Recombinant proteins in rheumatology - recent advances.

New targeted anti-inflammatory drugs have revolutionized the therapeutic strategies in rheumatology. Recombinant DNA selection technologies have enabled the isolation and humanization of specific antibody fragments of any specificity that can be 'armed' to deliver effective anti-inflammatory 'payloads'. Antibodies and other targeted provide the opportunity to block key soluble mediators of inflammation in their milieu, or alternatively to block intracellular inflammation-triggering pathways by binding to an upstream cell-surface receptor. Designed proteins can be improved with respect to desired pharmacokinetic and pharmacodynamic effects. They facilitate the delivery of the required immunosuppressive effect. However, the individual extent of desired and undesired effects of a particular biologic therapy can be broader than initially predicted and requires careful evaluation during clinical trials. This review highlights advances in the application of recombinant antibody technology for novel biologic therapies in rheumatology.

Soluble CSF interleukin 2 receptor as indicator of neurosarcoidosis.

Neurosarcoidosis (NS) represents an important differential diagnosis of multiple sclerosis (MS). However, thus far no reliable laboratory marker of neurosarcoidosis exists. The objective of this study was to evaluate whether cerebrospinal fluid (CSF) levels of soluble interleukin 2 receptor (sIL2-R) distinguish NS and other inflammatory disorders of the central nervous system. For this purpose, 139 paired CSF and serum samples from 11 patients with NS, 21 with MS, 10 with CNS vasculitis, 22 with bacterial meningitis, 17 with viral meningitis/encephalitis, seven with neurotuberculosis, and 18 healthy donors were assessed for sIL2-R using an enzyme-linked immunosorbent assay. We found that sIL2-R CSF levels above 150 pg/ml identified untreated NS patients with an overall accuracy of 93% against a group of non-infectious CNS-diseases. Furthermore, an increase in sIL2-R in the CSF was associated with and preceded the outbreak of new neurological symptoms. In conclusion, these findings suggest that sIL2-R measurement in the CSF may be a valuable tool in the diagnosis and follow-up of patients with suspected and proven neurosarcoidosis.

An open-label pilot study of the efficacy and safety of anakinra in patients with psoriatic arthritis refractory to or intolerant of methotrexate (MTX).

Only limited data have been published about the therapeutic use of anakinra in patients with psoriatic arthritis. We undertook this study to evaluate the efficacy and safety of anakinra in patients with active psoriatic arthritis. In a prospective open-label single-center study, 20 patients were treated with 100 mg anakinra everyday either alone or in combination with ongoing methotrexate over 6 months. Safety and efficacy was evaluated using Psoriasis Arthritis Response Criteria (PsARC), Disease Activity Score (DAS) 28, American College of Rheumatology (ACR), European League Against Rheumatism (EULAR), Psoriasis Area and Severity Index Score, Dactylitis Score and Health Assessment Questionnaire (HAQ), and the C-reactive protein, and erythrocyte sedimentation rate. Of the 20 patients enrolled, six completed 24 weeks, 18 completed 12 weeks, and 19 completed 4 weeks of treatment. Early-treatment termination was mainly due to inefficacy (13 patients) and only one drop-out occurred because of an unrelated adverse event. Six patients fulfilled continuously the PsARC until week 24. A moderate EULAR response was achieved by four patients and a good EULAR response by three patients in week 24. Five patients reached ACR 20, four patients ACR 50, and two patients ACR 70 in week 24. HAQ improved slightly throughout the study (n = 19, mean (SD); baseline, 1.127 (0.671); week 24, 1.055 (0.812)) just as DAS 28 (n = 16; baseline, 4.7(1.5); week 24, 4.0(2.0)). Only nine patients showed skin manifestations affecting >3% of their body surface area which improved in two, worsened in four, stabilized in two patients, and newly evolved in one patient. Adverse events were mainly mild (95%). Fifteen (75%) patients showed injection site reactions. No serious infections occurred. Anakinra was well tolerated with no occurrence of serious drug-associated adverse events and lead to improvement of signs and symptoms in nine out of 19 patients, therefore providing a potential therapeutic option in patients with active psoriatic arthritis.

Effect of anakinra on functional status in patients with active rheumatoid arthritis receiving concomitant therapy with traditional disease modifying antirheumatic drugs: evidence from the OMEGA Trial.

