Immunoglobulin G/immunoglobulin M autoantibody ratios in incomplete systemic lupus erythematosus.

OBJECTIVE: Immunoglobulin G (IgG) autoantibodies in systemic lupus erythematosus (SLE) are considered pathogenic, whereas immunoglobulin M (IgM) autoantibodies may have protective effects. The aim of this study was to identify whether IgG/IgM autoantibody ratios differ between patients with incomplete systemic lupus erythematosus (iSLE), patients with SLE, and healthy controls (HCs), and whether IgG/IgM autoantibody ratios relate to progression from iSLE to SLE. METHOD: This prospective cohort study included 34 iSLE patients, 41 SLE patients, and 11 HCs. IgG and IgM anti-dsDNA, anti-Ro52, and anti-Ro60 were measured by fluoro-enzyme immunoassay in serum samples obtained at baseline in all groups and in follow-up samples of up to 5 years for iSLE patients. Correlations between IgG/IgM autoantibody ratios, interferon signature, and clinical parameters were also assessed. RESULTS: At baseline, IgG anti-dsDNA, anti-Ro52, anti-Ro60, and IgM anti-dsDNA were elevated in iSLE and SLE patients. IgG/IgM anti-dsDNA and anti-Ro52 ratios were similar between groups, while IgG/IgM anti-Ro60 ratios were significantly elevated in iSLE and SLE patients compared to HCs. IgG/IgM autoantibody ratios were not correlated with interferon signature or clinical parameters. IgG/IgM ratios at baseline were similar and remained relatively stable during a median follow-up of 18 months in non-progressors and six iSLE patients who progressed to SLE. CONCLUSION: IgG anti-dsDNA, anti-Ro52, anti-Ro60, and IgM anti-dsDNA were elevated in iSLE and SLE patients, which was not apparent from the respective IgG/IgM ratios only. IgG/IgM autoantibody ratios remained relatively stable over up to 5 years in iSLE non-progressors and six patients who progressed to SLE.

Differential effects of donor-specific HLA antibodies in living versus deceased donor transplant.

The presence of donor-specific anti-HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long-term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement-dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10-year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10-year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10-year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence.

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.

Evaluation of the XRCC1 gene as a phenotypic modifier in BRCA1/2 mutation carriers. Results from the consortium of investigators of modifiers of BRCA1/BRCA2.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in genes involved in DNA repair are good candidates to be tested as phenotypic modifiers for carriers of mutations in the high-risk susceptibility genes BRCA1 and BRCA2. The base excision repair (BER) pathway could be particularly interesting given the relation of synthetic lethality that exists between one of the components of the pathway, PARP1, and both BRCA1 and BRCA2. In this study, we have evaluated the XRCC1 gene that participates in the BER pathway, as phenotypic modifier of BRCA1 and BRCA2. METHODS: Three common SNPs in the gene, c.-77C>T (rs3213245) p.Arg280His (rs25489) and p.Gln399Arg (rs25487) were analysed in a series of 701 BRCA1 and 576 BRCA2 mutation carriers. RESULTS: An association was observed between p.Arg280His-rs25489 and breast cancer risk for BRCA2 mutation carriers, with rare homozygotes at increased risk relative to common homozygotes (hazard ratio: 22.3, 95% confidence interval: 14.3-34, P<0.001). This association was further tested in a second series of 4480 BRCA1 and 3016 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. CONCLUSIONS AND INTERPRETATION: No evidence of association was found when the larger series was analysed which lead us to conclude that none of the three SNPs are significant modifiers of breast cancer risk for mutation carriers.

