Lower limb ischaemia and reperfusion injury in healthy volunteers measured by oxidative and inflammatory biomarkers.

OBJECTIVE: Ischaemia-reperfusion (IR) injury is partly caused by the release of reactive oxygen species and cytokines and may result in remote organ injury. Surgical patients are exposed to surgical stress and anaesthesia, both of which can influence the IR response. An IR model without these interfering factors of surgery is, therefore, useful to test the potential of antioxidant and cytokine-modulatory treatments. The aim of this study was to characterize a human ischaemia-reperfusion model with respect to oxidative and inflammatory biomarkers. MATERIALS AND METHODS: Ten male volunteers were exposed to 20 minutes of lower limb ischaemia. Muscle biopsies and blood samples were taken at baseline and 5, 15, 30, 60 and 90 minutes after tourniquet release and analysed for malondialdehyde (MDA), ascorbic acid, dehydroascorbic acid, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-1 receptor antagonist (IL-1Ra), IL-6, IL-10, TNF-receptor (TNF-R)I, TNF-RII and YKL-40. RESULTS: We found no significant increase in MDA in the muscle biopsies after reperfusion. Plasma levels of oxidative and pro- and anti-inflammatory parameters showed no significant differences between baseline and after reperfusion at any sampling time. CONCLUSION: Twenty minutes of lower limb ischaemia does not result in an ischaemia-reperfusion injury in healthy volunteers, measurable by oxidative and pro- and anti-inflammatory biomarkers in muscle biopsies and in the systemic circulation.

Single nucleotide polymorphisms in genes encoding toll-like receptors 7, 8 and 9 in Danish patients with systemic lupus erythematosus.

Several studies indicate a role for toll-like receptors (TLRs) in the pathogenesis of systemic lupus erythematosus (SLE). We aimed to investigate the risk of SLE and typical clinical and serological manifestations of SLE potentially conferred by selected single nucleotide polymorphisms (SNPs) of genes encoding TLR7, TLR8, and TLR9. Using a multiplexed bead-based assay, we analyzed eight SNPs in a cohort of 142 Danish SLE patients and a gender-matched control cohort comprising 443 individuals. Our results showed an association between the rs3853839 polymorphism of TLR7 and SLE (G vs. C, P = 0.008, OR 1.60, 95 % CI 1.12-2.27 in females; P = 0.02, OR 4.50, 95 % CI 1.18-16.7 in males) confirming recent findings in other populations. Additionally, an association between the rs3764879 polymorphism of TLR8 and SLE (G vs. C, P < 0.05, OR 1.36, 95 % CI 0.99-1.86 in females; P = 0.06, OR 4.00, 95 % CI 0.90-17.3 in males) was found. None of the other investigated SNPs were associated with SLE but several SNPs were associated with clinical and serological manifestations. In summary, a previously shown association between the rs3853839 SNP of TLR7 and SLE in Asian patients was also found in Danish patients. Together with the association of several other SNPs of TLR8 and TLR9 with various clinical and serological manifestations of SLE these findings corroborate the pathogenic significance of TLRs in SLE.

Genetic polymorphisms of dsRNA ligating pattern recognition receptors TLR3, MDA5, and RIG-I. Association with systemic lupus erythematosus and clinical phenotypes.

