Incidence of anti-neutrophil cytoplasmic antibody-associated vasculitis before and after the February 2011 Christchurch Earthquake.

BACKGROUND: It has been suggested that environmental pollution from an earthquake might be associated with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). AIM: To determine the incidence of AAV during the 3-year period before (period 1), and the 3 years following (period 2), the earthquake that occurred on 22 February 2011 in Christchurch, New Zealand. METHODS: All ANCA tests performed in the Canterbury region for 3 years before the earthquake (period 1, 2007-2010), and for 3 years after the earthquake (period 2, 2011-2014) were examined. AAV was defined according to The European Medicines Agency classification algorithm. Medical records were reviewed and cases were included if they were newly diagnosed within the study period. Incidence was calculated using population data from the 2013 New Zealand census. RESULTS: A total of 52 new cases of AAV was identified. The incidence in period 1 was 1.87/100 000/annum (95% C.I. 1.23-2.72), and for period 2 was 1.73/100 000/annum (95% C.I. 1.12-2.55). There was no statistically significant difference in incidence between the two study periods. There was no difference when analysing by myeloperoxidase (MPO) or proteinase-3 status, or restricting the analyses to those residing in an urban environment. The mean age at diagnosis for MPO AAV was significantly younger in period 2 than period 1 (61 years vs 71 years, P = 0.05). There were no other clinically important differences between the two groups. CONCLUSION: This study does not support the hypothesis that an environmental agent, caused by dust pollution related to earthquake damage, has a causative role in the pathogenesis of AAV.

Wegener's granulomatosis: treatment and survival characteristics in a high-prevalence southern hemisphere region.

Our aim was to describe the treatment and survival characteristics of Wegener's granulomatosis (WG) in Canterbury, New Zealand. The medical records of 48 patients diagnosed between 1 July 1997 and 31 December 2008 and fulfilling validated classification criteria for WG were reviewed to characterise survivorship in the province of Canterbury, New Zealand. The age at diagnosis was 61 years (range 20 to 83 years) with an equal number of males and females. Using Kaplan-Meier product-limit analysis, the probability of survival at 1 and 10 years was 91% and 62% respectively. Of the 12 deaths in the cohort, four occurred within 12 months of diagnosis: two from pulmonary haemorrhage and two from renal failure. Beyond 12 months, two patients died of renal failure, two of myocardial infarction and one from cardiac arrhythmia, one from cerebrovascular disease and two from colorectal carcinoma. The median time to relapse was 6.75 years, and the probability of relapse within 10 years was 67%. Survivorship, treatment response rate and the rate of relapse from WG in a cohort of patients from this high-prevalence southern hemisphere region were similar to that reported for northern hemisphere cohorts with a similar prevalence.

Relationship between serum urate and plasma oxypurinol in the management of gout: determination of minimum plasma oxypurinol concentration to achieve a target serum urate level.

The treatment of gout requires a lowering of serum urate (SU) levels, and allopurinol is the drug that is most commonly used for this purpose. The objectives of this study were to define the relationships between allopurinol dose on the one hand and plasma oxypurinol, renal function, and SU levels on the other and to determine the minimum plasma oxypurinol concentration that would result in a target level of <6 mg/dl (0.36 mmol/l) of SU. For this purpose, 82 patients who had been receiving allopurinol for at least 1 month were recruited. Patients with SU <6 mg/dl were followed up quarterly for 12 months. In patients with SU ≥6 mg/dl, the dose of allopurinol was increased to bring the level of SU to <6 mg/dl. These patients were followed up once a month until the SU level remained at <6 mg/dl for 3 consecutive months; thereafter they were seen quarterly. SU, creatinine, and plasma oxypurinol were measured 6-9 hours after administration of the allopurinol dose. There were significant inverse correlations between creatinine clearance (CrCl) and plasma oxypurinol (P = 0.002), between allopurinol dose and SU (P < 0.0001) and between plasma oxypurinol and SU (P < 0.0001). Using receiver operating characteristic analysis, the target SU of <6 mg/dl was achieved in 75% of serum samples with plasma oxypurinol levels of >100 µmol/l (15.2 mg/l). Increasing the allopurinol dose resulted in increased plasma oxypurinol and reduced SU concentrations. Plasma oxypurinol concentrations >100 µmol/l were required to achieve SU <6 mg/dl.

