Antigen-Specific Detection of Autoantibodies Against Myeloperoxidase (MPO) and Proteinase 3 (PR3).

ANCA testing was introduced in many laboratories throughout the world when it was recognized that a significant subset of patients with small vessel vasculopathies presented with such antibodies. Many laboratories developed and introduced in-house testing methods for antigen-specific ANCA detection complementary to indirect immune fluorescence screening. Such in-house tests have proven their merit in diagnosing vasculitis and were important to identify critical steps in the development of antigen-specific assays with high sensitivity and specificity. In the meantime various commercial assays became available for antigen-specific ANCA testing. Because of the high diagnostic accuracy of such assays it can be anticipated that commercial, antigen-specific tests will completely replace in-house testing for MPO- and PR3-ANCA. Furthermore, such tests will replace the need for IIF in the diagnostic workup of AAV. In this light it can be foreseen that the knowledge that underlies the development of in-house ANCA testing will gradually disseminate over time. Therefore we describe the current antigen-specific ANCA ELISAs (direct and capture) with the intention to maintain the knowledge and the identification of the critical steps in the development of robust assays.

Autoantibodies to box A of high mobility group box 1 in systemic lupus erythematosus.

Autoantibodies to nuclear structures are a hallmark of systemic lupus erythematosus (SLE), including autoantibodies to nuclear protein high mobility group box 1 (HMGB1). HMGB1 consists of three separate domains: box A, box B and an acidic tail. Recombinant box A acts as a competitive antagonist for HMGB1 and might be an interesting treatment option in SLE. However, antibodies to box A might interfere. Therefore, levels of anti-box A were examined in SLE patients in association with disease activity and clinical parameters. Serum anti-box A was measured in 86 SLE patients and 44 age- and sex-matched healthy controls (HC). Serum samples of 28 patients with primary Sjögren's syndrome and 32 patients with rheumatoid arthritis were included as disease controls. Anti-HMGB1 and anti-box B levels were also measured by enzyme-linked immunosorbent assay during quiescent disease [SLE Disease Activity Index (SLEDAI) ≤ 4, n = 47] and active disease (SLEDAI ≥ 5, n = 39). Anti-box A levels in active SLE patients were higher compared to quiescent patients, and were increased significantly compared to HC and disease controls. Anti-box A levels correlated positively with SLEDAI and anti-dsDNA levels and negatively with complement C3 levels. Increased levels of anti-box A antibodies were present in the majority of patients with nephritic (73%) and non-nephritic exacerbations (71%). Antibodies to the box A domain of HMGB1 might be an interesting new biomarker, as these had a high specificity for SLE and were associated with disease activity. Longitudinal studies should be performed to evaluate whether these antibodies perform better in predicting an exacerbation, especially non-nephritic exacerbations.

Statistical controversies in clinical research: Value of adverse events relatedness to study treatment: analyses of data from randomized double-blind placebo-controlled clinical trials.

BACKGROUND: Collection and reporting of adverse events (AEs) and their relatedness to study treatment, known commonly as attribution, in clinical trials is mandated by regulatory agencies (the National Cancer Institute and the Food and Drug Administration). Attribution is assigned by the treating physician using judgment based on various factors including patient's baseline status, disease history, and comorbidity as well as knowledge about the safety profile of the study treatments. We evaluate the patterns of AE attribution (unrelated, unlikely, possibly, probably, and definitely related to the treatment) in treatment, symptom intervention (cancer patients) and cancer prevention (participants at high risk for cancer) setting. MATERIALS AND METHODS: Nine multicenter placebo-controlled trials (two treatment, two symptom intervention, and five cancer prevention) were analysed separately (2155 patients). Frequency and severity of AEs were summarized by arm. Attribution and percentage of repeated AEs whose attribution changed overtime were summarized for the placebo arms. Percentage of physician over- or under-reporting of AE relatedness was calculated for the treatment arms using the placebo arm as the reference. RESULTS: Across all trials and settings, a very high proportion of AEs reported as related to treatment were classified as possibly related, a significant proportion of AEs in the placebo arm were incorrectly reported as related to treatment, and clinician-reported attribution over-estimated the rate of AEs related to treatment. Fatigue, nausea, vomiting, diarrhea, constipation, and neurosensory were the common AEs that were over reported by clinician as related to treatment. CONCLUSIONS: These analyses demonstrate that assigning causality to AE is a complex and difficult process that produces unreliable and subjective data. In randomized double-blind placebo-controlled trials where data are available to objectively assess relatedness of AE to treatment, attribution assignment should be eliminated.