This study aimed to demonstrate possible associations between genetic polymorphisms in Toll-like receptor 3, interferon induced with helicase C domain 1 (IFIH1) and DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 and systemic lupus erythematosus (SLE), including the phenotypes lupus nephritis and malar rash, as well as the presence of autoantibodies against nucleic acid-containing complexes. Genotyping was carried out in two Danish cohorts [Copenhagen (CPH) and Odense (ODE)] totaling 344 patients and was compared with 641 previously genotyped healthy controls. In the ODE cohort, the patients were only genotyped for the rs1990760 polymorphism of IFIH1. Single nucleotide polymorphisms (SNPs) were determined by a multiplex bead-based assay (CPH cohort) or real-time PCR (ODE cohort). Associations were investigated using the Cochran-Armitage trend test. The odds ratio (OR) for minor allele homozygotes versus major allele homozygotes suggested a protective effect of the IFIH1 rs1990760 SNP for SLE in the ODE cohort [OR 0.52, 95 % confidence intervals (95 % CI) 0.31-0.88, Pcorr. = 0.05] but not in the CPH cohort, although the OR suggested a trend in the same direction, and when combining the two patient cohorts, ORs were 0.57, 95 % CI 0.37-0.88. None of the other investigated polymorphisms showed any association with SLE. Regarding phenotypes, we found a statistically significant association between rs1990760 and malar rash in the CPH cohort, with ORs suggesting a protective effect (OR 0.28, 95 % CI 0.13-0.62 for heterozygotes and OR 0.11, 95 % CI 0.03-0.41 for homozygotes, Pcorr. = 0.0001). There were no significant associations between rs1990760 and presence of anti-dsDNA, anti-U1RNP, or anti-Smith antibodies. Our study supports previous findings of an association between the rs1990760 polymorphism of IFIH1 and SLE and indicates that this SNP may also be associated with malar rash in SLE patients although this finding needs confirmation.

In vitro cytokine responses to periodontal pathogens: generalized aggressive periodontitis is associated with increased IL-6 response to Porphyromonas gingivalis.

Generalized aggressive periodontitis (GAgP) is an inflammatory condition resulting in destruction of tooth-supporting tissues. We examined the production of IL-1beta, IL-6, tumour necrosis factor (TNF)-alpha, IL-12 and IL-10 in cultures of peripheral mononuclear cells (MNC) from 10 patients with GAgP and 10 controls stimulated with periodontal pathogens or a control antigen, tetanus toxoid (TT) in the presence of autologous serum. The pathogens used were Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum, either as type strains or bacteria isolated from the participants' inherent oral flora. The P. gingivalis -induced production of IL-6 was approximately 2.5-fold higher in patients with GAgP than in healthy controls (P < 0.05), while the corresponding TNF-alpha production was non-significantly elevated. IL-1beta production induced by P. gingivalis, as all cytokine responses induced by Pr. intermedia, F. nucleatum and TT was similar in the two groups. A reduced IL-12p70 response to Pr. intermedia and F. nucleatum was observed in smokers compared to non-smoking patients (P < 0.02). To assess the role of serum factors in the elevated IL-6 response to P. gingivalis, MNC from two donors free of disease were stimulated with this bacterium in the presence of the various patient and control sera. An elevated IL-6 and TNF-alpha response was observed in the presence of patient sera (P < 0.01 and P < 0.04, respectively). The data suggest that an exaggerated production of IL-6 occurs in GAgP, and that pro-inflammatory serum factors play an essential role in the response.

Specific and strain-independent effects of dexamethasone in the prevention and treatment of experimental autoimmune encephalomyelitis in rodents.

Experimental autoimmune encephalomyelitis in rodents (EAE) is a generally accepted in vivo model for immunopathogenic mechanisms underlying multiple sclerosis (MS). There are, however, different forms of rodent EAE, and therapeutic regimens may affect these forms differently. We have therefore tested the effects of dexamethasone (Dex) and found that both prophylactic and early therapeutic regimens were effective in suppressing the development of monophasic EAE in myelin basic protein-immunized Lewis rats, the relapsing-remitting forms of EAE induced in SJL mice by proteolipid protein and in DA rats by syngeneic spinal cord homogenate, and the progressive forms induced in C57BL/6 and DBA/1 mice by immunization with myelin oligodendrocyte glycoprotein. In addition, prophylactically administered Dex suppressed histological and immunological features of EAE such as spinal cord infiltration of inflammatory cells and the increased frequency of autoantigen-specific interferon-gamma-secreting lymph node mononuclear cells. The present data reproduced in rodent EAE models some of the beneficial effects observed with glucocorticoids in MS. This strengthens the validity of these five models as in vivo predictors of drug efficacy in at least some variants of human MS. Better understanding of the clinical and immunopharmacologic features of these models might prove useful when testing new drug candidates for MS treatment.

Formation of antibodies against infliximab and adalimumab strongly correlates with functional drug levels and clinical responses in rheumatoid arthritis.

