Peter Charles

Repeated therapy with monoclonal antibody to tumour necrosis factor α (cA2) in patients with rheumatoid arthritis

Our in-vitro, animal, and early clinical data suggest that tumour necrosis factor alpha (TNF alpha) is an important target for specific biological therapy in rheumatoid arthritis. We report the results of repeated treatment with a chimeric monoclonal antibody to TNF alpha (cA2) in patients having disease flares. 7 patients originally enrolled in an open-label trial completed two to four cycles, each of which was followed by a good clinical response, with median improvements in the swollen-joint count and C-reactive protein exceeding 80%. cA2 may be useful therapy in the control of acute disease flares in rheumatoid arthritis and treatment programmes including cA2 may be effective in the long-term management of this disease.

Antibodies to citrullinated α‐enolase peptide 1 are specific for rheumatoid arthritis and cross‐react with bacterial enolase

OBJECTIVE To map the antibody response to human citrullinated alpha-enolase, a candidate autoantigen in rheumatoid arthritis (RA), and to examine cross-reactivity with bacterial enolase. METHODS Serum samples obtained from patients with RA, disease control subjects, and healthy control subjects were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with citrullinated alpha-enolase peptides. Antibodies specific for the immunodominant epitope were raised in rabbits or were purified from RA sera. Cross-reactivity with other citrullinated epitopes was investigated by inhibition ELISAs, and cross-reactivity with bacterial enolase was investigated by immunoblotting. RESULTS An immunodominant peptide, citrullinated alpha-enolase peptide 1, was identified. Antibodies to this epitope were observed in 37-62% of sera obtained from patients with RA, 3% of sera obtained from disease control subjects, and 2% of sera obtained from healthy control subjects. Binding was inhibited with homologous peptide but not with the arginine-containing control peptide or with 4 citrullinated peptides from elsewhere on the molecule, indicating that antibody binding was dependent on both citrulline and flanking amino acids. The immunodominant peptide showed 82% homology with enolase from Porphyromonas gingivalis, and the levels of antibodies to citrullinated alpha-enolase peptide 1 correlated with the levels of antibodies to the bacterial peptide (r2=0.803, P<0.0001). Affinity-purified antibodies to the human peptide cross-reacted with citrullinated recombinant P gingivalis enolase. CONCLUSION We have identified an immunodominant epitope in citrullinated alpha-enolase, to which antibodies are specific for RA. Our data on sequence similarity and cross-reactivity with bacterial enolase may indicate a role for bacterial infection, particularly with P gingivalis, in priming autoimmunity in a subset of patients with RA.

Monoclonal anti-TNF-alpha antibody as a probe of pathogenesis and therapy of rheumatoid disease

Rheumatoid arthritis is a common cause of chronic disability for which current therapies are of limited value in controlling the disease process and outcome. Our initial approach to understanding the pathogenesis of RA and defining a novel therapeutic target was to investigate the role of cytokines by blocking their action with antibodies on cultured synovial-derived mononuclear cells in vitro. These investigations suggested that neutralization of TNF alpha with antibodies significantly inhibited the generation of other pro-inflammatory cytokines also over-produced, such as, IL-1, GM-CSF, IL-6 and IL-8. The implication that blockade of a single cytokine, TNF alpha might have far-reaching effects on multiple cytokines and thereby exert significant anti-inflammatory and protective effects on cartilage and bone of joints, was tested in arthritic DBA/1 mice immunized with collagen II. Impressive amelioration of joint swelling and joint erosions in this model encouraged clinical trials with a monoclonal anti-TNF alpha antibody. The cA2 chimeric anti-TNF alpha high-affinity antibody was initially tested in an open-label study at a dose of 20 mg/kg on 20 patients, with substantial and universal benefit. Subsequently, a randomized placebo-controlled double-blind trial was performed on 73 patients comparing a single intravenous injection of placebo (0.1% human serum albumin) with two doses of cA2. Using a composite disease activity index, at 4 weeks post infusion, 8% of patients receiving placebo improved compared with 44% receiving 1 mg/kg cA/2 and 79% receiving 10 mg/kg. Between 2 to 4 repeated cycles of cA2 were administered to 7 patients and all patients showed improvement of a similar magnitude with each cycle. These data support our proposition that TNF alpha is implicated in the pathogenesis of RA, and is thus a key therapeutic target. Monoclonal anti-TNF alpha antibodies control disease flares and are candidate agents for longer-term control of RA, although repeated therapy with cA2 is associated with anti-idiotypic responses in 50% of patients and a trend toward shortening of the duration of response. In the DBA/1 arthritic mice, synergy of action of anti-TNF and anti-CD4 is observed together with suppression of an anti-globulin response, indicating one way in which benefit might be augmented in the future.