Presence of anticitrullinated protein antibodies in a large population-based cohort from the Netherlands.

OBJECTIVES: To determine the prevalence of anticitrullinated protein antibodies (ACPAs) and their association with known rheumatoid arthritis (RA) risk factors in the general population. METHODS: Lifelines is a multidisciplinary prospective population-based cohort study in the Netherlands. Cross-sectional data from 40 136 participants were used. The detection of ACPA was performed by measuring anti-CCP2 on the Phadia-250 analyser with levels ≥6.2 U/mL considered positive. An extensive questionnaire was taken on demographic and clinical information, including smoking, periodontal health and early symptoms of musculoskeletal disorders. RA was defined by a combination of self-reported RA, medication use for the indication of rheumatism and visiting a medical specialist within the last year. RESULTS: Of the total 40 136 unselected individuals, 401 (1.0%) had ACPA level ≥6.2 U/mL. ACPA positivity was significantly associated with older age, female gender, smoking, joint complaints, RA and first degree relatives with rheumatism. Of the ACPA-positive participants, 22.4% had RA (15.2% had defined RA according to our criteria and 7.2% self-reported RA only). In participants without RA, 311 (0.8%) were ACPA-positive. In the non-RA group, older age, smoking and joint complaints remained significantly more frequently present in ACPA-positive compared with ACPA-negative participants. CONCLUSIONS: In this large population-based study, the prevalence of ACPA levels ≥6.2 U/mL was 1.0% for the total group and 0.8% when excluding patients with RA. Older age, smoking and joint complaints were more frequently present in ACPA-positive Lifelines participants. To our knowledge, this study is the largest study to date on ACPA positivity in the general, mostly Caucasian population.

HDAC8 and STAT3 repress BMF gene activity in colon cancer cells.

Histone deacetylase (HDAC) inhibitors are undergoing clinical trials as anticancer agents, but some exhibit resistance mechanisms linked to anti-apoptotic Bcl-2 functions, such as BH3-only protein silencing. HDAC inhibitors that reactivate BH3-only family members might offer an improved therapeutic approach. We show here that a novel seleno-α-keto acid triggers global histone acetylation in human colon cancer cells and activates apoptosis in a p21-independent manner. Profiling of multiple survival factors identified a critical role for the BH3-only member Bcl-2-modifying factor (Bmf). On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300. Treatment with a p300 inhibitor or transient overexpression of exogenous HDAC8 interfered with BMF induction, whereas RNAi-mediated silencing of STAT3 activated the target gene. This is the first report to identify a direct target gene of HDAC8 repression, namely, BMF. Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis. These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy.

Are urinary levels of high mobility group box 1 markers of active nephritis in anti-neutrophil cytoplasmic antibody-associated vasculitis?