BACKGROUND: Tumour necrosis factor alpha (TNFalpha) neutralising antibody constructs are increasingly being used to treat rheumatoid arthritis (RA). OBJECTIVE: To determine potential differences in clinical responses, soluble drug levels and antibody formation between patients with RA receiving infliximab and adalimumab. METHODS: 69 patients with RA fulfilling the 1987 American College of Rheumatology criteria and about to start treatment with infliximab or adalimumab, were enrolled consecutively. All patients had active disease (28-joint count Disease Activity Score >3.2). Infliximab was given intravenously at 3 mg/kg at baseline and after 2, 6 and 14 weeks. Adalimumab was administered as 40 mg biweekly subcutaneously. Concomitant drug treatment was monitored and continued at constant dosage during the study. All serum samples were tested for infliximab/adalimumab levels and anti-infliximab/anti-adalimumab antibodies. RESULTS: 35 patients received infliximab, 34 received adalimumab. At 6 months, 15 (43%), 6 (17%) and 14 (40%) of the infliximab-treated patients fulfilled the EULAR criteria for good, moderate and non-responders, respectively, whereas the corresponding figures for adalimumab-treated patients were 16 (47%), 8 (24%) and 10 (29%). Clinical responses correlated with the levels of S-infliximab/adalimumab and the formation of anti-infliximab/anti-adalimumab antibodies. CONCLUSION: The clinical response to two anti-TNFalpha biological agents closely follows the trough drug levels and the presence of antibodies directed against the drugs. Further studies that focus on the underlying pathways leading to antibody formation are warranted to predict immunogenicity of these expensive biological agents and treatment outcomes.

Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans.

Activated mononuclear cells appear to be important effector cells in autoimmune beta cell destruction leading to insulin-dependent (type 1) diabetes mellitus. Conditioned medium from activated mononuclear cells (from human blood) is cytotoxic to isolated rat and human islets of Langerhans. This cytotoxic activity was eliminated from crude cytokine preparations by adsorption with immobilized, purified antibody to interleukin-1 (IL-1). The islet-inhibitory activity and the IL-1 activity (determined by its comitogenic effect on thymocytes) were recovered by acid wash. Purified natural IL-1 and recombinant IL-1 derived from the predominant pI 7 form of human IL-1, consistently inhibited the insulin response. The pI 6 and pI 5 forms of natural IL-1 were ineffective. Natural and recombinant IL-1 exhibited similar dose responses in their islet-inhibitory effect and their thymocyte-stimulatory activity. Concentrations of IL-1 that inhibited islet activity were in the picomolar range. Hence, monocyte-derived pI 7 IL-1 may contribute to islet cell damage and therefore to the development of insulin-dependent diabetes mellitus.

Methylprednisolone does not restore biological response in multiple sclerosis patients with neutralizing antibodies against interferon-ß.

BACKGROUND AND PURPOSE: Neutralizing antibodies (NAbs) appearing during treatment with Interferon-beta (IFN-beta) reduce or abolish bioactivity and therapeutic efficacy. Initial combination therapy with methylprednisolone (MP) may reduce the frequency of NAb positive patients. We hypothesized that MP treatment might also reduce NAb levels and re-establish IFN-beta bioactivity in patients already NAb+, who discontinue IFN-beta therapy. METHODS: In a 6-month open-label trial, we compared monthly high-dose pulsed MP treatment in 38 Nab positive patients with 35 NAb+, MP-untreated control patients discontinuing any therapy or switching to glatiramer acetate. All patients were NAb+ with an absent in vivo response to IFN-beta. NAbs were measured using a cytopathic effect assay and expressed as neutralizing capacity (NC) in percentage of added IFN-beta. Bioactivity was expressed as in vivo Myxovirus Resistance Protein A (MxA) mRNA induction in whole blood using real time PCR. RESULTS: At the end of study, median NAb NC was 92% in both groups. Eight patients (21%) in the MP group and four patients (11%) in the control group had regained an in vivo MxA response to IFN-beta (P = 0.35). CONCLUSIONS: Monthly pulsed MP treatment in NAb positive patients has no beneficial effect on NAb status or IFN-beta bioactivity.

Lewis and AB0 blood group-phenotypes in periodontitis, cardiovascular disease, obesity and stroke.

