Autoantibody Specificities and Type I Interferon Pathway Activation in Idiopathic Inflammatory Myopathies.

Myositis is a heterogeneous group of autoimmune diseases, with different pathogenic mechanisms contributing to the different subsets of disease. The aim of this study was to test whether the autoantibody profile in patients with myositis is associated with a type I interferon (IFN) signature, as in patients with systemic lupus erythematous (SLE). Patients with myositis were prospectively enrolled in the study and compared to healthy controls and to patients with SLE. Autoantibody status was analysed using an immunoassay system and immunoprecipitation. Type I IFN activity in whole blood was determined using direct gene expression analysis. Serum IFN-inducing activity was tested using peripheral blood cells from healthy donors. Blocking experiments were performed by neutralizing anti-IFNAR or anti-IFN-α antibodies. Patients were categorized into IFN high and IFN low based on an IFN score. Patients with autoantibodies against RNA-binding proteins had a higher IFN score compared to patients without these antibodies, and the IFN score was related to autoantibody multispecificity. Patients with dermatomyositis (DM) and inclusion body myositis (IBM) had a higher IFN score compared to the other subgroups. Serum type I IFN bioactivity was blocked by neutralizing anti-IFNAR or anti-IFN-α antibodies. To conclude, a high IFN score was not only associated with DM, as previously reported, and IBM, but also with autoantibody monospecificity against several RNA-binding proteins and with autoantibody multispecificity. These studies identify IFN-α in sera as a trigger for activation of the type I IFN pathway in peripheral blood and support IFN-α as a possible target for therapy in these patients.

Gene expression analysis in RA: towards personalized medicine.

Gene expression has recently been at the forefront of advance in personalized medicine, notably in the field of cancer and transplantation, providing a rational for a similar approach in rheumatoid arthritis (RA). RA is a prototypic inflammatory autoimmune disease with a poorly understood etiopathogenesis. Inflammation is the main feature of RA; however, many biological processes are involved at different stages of the disease. Gene expression signatures offer management tools to meet the current needs for personalization of RA patients' care. This review analyses currently available information with respect to RA diagnostic, prognostic and prediction of response to therapy with a view to highlight the abundance of data, whose comparison is often inconclusive due to the mixed use of material source, experimental methodologies and analysis tools, reinforcing the need for harmonization if gene expression signatures are to become a useful clinical tool in personalized medicine for RA patients.

Gene-expression signatures: biomarkers toward diagnosing multiple sclerosis.

Identification of biomarkers contributing to disease diagnosis, classification or prognosis could be of considerable utility. For example, primary methods to diagnose multiple sclerosis (MS) include magnetic resonance imaging and detection of immunological abnormalities in cerebrospinal fluid. We determined whether gene-expression differences in blood discriminated MS subjects from comparator groups, and identified panels of ratios that performed with varying degrees of accuracy depending upon complexity of comparator groups. High levels of overall accuracy were achieved by comparing MS with homogeneous comparator groups. Overall accuracy was compromised when MS was compared with a heterogeneous comparator group. Results, validated in independent cohorts, indicate that gene-expression differences in blood accurately exclude or include a diagnosis of MS and suggest that these approaches may provide clinically useful prediction of MS.

Interferon regulatory factor 5 gene variants and pharmacological and clinical outcome of Interferonß therapy in multiple sclerosis.

Interferon-ß (IFNß) therapy is effective in approximately half of the patients with relapsing-remitting multiple sclerosis (RRMS). Clinical non-responders were characterized by an increased expression of IFN response genes before the start of therapy, and a lack of a pharmacologically induced increase in IFN response gene activity. Because Interferon Regulatory Factor 5 (IRF5) is a master regulator of IFN-activity, we carried out a candidate gene study of IRF5 gene variants in relation to the pharmacological and clinical response upon IFNß treatment. We found that patients with the IRF5 rs2004640-TT and rs47281420-AA genotype exerted a poor pharmacological response to IFNß compared with patients carrying the respective G-alleles (P=0.0006 and P=0.0023, respectively). Moreover, patients with the rs2004640-TT genotype developed more magnetic resonance imaging (MRI)-based T2 lesions during IFNß treatment (P=0.003). Accordingly, an association between MRI-based non-responder status and rs2004640-TT genotype was observed (P=0.010). For the rs4728142-AA genotype a trend of an association with more T2 lesions during IFNß treatment and MRI-based non-responder status was observed (P=0.103 and P=0.154, respectively). The clinical relevance of the rs2004640-TT genotype was validated in an independent cohort wherein a shorter time to first relapse was found (P=0.037). These findings suggest a role for IRF5 gene variation in the pharmacological and clinical outcome of IFNß therapy that might have relevance as biomarker to predict the response to IFNß in multiple sclerosis.