Antinuclear autoantibodies are more prevalent in COPD in association with low body mass index but not with smoking history.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with a higher prevalence of antinuclear autoantibodies (ANAs). However, a significant subgroup of patients is ANA negative. It remains to be determined which patient groups carry autoantibodies. METHODS: The association of smoking behaviour, disease status, gender, age and body mass index (BMI) with the presence of autoantibodies in the serum was determined in 124 patients with COPD and 108 non-COPD control subjects. In addition, the role of B cells in autoantibody generation in COPD was investigated by sequencing the antibody repertoire of B cells in the lungs of patients with COPD and of ex-smoking and never-smoking control subjects. RESULTS: Patients with COPD had a significantly higher risk of being serum positive for ANAs (OR 3.12, 95% CI 1.68 to 5.76, p<0.001). ANAs were not significantly associated with age, smoking status, gender or pack-years of smoking. Within the COPD population, subjects with BMI <22 kg/m2 had a significantly higher risk of ANAs (OR 4.93, 95% CI 1.50 to 16.50, p=0.009) than those with normal or high BMI. The antibody repertoire of B cells in the lungs of patients with COPD had a high frequency of positively charged CDR3 residues, a feature which is associated with self-reactive antibodies. CONCLUSION: The results show that COPD is a heterogeneous disease with respect to the prevalence of ANAs. ANAs are primarily associated with the presence of COPD and with low BMI, but not with smoking and forced expiratory volume in 1 s.

Pathogenic neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq.

Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1.

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.

Detection of Anti-neutrophil Cytoplasmic Antibodies (ANCA) by Indirect Immunofluorescence.

The eventual presence of anti-neutrophil cytoplasmic antibodies (ANCA) can initially be screened with indirect immunofluorescence (IIF). The majority of laboratories that facilitate ANCA testing use commercial kits. Although in-house assays are not encouraged in routine clinical laboratories, knowledge on the methodological aspects of the assay remains of importance. These aspects include choice of substrate, choice of fixative, staining procedure, and interpretation procedure. In this paper details on the methodology are provided and discussed in the context of the clinical application.

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.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments.

Loss of imprinting of IGF2 and not H19 in breast cancer, adjacent normal tissue and derived fibroblast cultures.

Insulin-like growth factors are involved in the paracrine growth regulation of human breast tumor cells. IGF2 is imprinted in most tissues, and shows expression of the paternal allele only. To investigate whether disruption of this monoallelic IGF2 expression is involved in breast cancer development, a series of primary tumors and adjacent, histologically normal, breast tissue samples, as well as matched primary in vitro fibroblast cultures were studied. Biallelic expression (partial) of IGF2 was found in the majority of in vivo samples, and corresponding fibroblast cultures, while monoallelic expression was found in a normal breast sample. In contrast, H19, a closely apposed, but reciprocally imprinted gene, assumed to be regulated by a common control element, showed retention of monoallelic H19 expression in all in vivo and in the majority of in vitro samples. These data indicate that IGF2, but not H19, is prone to loss of imprinting in breast cancer.

A new photometric method for the quantitation of Fc receptors for murine IgG1 on human monocytes and cell lines.

We have previously shown a polymorphism of human Fc receptors for mouse IgG1 using an EA rosette technique in which human erythrocytes sensitized with a murine IgG1 monoclonal antibody against glycophorin A acted as indicator cells. We now describe a method to quantitate this EA rosetting using the pseudoperoxidase activity present in erythrocytes. This photometric assay allows the sensitive quantitative determination of Fc receptor expression on human monocytes and cell lines. Not only the human Fc receptor for murine IgG1 can be studied in this way, but the method can also be applied to other Fc receptors. An important factor in this type of rosette assay appears to be the amount of negative charge present on the surface of the indicator erythrocytes. Using alcian blue as a probe, we found that this negative charge is higher on human erythrocytes than on sheep erythrocytes, which may contribute to a better signal-to-noise ratio. The method described facilitates the characterization of Fc receptors and permits the rapid screening of monoclonal anti-Fc receptor antibodies.

Antineutrophil cytoplasmic antibodies in primary sclerosing cholangitis: defined specificities may be associated with distinct clinical features.