Lack of association between elevated mean red cell volume and haematological toxicity in patients receiving long-term methotrexate for rheumatoid arthritis.

AIMS: It has been suggested that elevated mean red cell volume (MCV) may be a predictor of haematological toxicity in rheumatoid arthritis (RA) patients receiving methotrexate (MTX). We wished to identify whether there was an association between MCV, red cell folate and haematological toxicity in patients on MTX monotherapy for the long-term management of RA. METHODS: Evidence of haematological toxicity was sought by note review of patients recruited in a cross-sectional study of MTX monotherapy in RA. Retrospective data included MCVs from before MTX initiation and after 3 and 6 months of treatment. Data were collected prospectively every 6 months for up to 2 years after enrolment. Any record of cytopenia or the development of haematological malignancy was recorded from commencement of MTX until the present day. Red cell folate concentrations were tested on enrolment to the study. RESULTS: A total of 165 patients was included, 74.5% female, median disease duration 7 years (range 3 months-57 years). The median duration of MTX treatment was 74.9 months (range 10-241 months) giving 1030.2 patient-years of MTX exposure. Twenty-four patients (14.5%) had a MCV > 98 fL on study entry. Evidence of haematological abnormality was found in six patients (3.6%); chronic lymphocytic leukaemia (1), persistent lymphocytosis (1), persistent monocytosis (1) and neutropenia (3). There was no association between red cell folate or MCV and haematological toxicity. CONCLUSION: Neutropenia and pancytopenia are rare side-effects of MTX therapy in this cohort. Elevated MCV or low mean red cell folate does not appear to be associated with haematological malignancy or toxicity in this cohort of patients on long-term MTX therapy.

Evidence for association of an interleukin 23 receptor variant independent of the R381Q variant with rheumatoid arthritis.

OBJECTIVE: The rare allele of a non-synonymous interleukin 23 receptor (IL23R) single nucleotide polymorphism (SNP) rs11209026 (p.Arg381Gln) confers strong protection against Crohn disease (CD) and psoriasis. Other IL23R variants also exhibit association with CD, genetically independent of rs11209026. In rheumatoid arthritis (RA), IL23 is an important determinant of the production of IL17A, a cytokine of consequence in inflammation and bone destruction. While there is no previous support for strong association of IL23R with RA, the possibility of a weaker role for IL23R variants in the aetiology of RA cannot be eliminated. METHODS: A New Zealand RA cohort was tested for association with six IL23R SNPs and the resulting data combined with a reanalysis of the Wellcome Trust Case Control Consortium data and a previously published Spanish data set. The combined data set totals over 3000 Caucasian cases and 3800 controls, which has sufficient power to detect a risk of as low as odds ratio (OR) = 1.2. RESULTS: Our data emphasise the lack of association of rs11209026 with RA (OR 1.01, 95% confidence interval (CI) 0.88 to 1.16, p = 0.86). However there was some evidence for association of rs1343151 with RA (OR 1.14, 95% CI 1.06 to 1.22, p = <0.001). CONCLUSIONS: While requiring further replication, these data further support a role for the IL17A/IL23 pathway in RA. Understanding how different variants of IL23R associate, at varying levels of strength, with contrasting groups of immune-mediated diseases (CD, psoriasis, ankylosing spondylitis, RA) will enhance knowledge on the aetiology of these diseases.

Evidence for an influence of chemokine ligand 3-like 1 (CCL3L1) gene copy number on susceptibility to rheumatoid arthritis.