The objective of this study is to evaluate urinary high mobility group box 1 (HMGB1) levels as markers for active nephritis in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) in comparison with urinary CD4(+) effector memory T cells and urinary monocyte chemoattractant protein-1 (MCP-1). Twenty-four AAV patients with active nephritis and 12 healthy controls (HC) were evaluated. In nine patients, samples were also obtained during remission. Urinary levels of HMGB1 were measured by Western blot. CD4(+) T cells and CD4(+) effector memory T cells (CD4(+) CD45RO(+) CCR7(-) ) were determined in urine and whole blood by flow cytometry. Measurement of urinary levels of MCP-1 and serum HMGB1 levels were performed by enzyme-linked immunosorbent assay (ELISA). AAV patients with active nephritis had higher median intensity of HMGB1 in urine than HC [10·3 (7·05-18·50) versus 5·8 (4·48-7·01); P = 0·004]. Both urinary HMGB1 and MCP-1 levels decreased significantly from active nephritis to remission. The urinary MCP-1/creatinine ratio correlated with Birmingham Vasculitis Activity Score (BVAS) (P = 0·042). No correlation was found between the HMGB1/creatinine ratio and 24-h proteinuria, estimated glomerular filtration rate (eGFR), MCP-1/creatinine ratio, BVAS and serum HMGB1. A positive correlation was found between urinary HMGB1/creatinine ratio and CD4(+) T cells/creatinine ratio (P = 0·028) and effector memory T cells/creatinine ratio (P = 0·039) in urine. Urinary HMGB1 levels are increased in AAV patients with active nephritis when compared with HC and patients in remission, and urinary HMGB1 levels are associated with CD4(+) T cells and CD4(+) effector memory T cells in urine. Measurement of urinary HMGB1 may be of additional value in identifying active glomerulonephritis in AAV patients.

High mobility group box1 (HMGB1) in relation to cutaneous inflammation in systemic lupus erythematosus (SLE).

SUMMARY: Photosensitivity is characteristic of systemic lupus erythematosus (SLE). Upon ultraviolet B (UVB) exposure, patients develop inflammatory skin lesions in the vicinity of sunburn cells (SBCs). High mobility group box 1 (HMGB1) is released from apoptotic and activated cells and exerts inflammatory actions through ligation to its receptors. METHODS: Eleven SLE patients and 10 healthy controls (HCs) were exposed to UVB. Skin biopsies were taken before and at one, three and 10 days after irradiation. Sections were stained for SBC, HMGB1, CD3, CD68, interferon-induced protein MxA and cleaved caspase 3. In vitro experiments with UVB-irradiated keratinocytes were also performed. Higher numbers of cells that had released HMGB1 were seen in the skin of SLE patients compared to HCs before and after irradiation. HMGB1-negative nuclei correlated with the presence of SBCs, and with the number of cleaved caspase 3 positive cells in lupus skin. RESULTS: HMGB1 release is increased in the skin of SLE patients compared to HCs. Upon UVB exposure, HMGB1 release further increases in SLE patients and is related to the number of apoptotic cells. Our data suggest that HMGB1, probably released from apoptotic keratinocytes, contributes to the development of inflammatory lesions in the skin of SLE patients upon UVB exposure.

HMGB1 in vascular diseases: Its role in vascular inflammation and atherosclerosis.

The nuclear protein high mobility group box 1 (HMGB1) has been suggested to be involved in the pathogenesis of several vascular diseases such as systemic vasculitis and atherosclerosis. In systemic vasculitides including ANCA-associated vasculitis and Kawasaki disease, serum HMGB1 levels are higher in patients with active disease compared to healthy controls. In atherosclerotic disease, HMGB1 displays increased expression in nuclei and cytoplasm of macrophages and smooth muscle cells in the atherosclerotic lesions, and is implicated in the progression of the atherosclerotic plaque. Experimental models of acute coronary syndromes and cerebrovascular accidents show that HMGB1 is not only involved in the amplification of the inflammatory response during acute ischemic injury, but also in the recovery and remodeling process after ischemia. Patients with acute coronary syndromes or stroke present significantly higher serum levels of HMGB1 than healthy controls and levels are associated with disease severity and mortality. Here we review clinical and experimental studies dealing with the role of HMGB1 in vascular diseases.

The formation of autoantibodies and antibodies to TNF-α blocking agents in relation to clinical response in patients with ankylosing spondylitis.