The AB0 blood group has been linked to ischaemic heart disease, stroke, and periodontal disease, while the Lewis blood group has been linked to ischaemic heart disease and obesity, all of which have been associated with periodontitis. AB0 or Lewis blood group phenotype may therefore constitute common hereditary components predisposing to these disorders. In this study, we investigated if blood group phenotype associated with periodontitis in a subpopulation consisting of 702 participants from a Danish cross-sectional cohort and, secondarily, attempted to confirm their association with hypertension, ischaemic heart disease, stroke, and obesity. No significant association between blood group phenotype and periodontitis was detected, nor were previously reported associations between blood group phenotype and hypertension, ischaemic heart disease, stroke, and obesity confirmed. This may, at least partly, be attributed to differences in study type, outcome definitions, cohort sizes, and population attributable factors. However, our results suggested a strong association between self-reported stroke and the Lewis (a-b-) phenotype (P = 0.0002, OR: 22.28; CI 95: 4.72-131.63).

Cytokines, autoantibodies and viral antibodies in premorbid and postdiagnostic sera from patients with rheumatoid arthritis: case-control study nested in a cohort of Norwegian blood donors.

OBJECTIVE: To assess the timing of changes in cytokines, cytokine-related markers, autoantibodies and viral antibodies in the pathogenesis of rheumatoid arthritis (RA). METHODS: Case-control study nested in a prospective cohort of 31 330 blood donors in Oslo, Norway. Forty-nine donors developed RA up to 23 years after their most recent blood donation. Stored sera from these donors (case sera) and a sex- and age-matched sample of 245 healthy donors (control sera), and postdiagnostic sera from 33 of the 49 RA cases, were analysed for a panel of cytokines and cytokine-related markers, autoantibodies and antibodies against Epstein-Barr virus and parvovirus B19. RESULTS: Cytokines and cytokine-related markers were generally negative in case sera from >5 years before the diagnosis of RA. In the 5-year interval immediately before the diagnosis of RA, more case than control sera were positive (odds ratios >2) for interleukin (IL)-1 alpha, IL-1 beta, IL-1 receptor antagonist, IL-4, IL-10, tumour necrosis factor-alpha and soluble tumour necrosis factor receptor I. In postdiagnostic sera, however, 11 of 16 examined cytokines and cytokine-related markers were statistically significantly elevated compared with control sera. Seropositivity for IgG antibodies against cyclic citrullinated peptides and for IgM and IgA rheumatoid factors were seen in case sera from up to 18 years before the diagnosis of RA. IgG antibodies against Epstein-Barr virus and parvovirus B19 did not differ significantly between case and control sera. CONCLUSIONS: Cytokines and cytokine-related markers appear to be upregulated rather late in RA pathogenesis. In contrast, IgM rheumatoid factor and IgG anti-cyclic citrullinated peptide autoantibodies may precede the diagnosis of RA by up to two decades.

Infections and treatment of patients with rheumatic diseases.

Glucocorticoids (GCs) have many complex quantitative and qualitative immunosuppressive effects which induce cellular immunodeficiency and increase host susceptibility to various viral, bacterial, fungal and parasitic infections. As cortisol secretion is inadequate in chronic immune/inflammatory conditions, and current therapies have the aim of providing adequate (low) compensatory doses, the timing of GC administration, such as during the nocturnal turning-on phase of tumour necrosis factor (TNF) secretion, can be extremely important. The use of the lowest possible GC dose, at night, and for the shortest possible time should therefore greatly reduce the risk of infections. Infection is a major co-morbidity in rheumatoid arthritis (RA), and conventional disease-modifying anti-rheumatic drugs (DMARDs) can increase the risk of their occurrence, including tuberculosis. TNF-alpha plays a key role in the pathogenesis of RA, and the data concerning infections in RA patients treated with anti-TNF agents are controversial. Patients and physicians should vigilantly monitor for signs of infection when using anti-TNF agents. Recombinant gene technologies now make it possible to produce protein drugs that are almost identical to naturally occurring human polypeptides, including antibody (Ab) constructs; unfortunately, all human biological agents are potentially immunogenic. An increasing number of recent studies have demonstrated the safety of influenza and pneumococcal vaccines administered to patients with systemic lupus erythematosus (SLE) or RA. These vaccinations are generally immunogenic (i.e., capable of inducing a protective level of specific antibodies) but may not induce an adequate response in a substantial proportion of patients.