Biomarkers and personalised medicine in rheumatoid arthritis: a proposal for interactions between academia, industry and regulatory bodies.

Rheumatoid arthritis (RA) is one of the most appropriate conditions for the application of personalised medicine as a high degree of heterogeneity has been recognised, which remains to be explained. Such heterogeneity is also reflected in the large number of treatment targets and options. A growing number of biologics as well as small molecules are already in use and there are promising new drugs in development. In order to make the best use of treatment options, both targeted and non-targeted biomarkers have to be identified and validated. To this aim, new rules are needed for the interaction between academia and industry under regulatory control. Setting up multi-centre biosample collections with clear definition of access, organising early, possibly non-committing discussions with regulatory authorities, and defining a clear route for the validation, qualification and registration of the biomarker-drug combination are some of the more critical areas where effective collaboration between the drug industry, academia and regulators is needed.

Pharmacogenomics of infliximab treatment using peripheral blood cells of patients with rheumatoid arthritis.

To provide insight into the pharmacological changes in the peripheral blood (PB) molecular profile induced by tumor necrosis factor (TNF)-blockade in patients with rheumatoid arthritis (RA), blood was obtained in PAXgene tubes from 33 RA patients before and 1 month after TNF-blocking therapy (infliximab). From 15 randomly chosen patients pre- and post-treatment gene expression profiles were determined. The remaining 18 RA patients served as validation cohort. A group-based paired analysis of the gene expression profiles from the post- vs pre-treatment samples revealed a signature of genes significantly regulated by TNF-blockade. Downregulated genes reflected several biological pathways such as inflammation, angiogenesis, B- and T-cell activation. Further analysis revealed that the pharmacological response signature was significantly regulated in all treated patients, irrespective of clinical response, which is indicative for the presence of an active TNF pathway in all RA patients. The data imply that all patients carried features of TNF bioactivity irrespective of clinical response. These results favor a model for the parallel presence of TNF-dependent and TNF-independent pathways in the individual RA patient. Clinical response status to TNF-blockade may be dependent on the relative contribution of TNF-independent effector pathways.

Molecular subtypes of systemic sclerosis in association with anti-centromere antibodies and digital ulcers.

The objective of this study was to identify molecular profiles that may distinguish clinical subtypes in systemic sclerosis (SSc). Large-scale gene expression profiling was performed on peripheral blood (PB) from 12 SSc patients and 6 healthy individuals. Significance analysis of microarrays, two-way hierarchical cluster analysis and PANTHER (Protein ANalysis THrough Evolutionary Relationships) ontology classification were used to analyze the data. Quantitative PCR was applied for validation in a cohort of 43 SSc patients. The results show that the expression of genes involved in immune defense, cell cycle and signal transduction was significantly elevated in PB of SSc patients (n=12) compared with healthy individuals (n=6). SSc patients could be stratified into subgroups based on differential expression of genes induced by type I interferon (IFN) and genes involved in antimicrobial (AM) activity. Differential expression of type I IFN or AM signature genes was validated and extended in an independent cohort of 31 patients by quantitative PCR. Low expression of IFN response genes was associated with the presence of anti-centromere antibodies, whereas increased expression was associated with the appearance of digital ulcers. In conclusion, patients with SSc can be classified on the basis of differential expression of immune defense genes. Differences in the activity of the type I IFN response program stratify patients into two clinically relevant subgroups.

Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation.