OBJECTIVE: There is increasing evidence that gene copy-number variation influences phenotypic variation. Chemokine ligand 3-like 1 (CCL3L1) is encoded by a variable copy-number gene, and binds to several pro-inflammatory cytokine receptors, including chemokine receptor 5 (CCR5). Considering lymphocyte recruitment by beta-chemokines is a feature of autoimmunity, and that the CCR5Delta32 variant is associated with protection to rheumatoid arthritis (RA), we hypothesised that CCL3L1 copy-number influences susceptibility to RA and type 1 diabetes (T1D). METHODS: We measured CCL3L1 copy-number in 1136 RA cases from New Zealand (NZ) and the UK, 252 NZ T1D cases and a total of 1470 controls. All subjects were ancestrally Caucasian. RESULTS: A copy-number higher than 2 (the most common copy number) was a risk factor for RA in the NZ cohort (odds ratio (OR) 1.34, 95% CI 1.08-1.66, p = 0.009) but not the smaller UK RA cohort (OR 1.09, 95% CI 0.75-1.60, p = 0.643). There was evidence for association in the T1D cohort (OR 1.46, 95% CI 0.98-2.20, p = 0.064) and in the combined RA/T1D cohort (OR 1.30, 95% CI 1.00-1.54, p = 0.003). Genetic interaction between CCL3L1 dosage and CCR5 genotype was found; the increased genetic risk conferred by higher CCL3L1 copy-number was ablated by a dysfunctional CCR5 (CCR5Delta32). CONCLUSIONS: These data suggest that increased CCL3L1 expression may enhance inflammatory responses and increase the chance of autoimmune disease. Genetic interaction data were consistent with a biologically plausible model; CCR5Delta32 protects against RA and T1D by blocking signalling through the CCR5 pathway, mitigating the pro-inflammatory effects of excess CCL3L1.

Wegener's granulomatosis in New Zealand: evidence for a latitude-dependent incidence gradient.

BACKGROUND: The aim of the study was to determine whether there was evidence for a geographic gradient in the incidence of Wegener's granulomatosis (WG) and WG-like disease in New Zealand (NZ). METHODS: The National Minimum Dataset of the Ministry of Health, NZ was searched for individual patient discharges coded by the International Classification of Diseases 10th Revision, Australian Modification as either M301 (polyarteritis with lung involvement, including Churg Strauss and allergic granulomatous angiitis) or M313 (WG, necrotizing respiratory granulomatosis) for the period 1 January 1999 to 31 December 2003. Data were standardized using the 2001 NZ census. RESULTS: One hundred and ninety-five patients (95 men) were given a first-time discharge code of either M301 (40 patients) or M313 (155 patients). No gender bias was seen. The rate among Europeans was twice that of NZ Maoris or Asians. The rate of disease peaked in the age band 70-79 years and during winter months. A significant positive north-south geographic gradient was present for M313. No difference in the rate of readmission or time to relapse between geographic regions was found for M313. CONCLUSION: A north-south gradient in the rate of patient discharges given a diagnostic code of M313 (WG, necrotizing respiratory granulomatosis) was present in NZ. This finding supports the hypothesis that there is a latitude-dependent risk factor(s) for WG possibly common to both global hemispheres.

Emerging therapies in the long-term management of hyperuricaemia and gout.

Gout is a common chronic arthritis that can lead to significant disability. Gout is one of the few rheumatological conditions that can be diagnosed with certainty, has a known cause and can be cured with appropriate therapy. Hypouricaemic agents reduce uric acid concentrations through inhibiting uric acid production (allopurinol) or enhancing uric acid excretion (probenecid, benzbromarone). Allopurinol is the most commonly used hypouricaemic agent but at recommended doses often fails to reduce adequately uric acid concentrations and prevent acute attacks of gout. The use of probenecid is limited by lack of efficacy in renal impairment. In the last few years, new agents in the management of hyperuricaemia and gout have become available. Febuxostat, a new xanthine oxidase inhibitor, is an effective hypouricaemic agent although further data are required for patients with renal impairment and other significant medical conditions. Rasburicase, a recombinant uricase (which catalyses the conversion of uric acid to the more readily excreted allantoin) is available for prevention of tumour lysis syndrome. However, its repeated use, as would be required in chronic gout, is limited by antigenicity. A less antigenic PEGylated uricase can rapidly reduce serum uric acid concentrations and promote resorption of tophi. However, further information with regard to the long-term risks and benefits of these agents is required. These agents may ultimately be used in the short term to rapidly deplete urate stores (induction therapy) followed by long-term therapy with an alternative hypouricaemic agent to prevent subsequent accumulation of uric acid (maintenance therapy).