Infusion of hypertonic saline before elective hysterectomy: effects on cytokines and stress hormones.

BACKGROUND: Infusion of hypertonic saline provides early haemodynamic benefits and may affect the immune system. It is unknown if infusion of hypertonic saline affects plasma cytokines and stress hormones after surgery. METHODS: Sixty-two women undergoing abdominal hysterectomy were randomized in a double-blind study to infusion of NaCl 7.5% (HS), NaCl 0.9% (NS4), both 4 ml kg(-1), or NaCl 0.9% 32 ml kg(-1) (NS32) over 20 min. Blood was collected at baseline, 1, 4, and 24 h after surgery (n=34) for the determination of interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12, IL-1ra, and tumour necrosis factor-alpha. Serum cortisol and vasopressin were measured at these time points and 48 h after operation. Epinephrine and norepinephrine (n=26) were quantified at baseline, after infusion, 25 min after incision, 1, and 4 h after surgery. Finally, C-reactive protein was measured at baseline, 24, and 48 h after surgery. RESULTS: Surgery and anaesthesia induced well-reported changes in the concentrations of cytokines and hormones. The concentration of norepinephrine briefly increased after infusion of HS and NS32 but not NS4 (P<0.05). Epinephrine was increased 25 min after incision in Group NS32 compared with the other groups (P<0.05). No other differences were found between the groups. CONCLUSIONS: Infusion of a clinically relevant dose of hypertonic saline before hysterectomy appears to have limited effect on the postoperative concentration of selected plasma cytokines and the hormonal stress-response.

Binding of cytokines to pharmaceutically prepared human immunoglobulin.

Pharmaceutically prepared IgG, pooled from sera of over 2,000 normal individuals, contained both monomeric and dimeric IgG. Each type of IgG bound 125I-labeled interleukin (IL)-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha. Increased binding to IgG was observed if 125I-IL-1 beta was denatured by heating to 39 degrees C. However, the binding of both nondenatured and denatured 125I-IL-1 beta was not inhibited by unlabeled IL-1 beta. In contrast, binding of 125I-IL-1 alpha, 125I-IL-6, and 125I-TNF alpha was inhibited by the corresponding unlabeled cytokine. Papain-digestion of IgG abolished binding of 125I-TNF alpha but failed to influence the displaceable binding of 125I-IL-1 alpha and 125I-IL-6. 125I-TNF alpha was a mixture of trimeric and monomeric forms, the latter being the predominant form at lower concentrations. The apparent saturability of 125I-TNF alpha was explained by a higher nonspecific binding of monomeric than of trimeric 125I-TNF alpha to IgG. The amounts of cytokine antibodies in IgG preparations would contribute approximately 2 micrograms anti-IL-1 alpha IgG and 1 microgram anti-IL-6 IgG per kg body wt during high dose immune globulin therapy. In conclusion, pharmaceutical preparations of human IgG contain specific and neutralizing, high affinity antibodies against IL-1 alpha and IL-6, but not against TNF alpha or IL-1 beta. There are significant methodological pitfalls that hamper detection of IgG autoantibodies against cytokines.

Expression of an insulin/interleukin-1 receptor antagonist hybrid gene in insulin-producing cell lines (HIT-T15 and NIT-1) confers resistance against interleukin-1-induced nitric oxide production.