One action of cyclosporin A thought to be central to many of its immunosuppressive effects is its ability to inhibit the early events of T lymphocyte activation such as lymphokine gene transcription in response to signals initiated at the antigen receptor. Cyclosporin A was found to specifically inhibit the appearance of DNA binding activity of NF-AT, AP-3, and to a lesser extent NF-kappa B, nuclear proteins that appear to be important in the transcriptional activation of the genes for interleukin-2 and its receptor, as well as several other lymphokines. In addition, cyclosporin A abolished the ability of the NF-AT binding site to activate a linked promoter in transfected mitogen-stimulated T lymphocytes and in lymphocytes from transgenic mice. These results indicate that cyclosporin A either directly inhibits the function of nuclear proteins critical to T lymphocyte activation or inhibits the action of a more proximal member of the signal transmission cascade leading from the antigen receptor to the nucleus.

Epigenetic regulation and nucleosome positioning in the human TATA-less IL-1 alpha promoter.

Interleukin-1 alpha (IL-1 alpha) is secreted by a variety of cell types and is a major player in immune and inflammatory processes. Genes involved in immunological processes are known to be strictly regulated; however, how epigenetic mechanisms contribute to this regulation in not understood. To gain insight into the epigenetic regulation of the human TATA-less IL-1A gene, we show that active and silent chromatin modifications characterize the regulatory regions of IL-1 alpha in expressing and non-expressing cells, respectively, and that the DNA methylation in the proximal promoter is associated with the expression status of the cells. Interestingly, although nucleosome depletion in active promoters is found in yeast and fly genes, now it has been reported in human promoters. We here show on the level of single DNA molecules that in expressing cells, a nucleosome is absent in about half of the proximal IL-1 alpha promoters. This observation might reflect a more subtle regulation of nucleosome positioning in TATA-less genes or human genes in general.

Expression of a pathogen-response program in peripheral blood cells defines a subgroup of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a heterogeneous disease with unknown etiology. Here we aimed to distinguish RA subtypes based on peripheral blood (PB) gene expression profiles in comparison with a pathogen-response transcriptional program. PB was obtained from 35 RA patients and 15 healthy individuals. For expression profiling we used DNA microarrays. A combined cluster analysis of RA and control samples together with samples from a viral infection model revealed that the gene expression profile of a subgroup of RA patients (RA(A)) was reminiscent to that of poxvirus-infected macaques. Statistical analysis, followed by Gene Ontology analysis of the RA(A) patients confirmed that these patients form a distinct group, with activation of several host defense mechanisms that resemble a common host-pathogen response. Analysis of the promoter region of genes that were overexpressed in the RA(A) patients, revealed an enrichment of transcription factor binding sites for NF kappaB and interferon-activated transcription factors. Moreover, this subgroup of RA patients expressed significantly increased titers of anti-cyclic citrullinated peptide antibodies. We conclude that activation of a host-pathogen response defines a subgroup of RA patients characterized by increased autoreactivity against citrullinated proteins.

Responsiveness to anti-tumour necrosis factor alpha therapy is related to pre-treatment tissue inflammation levels in rheumatoid arthritis patients.

OBJECTIVE: The response of rheumatoid arthritis (RA) patients to treatment with neutralising antibodies to tumour necrosis factor alpha (TNFalpha) is highly variable. The underlying mechanism for therapy responsiveness is currently unknown. We therefore evaluated the relationship between baseline molecular profiles of synovial tissues from RA patients and the clinical response to treatment with infliximab. METHODS: Synovial biopsies were obtained by arthroscopy from 18 RA patients with active disease (28 joint count Disease Activity Score (DAS28) > or = 3.2) before initiation of treatment with infliximab. All patients were on stable methotrexate treatment. Clinical response at 16 weeks was defined as a reduction in DAS28 of > or = 1.2, non-response as reduction in DAS28 < 1.2. Large-scale gene expression profiling using microarrays was performed on synovial tissue samples. To identify biological processes in synovial biopsies that could discriminate between responders and non-responders, we performed pathway analysis on the expression profiles. RESULTS: A total of 12 patients responded to therapy, while 6 patients failed to fulfil the response criteria. We identified several biological processes, related to inflammation, which were up-regulated in patients who responded to therapy, compared to those who did not show clinical improvement. CONCLUSION: These results indicate that patients with a high level of tissue inflammation are more likely to benefit from anti-TNFalpha treatment.