PURPOSE: The clinical significance of antineutrophil cytoplasmic autoantibodies (ANCA) in primary sclerosing cholangitis has not been established. We investigated whether analysis of the antigenic specificities of ANCA is useful for delineating clinical subsets of the disease. METHODS: Sixty-nine patients with primary sclerosing cholangitis were studied. The presence of ANCA was analyzed by indirect immunofluorescence. Antibodies directed against specific antigens--proteinase 3, myeloperoxidase, elastase, bactericidal/permeability-increasing protein, cathepsin G, and lactoferrin--were identified by enzyme-linked immunosorbent assay. RESULTS: ANCA were detected by indirect immunofluorescence in 46 (67%) patients. In antigen-specific enzyme-linked immunosorbent assays, 37 (55%) of the 69 patients had antibodies to one or more antigens: 32 (46%) had antibodies to bactericidal/permeability-increasing protein, 16 (23%) had antibodies to cathepsin G, and 15 (22%) had antibodies to lactoferrin. Only 3 patients had antibodies to proteinase 3. Antibodies to myeloperoxidase or elastase were not detected. Twenty (29%) patients had antibodies to different antigens simultaneously. ANCA as detected by indirect immunofluorescence were not significantly associated with the presence of cirrhosis nor with the coexistence of inflammatory bowel disease. However, antibodies to bactericidal/permeability-increasing protein and cathepsin G were both associated with the presence of cirrhosis, and antibodies to lactoferrin were more frequently detected in patients with primary sclerosing cholangitis in conjunction with ulcerative colitis than in those without inflammatory bowel disease. CONCLUSION: Defined specificities of ANCA in primary sclerosing cholangitis may be related to particular clinical features of the disease.

Transforming growth factor beta secretion from primary breast cancer fibroblasts.

Transforming growth factor beta (TGF-beta) is a hormonally regulated growth inhibitor with autocrine and/or paracrine functions in human breast cancer. In vivo, enhanced immunohistochemical staining of extracellular TGF-beta 1 has been detected around stromal fibroblasts in response to the antiestrogen treatment. We have investigated the effects of tamoxifen on the production of TGF-beta by primary human breast fibroblast cultures in serum-free medium. Highly variable levels of mainly latent TGF-beta 1 were detected in conditioned media from both tumor and normal tissue derived fibroblasts. Hydroxy-tamoxifen was shown to increase latent TGF-beta 1 secretion in three of the eight tumor tissue-derived fibroblast cultures. Such effect of hydroxy-tamoxifen was not observed in fibroblast cultures established from normal adjacent breast tissue.

The platelet-derived growth factor alpha-receptor 1.5 kb transcript: target for molecular detection of testicular germ cell tumours of adolescents and adults.

An accurate and relatively simple method to detect testicular germ cell tumours of adolescents and adults (TGCTs) could be useful for early detection and may help to avoid unnecessary orchidectomies. We report a highly sensitive reverse-transcription polymerase chain reaction assay for the 1.5 kb transcript of the platelet-derived growth factor-alpha receptor for molecular diagnosis of TGCTs. As a simulation of the clinical application of the assay the approach was applied on through-cut-biopsies from orchidectomy specimens. In a series of 31 specimens, the 1.5 kb transcript was detected in all samples containing either carcinoma in situ, or an invasive TGCT, with mature teratoma as only exception. No expression was detected in normal parenchyma. On the basis of the through-cut-biopsies the assay shows a sensitivity of 0.87 and a specificity of 1.00. The positive and negative predictive values of the test are 1.00 and 1.00. For carcinoma in situ alone these values are 0.86, 1.00, 1.00, and 0.80, respectively. The figures at least equal the results obtained with the most sensitive morphological/enzyme-histochemical study of duplicate biopsies. We conclude that the 1.5 kb transcript of the platelet-derived growth factor-alpha receptor is a useful molecular marker for TGCTs, and therefore of interest in a clinical setting.

Titres of anti-neutrophil cytoplasmic antibodies in inflammatory bowel disease are not related to disease activity.