A hybrid gene consisting of the insulin gene enhancer/promoter region, the signal sequence, the insulin B- and C-chains, and the human interleukin-1 receptor antagonist (IL-1ra) gene was constructed. This hybrid gene was transfected together with the pSV2-neo construct into the insulin-producing cell lines HIT-T15 and NIT-1. One of the geneticin-selected clones, HITra2, expressed a 1.4-kb mRNA, which hybridized both to insulin and IL-1ra-cDNA in Northern blot analysis. Three proteins, with the mol wt 23, 17, and 14 kD, were immunoprecipitated with anti-IL-1ra antibodies from [35S]methionine-labeled HITra2 cells. Both at a low and at a high glucose concentration, 4-5 ng of IL-1ra/10(6) cells (ELISA) was released from these cells. On the other hand, a high glucose concentration evoked a three-fold increase in the release of insulin, suggesting that IL-1ra was released constitutively. Measured by nitrite production, transfected HIT, and NIT-1 cells exhibited a more than 10-fold decrease in IL-1 beta sensitivity. Since the conditioned culture media from the HITra2 cells exhibited an anti-IL-1 beta activity of only 0.5 U/ml, and mixed culture of HITra2 cells and isolated rat islets prevented IL-1 beta induced inhibition of insulin release, it is likely that IL-1ra acts locally at the cell surface. It is concluded that expression of a hybrid insulin/IL-1ra gene confers resistance to IL-1 and that this technique may be used to elucidate the role of IL-1 in autoimmune disorders such as insulin-dependent diabetes mellitus.

High avidity IFN-neutralizing antibodies in pharmaceutically prepared human IgG.

This paper demonstrates and characterizes naturally occurring antibodies to interferon (IFN) in human IgG preparations. In vitro neutralization of the antiviral effect of IFN alpha and IFN beta, but not IFN gamma, was observed in 12 of 15 normal IgG preparations. The neutralizing capacity was higher against rIFN alpha 2A and rIFN alpha 2C than against lymphoblastoid IFN alpha and IFN beta. Frühsommer meningoencephalitis hyperimmune IgG and hepatitis-B hyperimmune IgG showed potent neutralization, whereas anti-rhesus D-, anti-rabies-, and anti-tetanus IgG showed weak neutralization. Saturable binding of 125I-rIFN alpha 2A was demonstrated only in those IgG preparations found to neutralize the antiviral effect of IFN. Significant correlation between IFN binding and neutralization capacity was observed. The antibodies bound with Fab to rIFN alpha 2A with an avidity of approximately 30 pM; the majority was of the IgG1 subclass. Maximum binding capacity was 490 pg rIFN alpha 2A/mg IgG. Cross-binding of rIFN alpha 2C, lyIFN alpha N1 and IFN beta occurred with 10 and 100-200 times lower activities than that of rIFN alpha 2A. There was no cross-binding with rIFN gamma or rIL-6. IgG preparations containing anti-IFN antibodies blocked the binding of 125I-rIFN alpha 2A to A549 cells. In conclusion, pharmaceutically prepared human IgG preparations contain variable but significant levels of high-avidity IFN alpha and IFN beta neutralizing antibodies.

Cytokines suppress human islet function irrespective of their effects on nitric oxide generation.

Cytokines have been proposed as inducers of beta-cell damage in human insulin-dependent diabetes mellitus via the generation of nitric oxide (NO). This concept is mostly based on data obtained in rodent pancreatic islets using heterologous cytokine preparations. The present study examined whether exposure of human pancreatic islets to different cytokines induces NO and impairs beta-cell function. Islets from 30 human pancreata were exposed for 6-144 h to the following human recombinant cytokines, alone or in combination: IFN-gamma (1,000 U/ml), TNF-alpha (1,000 U/ml), IL-6 (25 U/ml), and IL-1 beta (50 U/ml). After 48 h, none of the cytokines alone increased islet nitrite production, but IFN-gamma induced a 20% decrease in glucose-induced insulin release. Combinations of cytokines, notably IL-1 beta plus IFN-gamma plus TNF-alpha, induced increased expression of inducible NO synthase mRNA after 6 h and resulted in a fivefold increase in medium nitrite accumulation after 48 h. These cytokines did not impair glucose metabolism or insulin release in response to 16.7 mM glucose, but there was an 80% decrease in islet insulin content. An exposure of 144 h to IL-1 beta plus IFN-gamma plus TNF-alpha increased NO production and decreased both glucose-induced insulin release and insulin content. Inhibitors of NO generation, aminoguanidine or NG-nitro-L-arginine, blocked this cytokine-induced NO generation, but did not prevent the suppressive effect of IL-1 beta plus IFN-gamma plus TNF-alpha on insulin release and content. In conclusion, isolated human islets are more resistant to the suppressive effects of cytokines and NO than isolated rodent islets. Moreover, the present study suggests that NO is not the major mediator of cytokine effects on human islets.