The epidemiology of Wegener's granulomatosis and microscopic polyangiitis in a Southern Hemisphere region.

OBJECTIVE: To determine the prevalence of Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA) in the province of Canterbury, New Zealand. METHOD: Three hospital clinical databases and the immunology laboratory database were searched and case notes reviewed for patients fulfilling either the 1990 American College of Rheumatology (ACR) criteria for WG or a modification of those criteria that allowed for antineutrophil cytoplasmic antibody (ANCA) positivity in the absence of granulomatous vasculitis. MPA was defined by the Chapel Hill consensus definition; however, in the absence of histological evidence of pauci-immune glomerulonephritis, ANCA positivity in association with evidence of active glomerular disease was included as a criterion. The point prevalence at 31 December 2003 and the 5-yr period prevalence for the interval 1 January 1999 to 31 December 2003 were calculated. RESULTS: Seventy-three patients with WG and 28 patients with MPA fulfilled the inclusion criteria. A 5-yr period prevalence of 152 WG cases/million [95% confidence interval (CI) 117-186] and 58 MPA cases/million (95% CI 37-80) was calculated using 2001 census data as denominator. Nineteen patients with WG died and 10 patients with MPA died during the study period, resulting in a point prevalence for survivors at 31 December 2003 of 112 cases/million (95% CI 82-142) and 37 cases/million (95% CI 20-55), respectively. Using unmodified ACR criteria the 5-yr period and point prevalence for WG were 131/million (95% CI 99-163) and 93.5/million (95% CI 66-121), respectively. Apart from respiratory tract involvement, which formed part of the case definition of WG, organ involvement was similar in both diseases. CONCLUSION: The prevalence of WG and MPA in Canterbury is the highest reported to date. Restricting the case definition of WG to the ACR classification criteria we found a prevalence equivalent to that described in northern Norway. The clinical severity and serological characteristics were similar to descriptions in other WG and MPA patient cohorts. Studies of disease prevalence in other Southern Hemisphere centres will determine if the observed north-south negative disease gradient in the Northern Hemisphere is reciprocated.

Adult-onset inflammatory myopathy: North Canterbury experience 1989-2001.

AIM: To perform a clinical audit of all patients diagnosed with inflammatory myopathy in the North Canterbury region. METHODS: A retrospective case note audit of patients with a discharge diagnosis of inflammatory myopathy from June 1989 to June 2001 was performed. The audit was based at Christchurch Hospital, New Zealand, which services a population of 430,000. RESULTS: Of 77 case notes reviewed, 44 patients were identified who were considered to fulfil clinical criteria for inflammatory myopathy. There was a female preponderance (80% female, 20% male). Diagnostic categories in descending order of frequency included: dermatomyositis (41%), polymyositis (39%), inclusion body myositis (IBM) (14%) and overlap syndromes (6%). Malignancy-associated myositis occurred in 20% overall (dermatomyositis 11%, polymyositis 9%). Delays in diagnosis and late age at presentation (average 72 years) were seen in the IBM group. Proximal limb weakness was common, but not universal at presentation (80%). A muscle biopsy was performed in all patients and electromyography in 82%. All were treated with high dose prednisone (0.5-1 mg/kg) of whom 29% were maintained on prednisone alone. Immunosuppressives/immunomodulators used included: azathioprine (58%), methotrexate (31%), intravenous immunoglobulin (13%), chlorambucil (13%), and cyclophosphamide (9%). Thirteen patients (42%) required more than one agent, with three trialling five agents. There were 59 relapses in 20 patients (45%), with mean time to first relapse of 7.8 months. At audit completion, 33% had deceased with malignancy and respiratory failure the main causes. CONCLUSION: Inflammatory myopathy is a challenging condition in both diagnosis and management. Our audit has shown delays in the diagnosis of IBM, a relatively high incidence of malignancy and a notable risk of relapse and mortality.

Scintigraphy using a technetium 99m-labelled anti-E-selectin Fab fragment in rheumatoid arthritis.