OBJECTIVE: To assess changes in functional status in patients with rheumatoid arthritis (RA) receiving the interleukin-1 receptor antagonist anakinra in addition to a disease modifying antirheumatic drug (DMARD). METHODS: In this large, multicenter, open-label, single-arm study, adult patients with RA receiving methotrexate, sulfasalazine, or hydroxychloroquine for > or = 3 months were given anakinra 100 mg once daily for up to 36 weeks. The primary objective was to evaluate changes from baseline to week 36 in the Health Assessment Questionnaire (HAQ) disability index and subscales. Changes in the 28-joint Disease Activity Score (DAS28), proportion of patients meeting European League Against Rheumatism (EULAR) response criteria, and the safety of each combination regimen were also assessed. RESULTS: A total of 1207 patients were enrolled, received > or = 1 dose of anakinra, and were included in the efficacy and safety analyses. A statistically significant change in the HAQ disability index was observed (p = 0.0001); no significant differences were seen among the 3 DMARD groups. A clinically meaningful improvement in HAQ (> 0.22) was observed in 51% of patients. Mean improvement in DAS28 was 1.5 (p < 0.0001), and 64% of patients achieved a good or moderate EULAR response score. Injection site reaction was the most frequently (62%) reported adverse event. The incidence of infections (24%), most commonly respiratory infection, was similar across treatment groups. No notable changes were observed in laboratory findings and vital signs. CONCLUSION: These findings indicate that anakinra 100 mg/day in combination with DMARD therapy safely improved functional status in patients with active RA.

Relevance of the organic cation transporters 1 and 2 for antiretroviral drug therapy in human immunodeficiency virus infection.

Carrier-mediated transport across cell membranes is an important determinant of activity, resistance, and toxicity of chemotherapeutic agents including antiretroviral (ARV) drugs (ARDs). The organic cation transporters (OCTs) 1 and 2 have been implicated in the translocation of different cationic drugs but so far were insufficiently tested for interactions with ARDs. Here, we assessed among cationic drugs commonly used in human immunodeficiency virus (HIV) therapy inhibitors and substrates of OCTs, and analyzed the tissue distribution of OCTs and their expression in lymph nodes (LNs), the primary intracellular target of HIV and ARDs. Inhibitors were identified by measuring the attenuated uptake of the radiolabeled model substrate 1-methyl-4-phenylpyridinium into OCT-transfected human embryonic kidney-293 cells in the presence of ARDs. Substrates were identified by measuring OCT-specific intracellular accumulation using liquid chromatography/tandem mass spectrometry. Inhibitory drugs were (in order of increasing potency): nelfinavir < ritonavir < saquinavir < indinavir < trimethoprim < pentamidine, with consistently lower IC(50) values determined for OCT1. Substrates with highest transport efficacy (V(max)/K(m)) were lamivudine (OCT1, 8 microl/mg protein/min; OCT2, 4.4 microl/mg protein/min) and zalcitabine (OCT1, 4.1 microl/mg protein/min; OCT2, 2.6 microl/mg protein/min). Using quantitative real-time polymerase chain reaction, a marked expression level of OCT1 was detected in human samples of liver, ovary, prostate, and testis, and of OCT2 in kidney, colon, heart, skeletal muscle, and testis. Expression of OCTs in LNs was low in HIV-negative control individuals but dramatically increased in HIV-infected persons. These data suggest that drug interactions about the OCTs may be relevant for the ARV therapy, in particular by influencing drug accession to infected tissues and hepatic or renal elimination.

Association of rheumatoid arthritis with ergothioneine levels in red blood cells: a case control study.

OBJECTIVE: The dietary thiol compound and erythrocyte ingredient ergothioneine (ET) is the preferential physiological substrate of the organic cation transporter OCTN1, found to be associated with rheumatoid arthritis (RA) in genetic studies, but the biological roles of ET and OCTN1 are unclear. We investigated the association between ET concentrations in peripheral blood erythrocytes and the occurrence of RA. METHODS: Erythrocyte ET concentrations in patients with mildly active RA (n = 73) were compared to ET levels in patients with coronary heart disease (CHD; n = 62) and osteoarthritis (OA; n = 148), serving as non-RA chronic inflammatory disease controls. Correlation of ET levels in erythrocytes with levels of ET and OCTN1 mRNA in CD14+ monocytes was determined in 10 healthy subjects. RESULTS: Erythrocyte ET levels were significantly higher in patients with RA, with a median (interquartile range) of 12.6 micromole/l of erythrocytes (IQR 8.1-18.3), compared to 7.7 (IQR 5.0-12.0; p < 0.001) in CHD and 7.8 (IQR 4.8-12.8; p < 0.001) in OA. The prevalence of RA compared to non-RA controls increased with increasing blood ET concentrations, with an odds ratio of 0.23 (95% CI 0.13-0.41; p < 0.001) in the lowest quartile of RA erythrocyte ET levels to 3.11 (95% CI 1.54-6.29; p = 0.002) in the highest quartile. The group differences in ET values were maintained after adjustment for disease-related anthropometric and clinical variables (age, sex, body mass index, smoking, duration of disease, hemoglobin, C-reactive protein, and medication) and were also independent of erythrocyte glutathione levels and of polymorphisms of the OCTN1 gene. ET levels in erythrocytes were linearly correlated with ET concentrations (R2 = 0.936, p < 0.001) and OCTN1 mRNA levels (R2 = 0.946, p < 0.001) in CD14+ cells. CONCLUSION: Mildly active cases of RA are associated with an unexplained high level of ET in red blood cells.

