD28 response elements IL-2 CD28RE, IL-8 CD28RE, and the human immunodeficiency virus-1 (HIV-1) NF-kappaB motif, the IL-6 NF-kappaB motif efficiently bound c-Rel, c-Rel/NFKB1, and the recently identified inducible T-cell factor NF-MATp35. However, the IL-6 NF-kappaB sequence together with the IL-8 CD28RE and HIV-1 NF-kappaB sequence differed from the IL-2 CD28RE in the binding of NF-kappaB/Rel family proteins. Although the IL-2 CD28RE exerted selective binding with c-Rel and c-Rel/NFKB1, the other CD28REs allowed efficient binding of a wide range of NF-kappaB/Rel family proteins. The difference in binding specificity correlated with the capacity of the distinct CD28 response elements to function in the context of the IL-6 promoter in response to T-cell activation. Domain swapping experiments revealed that the IL-8 CD28RE and HIV-1 NF-kappaB motif conferred similar responsiveness as the genuine IL-6 NF-kappaB motif in the transcriptional activation of the IL-6 promoter upon CD28 costimulation. In contrast, replacement of the IL-6 NF-kappaB sequence by the IL-2 CD28RE motif strongly reduced the responsiveness of the IL-6 promoter. These data indicate that despite the sequence similarity, two different classes of CD28 responsive elements exist that differ in their NF-kappaB binding capacity and the ability to confer CD28 costimulatory responsiveness toward a heterologous promoter.

A new technique is presented that allows measurement of protein concentrations in the picomolar range with an assay time of only 10–20min. The method is an enzyme-linked immunosorbent assay (ELISA), but uses in-situ ellipsometric measurement of a precipitating enzyme product instead of the usual colorimetric detection of accumulating enzyme product in solution. Quantitative validation was obtained by use of annexin V, a protein with high binding affinity for phosphatidylserine-containing phospholipid membranes, resulting in a transport-limited adsorption rate. This property was exploited to obtain a range of low surface concentrations of annexin V by timed exposures of phospholipid bilayers to known concentrations of annexin V. Using polyvinylchloride (PVC)-coated and silanized silicon slides, various versions of this technique were used for the rapid assay of fatty acid-binding protein (FABP), a recently introduced early marker for acute myocardial infarction with a normal plasma concentration below 1 nmol/l, interleukin 6 (IL-6), a cytokine with normal plasma concentrations below 1 pmol/l, and again, annexin V. A possible future application of the method in the development of a one-step ELISA is discussed.

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 In the last years, the “treat to target” strategy revealed the importance of tight control of disease activity in RA patients 1 . To reach clinical remission or at least low disease activity as soon as possible, this strategy, now part of the EULAR guidelines, requires therapeutic combinations and/or fast switches between different therapies 2 . Over the last years it became clear that drug immunogenicity is one of the main mechanism behind biologic therapeutic failure 3-4 . How to integrate the notion of drug immunogenicity in clinical practice remains to be formally established. We conducted a systematic review of the Literature with a meta-analysis evaluating the clinical implications of drug immunogenicity 5 . Based on the information retrieved we designed a new algorithm which introduces immunogenicity assessment in the EULAR guidelines for the management of RA patients receiving biologic therapy. Objectives We assessed the influence of adherence to our proposed algorithm on therapeutic responses and low disease activity rates in a prospective cohort of RA patients treated with biologics. Methods We conducted a prospective cohort study over 2-years evolving 106 consecutive RA patients, 92% female, with a mean age of 55±13 yrs and mean disease duration of 6±4 yrs. Patients had been submitted to biologic therapy by a mean period of 4±3 years. Serum drug trough levels were assessed by ELISA and anti-drug antibodies (ADA) by an optimized Bridging ELISA, which we previously compared with a fluid-phase RIA. Clinicians were blind for the tests results. Therapeutic response and low disease activity were defined according to the EULAR guidelines. Results At the study onset 22 patients were receiving Infliximab (36.4% anti-infliximab pos), 33 Adalimumab (27.3% anti-adalimumab pos) and 49 Etanercept (0% anti-etanercept pos). Patients with detectable ADAs had undetectable serum drug trough levels and lower therapeutic responses rates (37.5% vs 76.9%, p=0.001 for Infliximab and 33.3 vs 66.7%, p=0.02 for Adalimumab). Not a single patient with detectable ADAs had low disease activity. During follow-up period, therapeutic decision coincident with our proposed algorithm concerned 48.6% of the patients (Group 1), however with a decalage median (IQR) time of 249 days (116-388). Therapeutic decisions were discordant with our algorithm for 51.4% of the patients (Group 2). A significant higher proportion of patients in Group 1, when compared to Group 2, reached therapeutic response at 3 months (97.9% vs 52.2%, p=0.001) and at 12 months (71.4% vs 13% p=p=0.000). Moreover, a significant higher proportion of patients in Group 1 achieved low disease activity at 3 months (71.4% vs 13%, p=0.000) and at 12 months (58.8% vs 3.8%, p=0.000). Considering drug direct costs only, we evaluated that about 1 million euros were spent inefficiently in this cohort over 2 years. Conclusions Our study strongly suggests that including immunogenicity assessment in EULAR guidelines will allow the design of more cost-effective strategies, tailored to each RA patient receiving biologic therapy. References Schipper LG et al. Ann Rheum Dis 2011;Dec30. Smolen JS et al. Ann Rheum Dis 2010;69:964-75. Bartelds et al. JAMA 2011;305:1460-8. Jamnistski et al. Ann Rheum Dis 2011; 70:284-8. EULAR 2012 Abstract: EULAR12-3348. Disclosure of Interest None Declared

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