OBJECTIVE: We previously described a novel radiolabelled monoclonal antibody (1.2B6), which reacts with porcine E-selectin, for targeting activated endothelium as a means of imaging inflammatory disorders, and presented initial clinical work based on (111)In-labelled antibody. The aim of the present study was to evaluate a Fab fragment of 1.2B6 labelled with (99m)Tc in patients with rheumatoid arthritis (RA) by comparison with (i) (111)In-labelled 1.2B6 F(ab')(2) and (ii) conventional bone scanning. METHODS: (99m)Tc-1.2B6-Fab ( approximately 440 MBq) and (111)In-1.2B6-F(ab')(2) ( approximately 27 MBq) were compared in 10 patients using a double-isotope protocol. Images were obtained 4 and 20-24 h after injection. Two normal volunteers were also imaged. In a separate group of 16 patients, (99m)Tc-1.2B6-Fab and (99m)Tc-oxidronate ((99m)Tc-HDP) ( approximately 740 MBq) were compared on the basis of visual and semi-quantitative analysis of joint uptake (joint/soft tissue ratios) 4 h after injection. The respective biodistributions and blood clearances of the two 1.2B6 fragments were also compared. RESULTS: Image contrast was slightly better with (99m)Tc-Fab at 4 h but equal for the two tracers at 24 h. Diagnostic accuracy, taking joint tenderness or swelling as the clinical endpoint, was 76% for both fragments at 24 h. Plasma clearance of (99m)Tc-Fab was faster than that of (111)In-F(ab')(2) (t(1/2) 142 vs 421 min; P<0.0001). (99m)Tc-Fab appeared somewhat unstable in vivo, as shown by activity in the thyroid gland and bowel. The diagnostic accuracy of (99m)Tc-Fab was 88%, higher than that of (99m)Tc-HDP (57%) as a result of the low specificity of the latter in RA. Receiver operating characteristic (ROC) curve analysis using joint/soft tissue ratios as a variable cut-off showed that (99m)Tc-Fab discriminates better than (99m)Tc-HDP between actively inflamed and silent joints (Z=4.72; P<0.0001). No uptake of (99m)Tc-Fab was observed by inactive or normal joints, whereas (99m)Tc-HDP was taken up by all joints to a variable degree, making the decision as to whether a particular joint is actively involved or chronically damaged very difficult. CONCLUSION: (99m)Tc-anti-E-selectin-Fab scintigraphy can be used successfully to image synovitis with better specificity than (99m)Tc-HDP bone scanning. The advantages over (111)In-1.2B6-F(ab')(2) are easier availability of the radionuclide, improved physical properties and optimal imaging 4 h after injection.

Reduction of chemokine levels and leukocyte traffic to joints by tumor necrosis factor alpha blockade in patients with rheumatoid arthritis.

OBJECTIVE: To verify the hypothesis that in rheumatoid arthritis (RA), tumor necrosis factor alpha (TNFalpha) plays a critical role in regulating leukocyte trafficking and chemokine levels. METHODS: Ten patients with longstanding RA received a single 10 mg/kg infusion of anti-TNFalpha monoclonal antibody (cA2). The articular localization of autologous granulocytes, separated in vitro and labeled with 111In, was studied by analysis of gamma-camera images both before and 2 weeks after treatment. At the same sequential time points, synovial biopsy samples were assessed for infiltrating CD3+ T cells, CD22+ B cells, and CD68+ macrophages. Synovial tissue expression of the chemokines interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, Groalpha, and RANTES was also determined. Serum IL-8 and MCP-1 concentrations were measured by enzyme-linked immunosorbent assay. RESULTS: Anti-TNFalpha therapy in RA significantly reduced 111In-labeled granulocyte migration into affected joints. There was a simultaneous and significant reduction in the numbers of infiltrating synovial CD3+ T cells, CD22+ B cells, and CD68+ macrophages and in the expression of IL-8 and MCP-1, with a trend toward a reduction in serum concentrations of these chemokines. CONCLUSION: TNFalpha blockade reduces synovial expression of the chemokines IL-8 and MCP-1 and diminishes inflammatory cell migration into RA joints.

Pulmonary granulocyte kinetics in relation to endothelial and granulocyte activation.