OBJECTIVES: To investigate the influence of antibody formation to TNF-α blocking agents on the clinical response in AS patients treated with infliximab (IFX), etanercept (ETA), or adalimumab (ADA), and to investigate the development of ANA, ANCA, and anti-dsDNA antibodies in association with the formation of antibodies to TNF-α blocking agents. METHODS: Consecutive AS outpatients with active disease who started treatment with IFX (n=20), ETA (n=20), or ADA (n=20) were included in this longitudinal observational study. Clinical data were collected prospectively at baseline and after 3, 6, and 12 months of anti-TNF-α treatment. At the same time points, serum samples were collected. In these samples, antibodies to TNF-α blocking agents, serum TNF-α blocker levels, and ANA, ANCA, and anti-dsDNA antibodies were measured retrospectively. RESULTS: Anti-IFX, anti-ETA, and anti-ADA antibodies were induced in 20%, 0%, and 30% of patients, respectively. Although ANA, ANCA, and anti-dsDNA antibodies were detected during anti-TNF-α treatment, no significant association was found between the presence of these autoantibodies and the formation of antibodies to TNF-α blocking agents. Patients with anti-IFX or anti-ADA antibodies had significantly lower serum TNF-α blocker levels compared to patients without these antibodies. Furthermore, significant negative correlations were found between serum TNF-α blocker levels and assessments of disease activity. CONCLUSIONS: This study indicates that antibody formation to IFX or ADA is related to a decrease in efficacy and early discontinuation of anti-TNF-α treatment in AS patients. Furthermore, autoantibody formation does not seem to be associated with antibody formation to TNF-α blocking agents.

HMGB1 in systemic lupus Erythematosus: Its role in cutaneous lesions development.

The chromatin non-histone DNA binding protein high mobility group box one (HMGB1) has recently been extensively studied in autoimmune diseases. In addition to its nuclear functions, HMGB1 has been identified as alarmin that can 'alarm' both innate and adaptive immunity. HMGB1 can amplify inflammation and enhance immune responses by interacting with the receptor for Advanced Glycation End Products (RAGE) and Toll-like receptors 2,4 and 9 (TLRs) . Release of HMGB1 occurs during cell activation as well as cell death. Cells die by apoptosis and eventually necrosis which both are thought to lead to release of HMGB1 into the microenvironment. In the past years disturbed apoptosis or clearance of apoptotic cells has been put forward as a major pathophysiological feature in autoimmune diseases such as Systemic Lupus Erythematosus (SLE), which is a prototypic autoimmune disease that affects many organs. Accumulation of apoptotic cells has been found in SLE. Also, elevated levels of HMGB1 have been detected in the serum of SLE patients and increased expression of HMGB1 was demonstrated in skin lesions of lupus patients. In this review the general characteristics and activities of HMGB1 are highlighted and its role in SLE will be discussed with special attention to its involvement in the pathogenesis of skin lesions.

Is a solitary inguinal lymph node metastasis from adenocarcinoma of the rectum really a metastasis?

OBJECTIVE: Metastatic involvement of inguinal lymph nodes (ILN) from rectal adenocarcinoma is unusual, particularly without signs of distant spread to other organ sites. By current convention, ILN involvement, including solitary involvement, is classified as metastatic disease (M). However, anecdotal reports suggest that such patients are a distinct entity and should be managed differently. The aim of this study was to gain further insight regarding this seemingly distinct patient subset. METHOD: This case series provides a descriptive report of patients with rectal adenocarcinoma and solitary inguinal lymph node metastasis (SILNM). RESULTS: Upon retrospective review of medical records from 4480 patients with rectal adenocarcinoma seen at Mayo Clinic Rochester from 1995 to 2004, six patients (0.13%) with SILNM were identified. Three had metachronous and three had synchronous SILNM (four left sided, one right sided, and one bilateral). The mean age at SILNM diagnosis was 61.3 years, and three patients had originally stage II (and three had stage III disease). Five patients received concurrent chemo-radiation therapy and one declined treatment. Among those with metachronous SILNM, the mean survival after diagnosis of rectal cancer was 42 months. By comparison, all three patients with synchronous SILNM were still alive after a mean duration of 40 months of follow up. CONCLUSION: Solitary involvement of ILNs might represent a distinct subset of patients with metastatic rectal adenocarcinoma who have a more favourable prognosis. If confirmed by larger studies, our data suggest that alternate management algorithms might be reasonable for such patients.