Monitoring patients treated with anti-TNF-alpha biopharmaceuticals: assessing serum infliximab and anti-infliximab antibodies.

OBJECTIVES: Infliximab is an anti-tumour necrosis factor-alpha (TNF-alpha) mouse-human IgG1/kappa antibody used to treat patients with rheumatoid arthritis (RA) and other inflammatory diseases. Unfortunately, response failure and side-effects due to immunogenicity of the drug are not rare. In this study, we have compared different methods of assessing drug levels and anti-infliximab antibodies (Abs) and analysed the character of these Abs in sera of RA patients treated with infliximab for 1.5-18 months. METHODS: Functional serum infliximab levels and anti-infliximab Abs were measured by fluid-phase RIAs using 125I-labelled ligands in combination with molecular size and affinity chromatography, and immune complex precipitation. RESULTS: Anti-infliximab Abs were predominantly IgG, 36% being IgG4, and half the immune complexes were lambda-light-chain-positive. Ab titres were associated with inhibition of TNF binding to the drug, and low trough levels of infliximab were most frequent in anti-infliximab Ab-positive sera. Cross-binding to two other anti-TNF drugs was not observed. Detection of anti-infliximab Abs by solid-phase RIA using cross-binding of plastic-fixed and soluble infliximab exhibited low sensitivity and the data were inconsistent with results obtained from binding of the Abs to soluble infliximab. CONCLUSIONS: Specific and neutralizing anti-infliximab antibodies develop in RA patients treated with infliximab, and that low trough levels of functional infliximab are associated with the presence of such antibodies. The most sensitive antibody assay involved binding to soluble and intact infliximab. Assessments of bioavailability and immunogenicity of anti-TNF biologicals may be used to optimize dose regimens and prevent prolonged use of inadequate therapy.

Induction of interleukin-6 (IL-6) autoantibodies through vaccination with an engineered IL-6 receptor antagonist.

Neutralization of cytokine activity by monoclonal antibodies or receptor antagonists is beneficial in the treatment of immune and neoplastic diseases, but the necessity for continuous parenteral delivery of these anticytokine agents poses considerable practical limitations. A viable alternative is to induce a neutralizing antibody response. Using transgenic mice with high circulating levels of human interleukin-6 (hIL-6), we show that injection of the hIL-6 receptor antagonist Sant1 (an IL-6 variant with seven amino-acid substitutions) induces a strong anti-hIL-6 antibody response. The elicited antibodies bind circulating hIL-6 with very high affinity, totally masking it, and neutralize hIL-6 bioactivity both in vitro and in vivo.

Differential effect of conditioning regimens on cytokine responses during allogeneic stem cell transplantation.

The purpose of this study was to characterize cytokine responses during conditioning in patients undergoing allogeneic stem cell transplantation (SCT) with the aim to identify which markers that may reliably reflect inflammatory activity during conditioning. We investigated inflammatory and anti-inflammatory mediators in plasma samples drawn daily during the conditioning of 20 patients. Soluble tumour necrosis factor alpha receptor I (sTNFRI) increased during the conditioning reflecting the type of conditioning given. Antithymocyte globulin (ATG) was the most potent inducer of sTNFRI (288% increase (median) P=0.002), followed by VP-16 (184%, P=0.03), cyclophosphamide (129%, P=0.03) and total body irradiation (148%, P=0.0005). Administration of i.v. busulfan (Busilvex; BU) was not associated with significant changes in sTNFRI levels. At day 0 (the day of stem cell infusion) the sTNFRI levels were not only elevated compared with baseline (188% increase), P<0.0001), they also correlated with the baseline values (r=0.72, P=0.0003). The levels of tumour necrosis factor, interleukin (IL)-1beta, IL-6, IL-8, IL-10 and IL-12 stayed at low levels during the conditioning, except from a transient increase in the levels of IL-6, IL-8, IL-10 and IL- receptor antagonist (IL-Ra) seen after ATG infusion. These findings suggest that further investigation of circulating sTNFRI levels may be of interest in studies of prognostic factors in SCT.