The aim of the study was to measure the peripheral blood levels of soluble E-selectin in patients with systemic inflammation and compare them with in vivo granulocyte activation, pulmonary intravascular granulocyte pooling, pulmonary extravascular granulocyte migration and 99mTc-diethylenetriaminepenta-acetic acid (DTPA) aerosol clearance, an index of lung injury. The level of soluble E-selectin was measured by capture ELISA. Granulocytes were labelled with 111In and 99mTc for quantification of pulmonary granulocyte kinetics. The pulmonary vascular granulocyte pool (PGP) was expressed as a fraction of the total blood granulocyte pool. Pulmonary granulocyte migration was quantified on 24-h images using the 111In signal. Granulocyte activation was quantified as the percentage of circulating cells showing shape change ('primed'). Lung injury was assessed from the clearance rate of inhaled 99mTc-DTPA aerosol. Eighteen patients with systemic inflammation were studied: five with inflammatory bowel disease, eight with systemic vasculitis, four with graft versus host disease and one with a recent renal transplant. The peripheral blood levels of soluble E-selectin were significantly elevated in patients with systemic inflammation. The level of soluble E-selectin showed a significant association with granulocyte migration (Spearman rank correlation coefficient, Rs=0.53; P<0.05) but not with PGP or with the percentage of cells showing shape change (P>0.05 for both). Granulocyte migration was bimodal: patients were therefore subdivided into 'migrators' and 'non-migrators'. Soluble E-selectin level, 99mTc-DTPA clearance and PGP, but not the percentage of cells showing shape change, were significantly higher in migrators than in non-migrators. We conclude that pulmonary intravascular granulocyte pooling is increased in the presence of increased numbers of circulating primed granulocytes but increased pooling does not by itself promote granulocyte migration into the lung interstitium. Insofar as an elevated level of E-selectin in peripheral blood reflects vascular endothelial activation, the data are consistent with the notion that pulmonary endothelial activation is required, in addition to granulocyte activation and an expanded PGP, for granulocyte migration into lung parenchyma and, therefore, for lung injury to occur.

The inhibition of myeloperoxidase by ceruloplasmin can be reversed by anti-myeloperoxidase antibodies.

BACKGROUND: The purpose of this study was to characterize the recently reported inhibition of myeloperoxidase (MPO) by ceruloplasmin and to determine whether this may be disturbed in the presence of anti-MPO antibodies. METHODS: Specificity of the binding between ceruloplasmin and MPO was confirmed by Western blotting and enzyme-linked immunosorbent assay (ELISA), and the enzymatic activity of MPO was measured in the presence of ceruloplasmin, affinity-purified anti-MPO antibodies, or both. The affinity of the binding between MPO and ceruloplasmin and MPO and the anti-MPO antibodies was measured using a biosensor, with the results confirmed by chaotrope ELISA. RESULTS: Affinity-purified anti-MPO antibodies from patients with microscopic polyangiitis and florid renal vasculitis inhibited the binding between ceruloplasmin and MPO to a maximum of 72.9 +/- 12.8%, whereas those from patients with Wegener's granulomatosis and only minimal renal involvement inhibited the binding to a maximum of only 36.8 +/- 10.9% (P < 0. 001), with comparable reversal of the ceruloplasmin-mediated inhibition of MPO activity. Measurement of the affinity of the interactions demonstrated that binding between MPO and the anti-MPO antibodies is stronger than that between MPO and ceruloplasmin (1.61 x 107 to 1.33 x 108 vs. 7.46 x 106 m-1), indicating that binding to the autoantibody would be favored in vivo. CONCLUSIONS: This study confirms a role for ceruloplasmin as a physiological inhibitor of MPO, and demonstrates how the inhibition may be disrupted in the presence of anti-MPO antibodies. Because a majority (16 of 21) of the antibodies did not themselves inhibit MPO activity, their interference with the inhibition mediated by ceruloplasmin may be brought about by steric hindrance consequent upon the binding of the antibody to a dominant epitope at or near the active site.

Expression of vascular cell adhesion molecule-1 by vascular endothelial cells in immune and nonimmune inflammatory reactions in the skin.