Genetic linkage of two intragenic restriction fragment length polymorphisms with von Willebrand's disease type IIA. Evidence for a defect in the von Willebrand factor gene.

Restriction fragment length polymorphisms (RFLPs), using the enzymes Bgl II and Xba I in conjunction with human von Willebrand factor (vWF) cDNA probes, have been described previously. In the present study we demonstrate the localization of both genetic markers within the vWF gene. The RFLPs were used to study the segregation of alleles associated with von Willebrand's disease (vWD) type IIA in a comprehensive, affected family. Individuals of this family were tested for their bleeding time and their plasma was analyzed for vWF antigen concentration and vWF ristocetin-cofactor activity. Based on these data, the affected members were diagnosed as vWD type-IIA patients; this conclusion was confirmed by the analysis of the multimeric vWF pattern of some of the patients. It was demonstrated that both RFLPs are completely linked with the vWD type-IIA trait. From this finding, we conclude that the defect that causes the vWD type IIA is most likely due to a mutation in the vWF gene and not to a mutation in a gene involved in posttranslational processing of the vWF protein.

Detection of intracellular interferon-gamma by light microscopy using an immunoperoxidase technique: correlation with the corresponding mRNA and protein product.

Identifying individual cytokine-producing cells may help to acquire insight into immunological processes. This study was designed to adapt a technique for the detection of individual cytokine-producing cells from an immunofluorescence to an immunoperoxidase staining procedure. The production of interferon-gamma (IFN-gamma) by anti-CD3-activated cloned human T cells was used as a model system. After the conditions for the staining procedure were optimized, the immunoperoxidase technique was slightly more sensitive than the immunofluorescence technique. The intracellular staining for IFN-gamma was preceded or paralleled by IFN-gamma mRNA production and followed by accumulation of IFN-gamma in the supernatant. It is concluded that intracellular IFN-gamma can easily be detected using an immunoperoxidase procedure. This procedure is highly sensitive and allows quantification of the production of multiple cytokines by counting the percentage of positively staining cells.

[Molecular unraveling of disease by means of DNA-microarrays]. / Moleculaire ontrafeling van ziekte met DNA-microarrays.

Determination of the human genome sequence and the development of microarray technologies allowing the rapid measurement of all genes in the genome have generated new perspectives for our current biomedical research. Gene expression analysis will make a major contribution to our insight into the underlying biology of disease and will lead to improved methods for diagnostics, prognosis and treatment. Microarray studies create the possiblity to subclassify patients with diseases such as rheumatoid arthritis, diffuse large B-cell lymphoma and breast cancer, with both prognostic and therapeutic consequences. The simultaneous quantification of the activity of all genes in tissues or cells from patients by microarray technology, linked to the clinical parameters, creates a large number of data points, which cannot be analysed without the aid of the advanced application of bioinformatics. As a result, genomic research has become, in part, a bioinformatics discipline that will be integrated with clinical medicine. The microarray technology makes it possible to develop personalized medicine, with a more accurate diagnosis and prognosis for every patient and subsequently a tailored treatment strategy.

Alternative splicing of IgA Fc receptor (CD89) transcripts.

An alternatively spliced CD89 transcript is present in peripheral blood mononuclear cells (PBMC) and U937 cells. The alternatively spliced CD89 mRNA species lacks the exon 4 sequence, encompassing 288 nucleotides, that encodes the extracellular membrane-proximal immunoglobulin-like domain (EC2).

Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins.

The human serine proteases granzymes A and B are expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzymes A and B in the cytotoxic response in vivo.

TNF-alpha promoter polymorphisms, production and susceptibility to multiple sclerosis in different groups of patients.