Blockade of TNF does not alter oxygen burst and phagocytosis of human neutrophils in patients with rheumatoid arthritis.

Clinical trials evaluating tumor necrosis factor alpha (TNF-alpha) binding agents in patients with rheumatoid arthritis (RA) have demonstrated significant efficacy in reducing symptoms of disease and slowing radiographic progression. However, infectious complications are the most severe and common adverse effects of anti-TNF therapy. The functional capacities of neutrophils (PMNs) as the first line of defense in bacterial and fungal infections are enhanced by soluble TNF as a potent neutrophil primer. The aim of this study was to assess the influence of in vivo TNF blockade on oxygen burst (OB) and phagocytosis of human neutrophils. PMNs were derived from 20 patients with RA on anti-TNF-alpha therapy and 13 patients using conventional DMARDs. By flow cytometry we measured OB upon stimulation with Escherichia coli and N-formyl-1-methionyl-1-leucyl-phenylalanine (FMLP) with and without priming with granulocyte-colony stimulating factor (G-CSF) and/or TNF-alpha using dihydrorhodamine (DHR) 123. Phagocytosis of fluorescein isothiocyanate (FITC)-labeled E. coli was also assessed by flow cytometry. Thirty-three healthy volunteers served as controls. Upon stimulation with E. coli and FMLP, there was no significant difference in OB between the two patient groups and healthy controls. Priming was effective in all groups. Phagocytosis of E. coli by PMNs was equally effective in controls and patients independent from the treatment regimen. These data show that OB, phagocytosis and responsiveness to priming with TNF and G-CSF of PMNs are not impaired in patients with RA treated with anti-TNF agents in comparison with patients on conventional DMARDs or healthy controls. Thus, the infectious complications observed in patients with TNF blockade cannot be explained by functional impairment of PMNs; however, the neutralization of TNF as a potent primer of neutrophil response may increase the susceptibility for infections in these patients.

Discovery of the ergothioneine transporter.

Variants of the SLC22A4 gene are associated with susceptibility to rheumatoid arthritis and Crohn's disease. SLC22A4 codes for an integral membrane protein, OCTN1, that has been presumed to carry organic cations like tetraethylammonium across the plasma membrane. Here, we show that the key substrate of this transporter is in fact ergothioneine (ET). Human OCTN1 was expressed in 293 cells. A substrate lead, stachydrine (alias proline betaine), was identified by liquid chromatography MS difference shading, a new substrate search strategy. Analysis of transport efficiency of stachydrine-related solutes, affinity, and Na+ dependence indicates that the physiological substrate is ET. Efficiency of transport of ET was as high as 195 microl per min per mg of protein. By contrast, the carnitine transporter OCTN2 from rat did not transport ET at all. Because ET is transported >100 times more efficiently than tetraethylammonium and carnitine, we propose the functional name ETT (ET transporter) instead of OCTN1. ET, all of which is absorbed from food, is an intracellular antioxidant with metal ion affinity. Its particular purpose is unresolved. Cells with expression of ETT accumulate ET to high levels and avidly retain it. By contrast, cells lacking ETT do not accumulate ET, because their plasma membrane is virtually impermeable for this compound. The real-time PCR expression profile of human ETT, with strong expression in CD71+ cells, is consistent with a pivotal function of ET in erythrocytes. Moreover, prominent expression of ETT in monocytes and SLC22A4 polymorphism associations suggest a protective role of ET in chronic inflammatory disorders.

Treatment of recalcitrant cicatricial pemphigoid with the tumor necrosis factor alpha antagonist etanercept.

The treatment of cicatricial pemphigoid is generally regarded as difficult and usually relies on individual clinical experience. Corticosteroids, as drugs of first choice, often have to be combined with steroid-sparing agents to prevent hazardous, long-term side effects. We describe a 72-year-old woman with long-standing cicatricial pemphigoid recalcitrant to established treatment regimens who responded rapidly and lastingly to therapy with the tumor necrosis factor alpha antagonist etanercept. To our knowledge, this is the first report of its use in the treatment of a bullous autoimmune disease.