Antinuclear antibodies: A contemporary nomenclature using HEp-2 cells

The choice of terms used to describe indirect immunofluorescence (IIF) staining patterns of autoantibodies binding to HEp-2 cells is at present quite varied and disordered because no accurate consensus on names and descriptions exist. The aim of our study was to propose a logical and ordered IIF classification taxonomy based on 29 different selected IIF patterns. In a preliminary project carried out at Statens Serum Institut it was first shown by use of a software programme named DOORS developed by Percepton Ltd, that reading of digitized images of HEp-2 patterns on an LCD monitor could be used instead of traditional microscopy. Digitized images of HEp-2 patterns were then used in the EU supported project named CANTOR (June 1998-July 2000) aiming to reach consensus among three clinical immunology expert centres and collaborating to attain a classification version that could be used to qualitatively and quantitatively test and train image recognitions skills of laboratory technicians against expert consensus. The usability of this classification version was then tested in a course consisting of training and certification. The conclusion was that participants in the training programme clearly increased their perceptive skills using images, terms, descriptions and the graphic and statistic tools in the self-administered DOORS programme and that software-assisted training could achieve a common and accurate level of visual pattern interpretation. All results from this project were reported to the European Commission but have not previously been published in scientific literature. This communication presents the final results of agreed image classifications.

Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis

Background Rheumatoid arthritis (RA) is characterised by autoimmunity to citrullinated proteins, and there is increasing epidemiologic evidence linking Porphyromonas gingivalis to RA. P gingivalis is apparently unique among periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate antigens driving the autoimmune response. Objectives To examine the immune response to PPAD in patients with RA, individuals with periodontitis (PD) and controls (without arthritis), confirm PPAD autocitrullination and identify the modified arginine residues. Methods PPAD and an inactivated mutant (C351A) were cloned and expressed and autocitrullination of both examined by immunoblotting and mass spectrometry. ELISAs using PPAD, C351A and another P gingivalis protein arginine gingipain (RgpB) were developed and antibody reactivities examined in patients with RA (n=80), individuals with PD (n=44) and controls (n=82). Results Recombinant PPAD was a potent citrullinating enzyme. Antibodies to PPAD, but not to Rgp, were elevated in the RA sera (median 122 U/ml) compared with controls (median 70 U/ml; p<0.05) and PD (median 60 U/ml; p<0.01). Specificity of the anti-peptidyl citrullinated PPAD response was confirmed by the reaction of RA sera with multiple epitopes tested with synthetic citrullinated peptides spanning the PPAD molecule. The elevated antibody response to PPAD was abolished in RA sera if the C351A mutant was used on ELISA. Conclusions The peptidyl citrulline-specific immune response to PPAD supports the hypothesis that, as a bacterial protein, it might break tolerance in RA, and could be a target for therapy.

Efficacy and safety of apremilast, an oral phosphodiesterase 4 inhibitor, in ankylosing spondylitis

Objectives To evaluate the efficacy and safety of an oral phosphodiesterase 4 inhibitor, apremilast, in treatment of ankylosing spondylitis (AS) by monitoring symptoms and signs in a pilot study including exploratory investigation of effects of PDE4 inhibition on blood biomarkers of bone biology. Methods In this double-blind, placebo-controlled, single-centre, Phase II study, patients with symptomatic AS with active disease on MRI were randomised to apremilast 30 mg BID or placebo over 12 weeks. Bath Indices were monitored serially. Patients were followed for 4 weeks after stopping medication. Bone biomarkers were assessed at baseline and day 85. Results 38 subjects were randomised and 36 subjects completed the study. Although the primary end-point (change in BASDAI at week 12) was not met, apremilast was associated with numerically greater improvement from baseline for all clinical assessments compared with placebo with mean change in BASDAI (−1.59±1.48 vs −0.77±1.47), BASFI (−1.74±1.91 vs −0.28±1.61) and BASMI (−0.51±1.02 vs −0.21±0.67); however, differences did not achieve statistical significance. The clinical indices returned to baseline values by 4 weeks after cessation of apremilast. Six apremilast patients (35.3%) vs 3 placebo (15.8%) achieved ASAS20 responses (p=0.25). There were statistically significant decreases in serum RANKL and RANKL:osteoprotegrin ratio and plasma sclerostin but no significant changes in serum DKK-1, bone alkaline phosphatase, TRAP5b, MMP3, osteoprotegrin, or osteocalcin. Conclusions Although a small pilot study, these results suggest that apremilast may be effective and well tolerated in AS and modulates biomarkers of bone biology. These data support further research of apremilast in axial inflammation.

Anti-tumour necrosis factor specific antibody (infliximab) treatment provides insights into the pathophysiology of rheumatoid arthritis.