TNF-alpha production in whole blood cultures upon stimulation with LPS was determined in 179 individuals from 61 families in order to characterise the magnitude of inherited differences in TNF-alpha production. The three families characterised by highest TNF production showed 7.1 +/- 0.3 ng TNF/ml upon culture with 10 ng LPS and 10.2 +/- 0.2 ng TNF/ml upon culture with 1000 ng LPS. in contrast to the three families characterised by the lowest TNF production that showed a production of 1.6 +/- 0.1 ng TNF upon culture with 10 ng and 2.5 +/- 0.2 ng/ml upon culture with 1000 ng LPS/ml. This difference could not be attributed to the promoter polymorphisms -308 G to A. -238 G to A or -376 G to A, although the -238 GA donors produced 2.1 +/- 0.9 ng TNF upon culture with 10 ng endotoxin compared to 3.2 +/- 2.2 ng TNF for the -238 GG donors. In line with these results the frequency of the -238 GG genotype was increased in hospitalized MS patients in a nursing home (100% 238GG, n = 57) compared to MS patients in an outpatient's clinic (94% 238GG, n = 98) or Dutch controls (90% 238GG, n = 180). These results suggest that the -238 GG genotype is differently distributed in hospitalized MS patients in a nursing home.

Carrier detection in severe (type III) von Willebrand disease using two intragenic restriction fragment length polymorphisms.

DNA from a family with a female member affected with severe (type III) vWD was analysed using three restriction enzymes and a partial vWF cDNA probe. Two restriction fragment length polymorphisms (RFLPs) detected with the enzymes Bgl II and Xba I proved to be informative in this family. A 36.0 Kb allele demonstrated with the enzyme Xba I was rare in the general population but very important in this family for segregation analysis of the alleles and their association with the putative defective chromosome. The propositus was homozygous for the 36.0 Kb Xba I polymorphic band and heterozygous for the Bgl II polymorphism. She was the only member of the family showing this allelic pattern. The linkage of the alleles could be determined because her mother was homozygous for the 9.0 Kb Bgl II polymorphism but heterozygous for the Xba I polymorphism. The segregation of the alleles could be traced to the proband's son and a niece. The genotypic analysis revealed that her niece could be considered as carrying a defective gene for severe vWD.

Tumor necrosis factor alpha-308 gene variants in relation to major histocompatibility complex alleles and Felty's syndrome.

The location of the human TNF genes within the MHC complex has prompted much speculation about the role of TNF alleles in the etiology of MHC-associated autoimmune diseases. On sequencing the 5' regulatory region of the human TNFA gene a G (TNFA-308G) to A (TNFA-308A) transition polymorphism at position -308 was discovered. We have developed a simple PCR assay to facilitate the screening of the -308 polymorphism at the DNA level. In view of the possible linkage between the TNFA-308A allele and a certain MHC type, TNFA-308 genotypes in HLA-typed healthy individuals (n = 88) were determined. A statistically significant association between the TNFA-308A allele and HLA-DR3, DQB1*0201, DQA1*0501, A1, B8, and the NcoI 5.5-kb RFLP of the TNFB gene was observed. In addition, we determined the frequency of the TNFA-308A allele in patients with FS (n = 13), an HLA-DR4-associated disease. In this study, no association was found of Felty's syndrome with the TNFA-308A allele, indicating that this allele does not appear to be a susceptibility factor for FS.

Polymorphism within the tumor necrosis factor alpha (TNF) promoter region in patients with ankylosing spondylitis.

In addition to HLA-B27, other genetic factors are thought to be involved in the pathogenesis of ankylosing spondylitis (AS). Because of the location of the TNF gene in the vicinity of the HLA-B locus, and the prominent role in inflammation of its product, we investigated the association between AS and two G to A transition polymorphisms located at position -238 and -376 in the promoter region of the TNF gene. The distribution of the TNF alleles was determined in 86 HLA-B27+ AS patients and 163 healthy controls. From the 86 AS patients, 33 suffered from acute anterior uveitis (AAU). No significant difference for the TNF-376 polymorphism in AS and healthy controls was observed. The frequency of the TNF-238A allele in HLA-B27+ AS patients was significantly decreased compared to random controls (p = 0.021). However, the frequency of the TNF-238A allele in HLA-B27+ AS patients was not significantly different from that observed in HLA-B27+ healthy individuals (p = 0.6). Assessment of association showed that the TNF-238G allele is in linkage disequilibrium with the HLA-B27 allele (delta = 0.053; P = 0.008). Therefore, we conclude that the association between TNF-238G and AS is secondary to the HLA-B27 gene and that TNF-238 and-TNF-376 alleles are not likely to be involved in the susceptibility to AS.