In the systemic vasculitides, serial measurement of titres of anti-neutrophil cytoplasmic autoantibodies (ANCA) is useful for follow-up of disease activity and prediction of relapses. ANCA have been detected in patients with inflammatory bowel disease, but their relation to disease activity in these diseases is unclear. We analysed the relation between disease activity and ANCA titres as determined by indirect immunofluorescence in paired samples obtained during active disease and at remission from individual patients with ulcerative colitis (n=60) and Crohn's disease (n=101). In addition, patients were followed prospectively, to study the fluctuations of ANCA with time in relation to disease activity. We did not detect a correlation between disease activity and ANCA titres, either in paired samples from active disease and remission, or in serial samples, either in ulcerative colitis or in Crohn's disease. In contrast to the ANCA-associated systemic vasculitides, serial measurement of ANCA titres is not useful in the monitoring of disease activity in patients with inflammatory bowel disease.

Prevalence and clinical significance of anti-lactoferrin autoantibodies in inflammatory bowel diseases and primary sclerosing cholangitis.

Anti-neutrophil cytoplasmic antibodies (ANCA) are autoantibodies directed against cytoplasmic constituents of neutrophil granulocytes. Antibodies with specificity for proteinase 3 and myeloperoxidase are seromarkers for systemic vasculitides. ANCA with specificity for lactoferrin were described in patients with several idiopathic inflammatory diseases, such as the inflammatory bowel diseases and rheumatoid arthritis. However, the clinical significance of anti-lactoferrin autoantibodies is still unclear. In this study, we determined the clinical significance of anti-lactoferrin autoantibodies in sera from large groups of patients with ulcerative colitis (UC), Crohn's disease (CD), and primary sclerosing cholangitis (PSC). Antibodies to human lactoferrin were detected by ELISA and by immunoblotting, using an extract of sonicated neutrophils as antigen source. Autoantibodies to lactoferrin were found in 29% of patients with UC, 13% of patients with CD, and 22% of patients with PSC. In inflammatory bowel diseases, the presence of anti-lactoferrin antibodies was not related to treatment, disease activity, duration of disease, or disease extent. In PSC, the presence of autoantibodies to lactoferrin did not correlate with duration of disease or the presence of cirrhosis. However, patients with PSC and coexistent UC had significantly more frequently antibodies to lactoferrin than PSC patients without IBD. In conclusion, autoantibodies to lactoferrin are a common feature of inflammatory bowel diseases and PSC. However, the clinical significance of those autoantibodies is limited as they lack sensitivity and specificity for those disorders. Future research should address the pathophysiological role of anti-lactoferrin ANCA and the influence of anti-lactoferrin ANCA binding on the functional properties of the lactoferrin molecule.

Anti-neutrophil cytoplasm autoantibodies (ANCA) in autoimmune liver diseases.

Anti-neutrophil cytoplasm antibodies (ANCA) are autoantibodies directed against cytoplasmic constituents of neutrophil granulocytes and monocytes. ANCA have been detected in serum from patients with inflammatory bowel diseases (mainly ulcerative colitis) and autoimmune mediated liver diseases (mainly primary sclerosing cholangitis and autoimmune hepatitis-type 1). The antigens recognized by ANCA in these diseases are multiple and the clinical and pathophysiological significance of ANCA in these disorders has not been completely defined.

The suitability of dogs as guide dogs for the blind: criteria and testing procedures.

Criteria and testing procedures with regard to the suitability of dogs as guide dogs for the blind were developed on the basis of a literature study and own observations. A profile of the guide dog comprising physical characteristics, skillfulness, behaviour, and obedience was drawn up. As a rule, the testing procedures concern health and skills of the dogs. In the skill test some elements of the behavioural and obedience test were included. The final evaluation is based on the results of physical examination and the skill test, unless testing of behaviour and/or obedience appears necessary as well. A method for evaluating the performance of the dogs as objectively as possible is described. Some implications of using and testing guide dogs are discussed.