Coexpression of CD177 and membrane proteinase 3 on neutrophils in antineutrophil cytoplasmic autoantibody-associated systemic vasculitis: anti-proteinase 3-mediated neutrophil activation is independent of the role of CD177-expressing neutrophils.

OBJECTIVE: Wegener's granulomatosis (WG) is strongly associated with antineutrophil cytoplasmic autoantibodies (ANCAs) directed against proteinase 3 (PR3). Recent studies have shown that membrane-bound PR3 (mPR3) is differentially expressed and colocalizes with CD177/NB1 on circulating neutrophils. We undertook this study to assess the differential expression of CD177 on neutrophils from patients with ANCA-associated systemic vasculitis (ASV) in comparison with patients with systemic lupus erythematosus (SLE), patients with rheumatoid arthritis (RA), and healthy individuals, and to investigate whether colocalization of mPR3 and CD177 affects anti-PR3-mediated neutrophil activation. METHODS: Expression of CD177 and mPR3 was analyzed by flow cytometry on isolated neutrophils from patients with ASV (n=53), those with SLE (n=30), those with RA (n=26), and healthy controls (n=31). Neutrophil activation mediated by anti-PR3 antibodies was assessed by measuring the oxidative burst with a dihydrorhodamine assay. RESULTS: Percentages of CD177-expressing neutrophils were significantly higher in patients with ASV and those with SLE than in healthy controls. In 3 healthy donors, CD177 expression was not detected. After priming with tumor necrosis factor alpha, neutrophils remained negative for CD177 while mPR3 expression was induced. Neutrophils from CD177-negative donors or CD177- neutrophils sorted from donors with bimodal expression were susceptible to anti-PR3-mediated oxidative burst. Variation in the extent of anti-PR3-mediated neutrophil activation among different donors occurred independent of the percentage of CD177-expressing neutrophils. CONCLUSION: Membrane expression of CD177 on circulating neutrophils is increased in patients with ASV and in those with SLE, but not in RA patients. However, primed neutrophils from CD177-negative individuals also express mPR3 and are susceptible to anti-PR3-mediated oxidative burst, suggesting that recruitment of CD177-independent mPR3 is involved in anti-PR3-induced neutrophil activation.

Hypoxia inducible factor-1-alpha (HIF-1alpha) is related to both angiogenesis and inflammation in rheumatoid arthritis.

OBJECTIVES: Despite the important role of the transcription factor HIF-1alpha in angiogenesis and inflammation, only a few studies on HIF-1alpha expression have been performed in RA patients. The aim of the present study was to identify the layer in synovial tissue of RA patients where HIF1a is expressed and to find out whether HIF-1alpha expression is related to both angiogenesis and inflammation in synovium from RA patients. METHODS: A reproducible staining method for HIF-1alpha was developed. HIF-1alpha -positive cells were quantified in synovial tissue from patients with RA. As control we used synovial tissue from patients with osteoarthritis (OA). The number of HIF-1alpha-positive cells was compared with the number of blood vessels present and was correlated with the amount of inflammation. The amount of inflammation was determined by counting inflammatory cells, by estimating the proliferation marker Ki67 in inflamed tissue, and by using a recently published synovitis score which gives an accurate estimate of the amount of inflammation present. RESULTS: HIF-1alpha was expressed weakly in the lining layer and strongly in the sublining layer in RA synovial tissue. In contrast, HIF-1alpha was only weakly expressed in OA synovial tissue. The number of HIF-1alpha -positive cells correlated strongly with the number of blood vessels in RA synovial tissue and with inflammatory endothelial cell infiltration (blood vessels), cell proliferation (Ki67) and the synovitis score. CONCLUSIONS: HIF-1alpha expression is strongest in the sub-lining layer of RA synovium and is related to both angiogenesis and inflammation in synovium from RA patients. These results thus suggest that HIF-1alpha could serve as an important new therapeutic target in RA, targeting both angiogenesis and inflammation.