Expression of VCAM-1 was compared with that of E-selectin in cytokine-induced lesions and in delayed-type hypersensitivity reactions to tuberculin purified protein derivative (PPD) in pig skin. Lumenally expressed Ags were quantified by measuring localization in skin of i.v. injected (111)In-mAb 10.2C7 (anti-vascular cell adhesion molecule-1 (anti-VCAM-1), (125)I-mAb 1.2B6 (anti-E-selectin), and (99m)Tc-MOPC21 (control IgG1). Anti-VCAM-1 mAb uptake was greater following intradermal (i.d.) injection of TNF-alpha than following injection of IL-1, while the two cytokines induced similar uptake of anti-E-selectin. In immunologically naive pigs there was no detectable increase in anti-VCAM-1 after i.d. injection of PPD, although anti-E-selectin uptake was increased at 3 and 6 h. In contrast, i.d. injection of PPD in sensitized pigs led to increased uptake of both anti-VCAM-1 and anti-E-selectin at 6, 8, 24, and 48 h, each of which was significantly greater than the uptake of control IgG1 into the same lesions (each p < 0.01). Anti-TNF-alpha mAb abolished the increased uptake of anti-VCAM-1 3 and 8 h following i.d. injection of PPD in sensitized pigs and significantly inhibited uptake at 24 h (p = 0.0025), but did not significantly reduce uptake of anti-E-selectin. We conclude that in this delayed-type hypersensitivity model 1) E-selectin expression by endothelial cells follows sequential Ag nonspecific and immune-specific phases, 2) increased VCAM-1 expression by endothelial cells is only seen in sensitized animals, and 3) expression of VCAM-1 appears to be relatively more dependent on TNF-alpha than E-selectin. Differential expression of E-selectin and VCAM-1 may influence the leukocytic infiltrate during the course of nonspecific and immune-specific inflammatory reactions.

A comparison between 111In-anti-E-selectin mAb and 99Tcm-labelled human non-specific immunoglobulin in radionuclide imaging of rheumatoid arthritis.

We have developed and validated a method for imaging inflammation using a monoclonal antibody (1.2B6) against E-selectin, an endothelial-cell specific adhesion molecule. This study was undertaken to compare 111In-1.2B6 with 99Tcm-labelled non-specific IgG (99Tcm-HIG) in the detection of synovitis in 11 patients with rheumatoid arthritis (RA). Imaging was performed 4 h and 20-24 h post-injection (pi) of 555 MBq 99Tcm-HIG and 15 MBq 111In-1.2B6. Scintigraphic results were compared with clinical scores of joint involvement. Joint uptake was semiquantitated. The scintigraphic appearances with both tracers correlated well, although 111In-1.2B6 at 24 h showed the highest detection rate. Taking joint tenderness or swelling as evidence of clinical activity, the sensitivity of 111In-1.2B6 at 4 h and 24 h was 69% and 82%, respectively, compared with 69% and 62% for 99Tcm-HIG. 111In-1.2B6 also displayed abnormal activity over a number of joints that appeared silent on clinical examination. Joint-to-soft tissue ratios were higher for 111In-1.2B6 at 24 h (4.0 +/- 1.9; p < 0.0001 vs all) than at 4 h (2.4 +/- 1.4) or than for 99Tcm-HIG at 4 h and 24 h (1.6 +/- 0.5 and 2.3 +/- 0.7, respectively). Net 111In counts over joints increased significantly between 4 h and 24 h (mean change: 54 +/- 40%). This study demonstrates that 111In-1.2B6 scintigraphy is a sensitive method by which to assess RA activity and that targeting is more intense and specific than using 99Tcm-HIG. However, the optimum time for 111In-1.2B6 scintigraphy is 24 h whereas good results are already obtained with 99Tc-HIG at 4 h pi. Current efforts are directed at developing 99Tcm-labelled 1.2B6 for imaging endothelial activation.

Endothelial activation in monosodium urate monohydrate crystal-induced inflammation: in vitro and in vivo studies on the roles of tumor necrosis factor alpha and interleukin-1.