Preclinical studies based on in vitro cell systems and in vivo models had established a position for tumour necrosis factor (TNF) α as a pivotal molecule regulating cellular activation and interactions in rheumatoid inflammation by 1992. That neutralisation of TNFα has a profound impact on the biology of inflammation is reflected by the rapid reduction in the concentration of C reactive protein (CRP), an acute phase protein, associated with a fall in the level of its well known inducer, interleukin (IL) 6. This supports the hypothesis that TNFα is a critical part of a regulatory cytokine network. The reduction in clinical signs of inflammation were soon shown by arthroscopic examination and synovial biopsies of knee joints to be a consequence of reduction in the density of infiltrating lymphocytes and macrophages. Circulating numbers of lymphocytes increased transiently after infliximab in a dose dependent fashion, associated with a reduction in soluble adhesion molecules E selectin, ICAM-1, and density of cells staining for these and VCAM-1 in synovial biopsies. The dynamics of cell trafficking have been examined by tracking the fate of the indium111 labelled polymorphonuclear cells injected intravenously in rheumatoid arthritis (RA) patients. These experiments show a reduction in uptake of radioactivity in joints after anti-TNF treatment indicating reduced adhesiveness and retention of the leucocytes in joints. The results of anti-TNF treatment on the progression of damage to cartilage, bone and other connective tissue components is not yet established in RA, although in collagen induced arthritis in DBA/1 mice, joint protection was reported. Measurement of circulating matrix metalloproteinases, MMP-1 and MMP-3, in their inactive form, has been noted following infliximab in patients. Recently we have documented a reduction in raised concentrations of serum VEGF, an important angiogenic factor after infliximab treatment. More direct evidence of a reduction in angiogenesis has been …

Autoantibodies against aquaporin-4 in patients with neuropsychiatric systemic lupus erythematosus and primary Sjögren's syndrome.