Favorable glycemic response of type 2 diabetics to low-calorie cranberry juice.

Fruit and vegetable intake is typically low for type 2 diabetics, possibly due to a perceived adverse effect on glycemic control. Cranberry juice (CBJ) may represent an attractive means for increasing fruit intake and simultaneously affording positive health benefits. This single cross-over design compared metabolic responses of type 2 diabetics (n= 12) to unsweetened low-calorie CBJ (LCCBJ; 19 Cal/240 mL), carbohydrate sweetened normal calorie CBJ (NCCBJ; 120 Cal/240 mL), isocaloric low-calorie sugar water control (LCC), and isocaloric normal calorie sugar water control (NCC) interventions. CBJ flavonols and anthocyanins, and proanthocyanidins were quantified with HPLC, LC-MS, and MALDI-TOF that includes an original characterization of several large oligomeric proanthocyanidins. Blood glucose peaked 30 min postingestion after NCCBJ and NCC at 13.3 +/- 0.5 and 12.8 +/- 0.9 (mmol/L), and these responses were significantly greater than the LCCBJ and LCC peaks of 8.1 +/- 0.5 and 8.7 +/- 0.5, respectively. Differences in glycemic response remained significant 60 min, but not 120 min postingestion. Plasma insulin values 60 min postingestion for NCCBJ and NCC interventions were 140 +/- 19 and 151 +/- 18 (pmol/L), respectively, and significantly greater than the LCCBJ and LCC values of 56 +/- 10 and 54 +/- 10; differences were not significant 120 min postingestion. Metabolic responses within the 2 high and 2 low-calorie beverages were virtually identical; however, exposure to potentially beneficial nutrients was greater with CBJ. Relative to conventionally sweetened preparation, LCCBJ provides a favorable metabolic response and should be useful for promoting increased fruit consumption among type 2 diabetics or others wishing to limit carbohydrate intake.

Type I interferons are involved in the development of ultraviolet B-induced inflammatory skin lesions in systemic lupus erythaematosus patients.

OBJECTIVE: To investigate the involvement of type I interferons and endothelial cell adhesion molecules in the development of ultraviolet B (UVB)-induced systemic lupus erythaematosus (SLE) skin lesions. METHODS: A total of 19 SLE patients and 13 controls were irradiated with two minimal erythaemal doses(MED) of UVB. Subsequently, skin biopsies were analysed (immuno) histologically over 10 days, for expression of IFNalpha-induced MxA, numbers of plasmacytoid dendritic cells (pDC), and expression of endothelial cell adhesion molecules, namely E-selectin, ICAM-1, and L-selectin ligand. Additionally, MxA expression was compared to its expression in nine established cutaneous lupus erythaematosus(CLE) lesions of SLE patients. RESULTS: Before irradiation IFNalpha-induced MxA was expressed at significantly higher levels in non-lesional skin of SLE patients compared to healthy controls. In patients developing infiltrates upon UVB irradiation, MxA expression increased further, reaching expression levels similar to or exceeding levels in CLE-skin lesions. In these patients, MxA expression was sustained up to day 10, in contrast to patients not developing infiltrates in whom expression decreased. No noteworthy numbers of plasmacytoid dendritic cells (pDC) were detected in nonirradiated skin or at any time after UVB exposure in SLE patients or controls. MxA expression correlated with influx of T-cells and monocytes/macrophages, and with expression of E-selectin and ICAM-1. CONCLUSION: Development of UVB-induced SLE skin lesions involves a skewing towards production of Th1-associated cytokines, such as IFNalpha. In turn, this may lead to up-regulation of E-selectin and ICAM-1 resulting in recruitment of T-cells and macrophages.