OBJECTIVE: There is relatively little direct evidence for the roles of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) in activating endothelium in vivo. The aim of this study was to use in vitro and in vivo models to investigate the contribution of these cytokines to both E-selectin expression and the recruitment of polymorphonuclear cells (PMN) in monosodium urate monohydrate (MSU) crystal-induced inflammation. METHODS: MSU crystals were incubated with freshly isolated mononuclear cells, after which the harvested supernatants were tested for their ability to induce E-selectin expression during coculture with human umbilical vein endothelial cells. Subsequent experiments were performed with the addition of neutralizing anticytokine antibodies/antisera. The role of TNF alpha was then studied in an MSU crystal-induced monarthritis model, in the presence or absence of anti-TNF alpha (5 mg/kg intravenously). 99mtechnetium (99mTc)-labeled PMN cells and (111)indium (111In)-labeled anti-E-selectin monoclonal antibody (MAb) 1.2B6 were intravenously administered 4 hours after intraarticular injection to quantify PMN recruitment and E-selectin expression in inflamed joints. RESULTS: MSU crystals were a potent stimulus for IL-1 and TNF alpha production by monocytes in vitro, and these cytokines fully accounted for MSU crystal-stimulated, monocyte-mediated endothelial activation. In the MSU crystal-induced monarthritis model, TNF alpha blockade was very effective in suppressing both E-selectin expression and PMN emigration into the inflamed joints, as judged by gamma-camera image analysis and postmortem tissue counting following the intravenous injection of 99mTc-PMN and 111In-anti-E-selectin MAb. CONCLUSION: IL-1 and TNF alpha appear to be the only factors released by monocytes following incubation with MSU crystals, which induce E-selectin expression in vitro. Anti-TNF alpha is effective in suppressing endothelial activation and PMN recruitment in vivo E-selectin imaging can be used to assess the endothelial response to therapy and may prove useful for clinical studies.

Use of a radiolabeled monoclonal antibody against E-selectin for imaging of endothelial activation in rheumatoid arthritis.

OBJECTIVE: To determine the potential of 111In-labeled anti-E-selectin monoclonal antibody (MAb) to image localized endothelial activation in rheumatoid arthritis (RA). METHODS: Fourteen patients with RA were studied after intravenous administration of 111In-labeled F(ab')2 fragments of MAb against the cytokine-inducible endothelial cell activation antigen E-selectin (MAb 1.2B6). To compare uptake of 1.2B6 with that of nonspecific immunoglobulin, 111In-labeled polyclonal human immunoglobulin (HIG) was separately administered to 6 of these patients and the relative uptake of each tracer was determined. RESULTS: Prominent and discrete uptake of the radiolabeled MAb 1.2B6 was clearly visible in inflamed joints of all patients. Compared with 111In-HIG, 111In-1.2B6 provided superior images in terms of sensitivity and image intensity. Furthermore, the distribution of uptake in inflamed joints was different for the 2 tracers, with 1.2B6 showing a more focal localization in synovium. CONCLUSION: This study demonstrates that it is possible to objectively assess E-selectin expression on activated endothelium in vivo in patients with RA, using a radiolabeled MAb. This technique has considerable potential for monitoring disease activity and response to therapy in inflammatory diseases.

Characterization of E-selectin expression, leucocyte traffic and clinical sequelae in urate crystal-induced inflammation: an insight into gout.

The self-limiting response to urate crystals allows the exploration of events involved in both the onset and resolution of gout. Using i.v. injected radiolabelled anti-E-selectin monoclonal antibody 1.2b6 together with differentially radiolabelled neutrophils, mononuclear cells and albumin, we have characterized the expression of E-selectin in relation to leucocyte traffic, microvascular permeability and clinical sequelae following intracutaneous injection of monosodium urate crystals. We found that the inflammatory response in this model involved several distinct phases. First, E-selectin expression increased over 2-6 h in the context of increases in neutrophil and mononuclear cell accumulation, and albumin leakage. Secondly, leucocyte accumulation rapidly declined despite persisting E-selectin expression. Thirdly, E-selectin expression peaked at approximately 8 h and then fell despite an increase in clinically detectable erythema and induration. Lastly, these clinical manifestations of inflammation resolved despite the continued presence of urate crystals in the tissues. The further dissection of mechanisms regulating these phases will lead to a better understanding of events in both the pathogenesis and resolution of gout. Of broader significance, this inflammatory model may yield information about the protective events that underly resolution of inflammation, and provide insights into factors which determine chronicity.