Neurologic manifestations occur in up to 70% of patients with systemic lupus erythematosus (SLE) and in 20% of patients with Sjögren’s syndrome (SS) and are associated with significant morbidity. There is as yet no biologic marker that specifically indicates neurologic involvement in rheumatic diseases. In the literature to date, the association between nervous system manifestations of SLE and autoantibodies against ribosomal P proteins and N-methyl-Daspartate receptor has been inconsistently demonstrated (1). Transverse myelopathy is a rare but serious condition reported in patients with SLE and SS (2,3). In sera from some of these patients, an IgG autoantibody marker, called NMOIgG, has been recently detected (4,5). NMO-IgG was initially identified by immunohistochemistry using brain tissue in sera from patients with neuromyelitis optica (NMO; also known as Devic’s disease), a severe demyelinating disorder of the central nervous system (CNS) that primarily affects the spinal cord and the optic nerves (6). In patients presenting with isolated longitudinally extensive transverse myelitis (LETM) involving at least 3 vertebral segments, as revealed using magnetic resonance imaging, and in patients with recurrent optic neuritis (ON), the presence of NMO-IgG indicates severe disease course with frequent relapses (7,8). NMO-IgG is not detectable in the sera of patients with multiple sclerosis (MS) (6). The antigenic target of NMO-IgG is aquaporin-4 (AQP-4), the most abundant water channel in the CNS (9). To detect these autoantibodies, assays employing recombinant AQP-4 have been repeatedly shown to be more sensitive than immunohistochemistry in CNS tissue (10). In order to evaluate the significance of NMO-IgG/ AQP-4 antibodies as potential markers of neurologic involvement in rheumatic conditions, we collected serum samples from patients with SLE (n 48) and SS (n 44), 28 and 22 of whom showed neurologic manifestations, respectively (Table 1). All patients fulfilled the American College of Rheumatology (ACR) classification criteria for SLE and SS (11,12). SLE patients with neurologic involvement fulfilled the ACR case definitions for neuropsychiatric lupus syndromes (13). Blood was drawn from 3 SLE patients (2 with LETM and 1 with LETM and recurrent ON) within 7 days of onset of a relapse. In the other SLE subsets, the time intervals between the serum sampling and the onset of the first neurologic symptoms or, in cases of relapses, the onset of the last relapse were as follows: TM, 1 year; LETM, 1–7 years (median 4 years); recurrent ON, 1 year; chorea, 1 year; psychosis/depression, 4–20 years (median 9.5 years); seizure disorders, 13 and 20 years. The duration of symptoms in SLE patients with polyneuropathy was 2–12 years (median 5 years). In patients with SS, time intervals were 7 and 8 years in 2 patients with TM and concomitant monophasic ON, 5 years in 1 patient with TM alone, and 13 years in both patients with LETM. Symptoms were present for 1–8 years (median 3 years) in SS patients with polyneuropathy. Samples were analyzed both by immunohistochemistry using native primate cerebellum, cerebrum, and optic nerve tissue sections and, for the first time in patients with SLE and SS, by means of a recombinant immunofluorescence assay (rIFA) using AQP-4–transfected HEK cells fixed in formalin. In direct comparison with the original procedure for the detection of NMO-IgG as described by Lennon et al (6), the rIFA exhibited an increase in sensitivity of 12.5% (overall sensitivity 78.1%; specificity 100%) in an earlier study based on 183 samples from NMO patients and relevant neurologic disease controls, including MS (14). Serum samples were classified as being positive or negative for NMO-IgG/AQP-4 antibodies by 2 independent investigators who were unaware of the clinical data. Testing was performed for diagnostic purposes in all cases. In SLE and SS, NMO-IgG/AQP-4 antibodies were found exclusively in patients with neurologic involvement comprising LETM or recurrent ON (Table 1). In the SLE cohort, autoantibodies against AQP-4 were detected in all of these patients, whereas NMO-IgG was found in 6 of 7 individuals, once more indicating a higher sensitivity of the recombinant cell substrate compared with immunohistochemistry. Antibody titers as determined by AQP-4–transfected HEK cells ranged from 1:10–1:1,000 in the 5 patients with LETM. The titer was 1:100 in the patient with recurrent ON and 1:3,200 in the patient with LETM and recurrent ON. In the SS group, 1 of the 2 patients with LETM was positive for NMO-IgG/AQP-4 antibodies, at a titer of 1:1,000. None of the SLE and SS patients without neurologic involvement or symptoms other than LETM or recurrent ON tested positive for NMO-IgG/AQP-4 antibodies (i.e., at a starting dilution of 1:10, antibodies were not detected in the serum samples from these patients). Our data demonstrate that autoantibodies against AQP-4 generally are not a marker for neurologic involvement in SLE and SS. Consistent with the findings of previous reports, our observations strongly support the notion that the presence of autoantibodies against AQP-4 is highly correlated with a distinct clinical phenotype, i.e., LETM and recurrent ON, which together are typical for NMO (6–8). Apparently, autoantibodies against AQP-4 are a marker for NMO that may occur either as an isolated syndrome or as part of a rheumatologic disease like SLE or SS. Of particular interest is the fact that 2 of the AQP-4–positive SLE patients (1 with LETM and 1 with recurrent ON) also had myasthenia gravis. This observation further supports the concept of coexisting independent, antibody-mediated autoimmune disorders in the same patient. It can be argued that the long intervals between the onset of neurologic symptoms and the time of blood sampling in most of the patients might have influenced the serologic findings. However, it is known from NMO patients with long-term followup that AQP-4 antibodies are detectable in the serum during relapse as well as during remission, suggesting that AQP-4 antibody testing can be of diagnostic relevance independently of disease activity (15). In clinical practice, myelitis and ON occurring in patients with SLE and SS can be

Anti-PL-7 (Anti-Threonyl-tRNA synthetase) Antisynthetase syndrome: Clinical manifestations in a series of patients from a european multicenter study (EUMYONET) and review of the literature

AbstractAutoantibodies against several aminoacyl-transfer-RNA synthetases have been described in patients with myositis; anti-threonyl-tRNA synthetase (anti-PL-7) is one of the rarest. We describe the clinical and laboratory characteristics of a cohort of European anti-PL-7 patients, and compare them with previously reported cases. This multicenter study of patients positive for anti-PL-7, identified between 1984 and 2011, derives from the EUMYONET cohort. Clinical and serologic data were obtained by retrospective laboratory and medical record review, and statistical analyses were performed with chi-squared and Fisher exact tests.Eighteen patients, 15 women, were anti-PL-7 antibody positive. Median follow-up was 5.25 years (interquartile range, 2.8–10.7 yr), and 4 patients died. All patients had myositis (12 polymyositis, 5 dermatomyositis, and 1 amyopathic dermatomyositis), 10 (55.6%) had interstitial lung disease, and 9 (50%) had pericardial effusion. Occupational exposure to organic/inorganic particles was more frequent in patients with interstitial lung disease than in the remaining patients (5 of 10 vs. 1 of 7; p = 0.152), although the difference was not significant. Concurrent autoantibodies against Ro60 and Ro52 were seen in 8 of 14 (57%) patients studied. In the literature review the most common manifestations of anti-PL-7 antisynthetase syndrome were interstitial lung disease (77%), myositis (75%), and arthritis (56%). As in other subsets of the antisynthetase syndrome, myositis and interstitial lung disease are common features of the anti-PL-7 antisynthetase syndrome. In addition, we can add pericarditis as a possible manifestation related to anti-PL-7 antibodies.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists.

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.