Transcription of proteinase 3 and related myelopoiesis genes in peripheral blood mononuclear cells of patients with active Wegener's granulomatosis.

OBJECTIVE: Wegener's granulomatosis (WG) is a systemic inflammatory disease that is associated with substantial morbidity. The aim of this study was to understand the biology underlying WG and to discover markers of disease activity that would be useful for prognosis and treatment guidance. METHODS: Gene expression profiling was performed using total RNA from peripheral blood mononuclear cells (PBMCs) and granulocyte fractions from 41 patients with WG and 23 healthy control subjects. Gene set enrichment analysis (GSEA) was performed to search for candidate WG-associated molecular pathways and disease activity biomarkers. Principal components analysis was used to visualize relationships between subgroups of WG patients and controls. Longitudinal changes in proteinase 3 (PR3) gene expression were evaluated using reverse transcription-polymerase chain reaction, and clinical outcomes, including remission status and disease activity, were determined using the Birmingham Vasculitis Activity Score for WG (BVAS-WG). RESULTS: Eighty-six genes in WG PBMCs and 40 in WG polymorphonuclear neutrophils (PMNs) were significantly up-regulated relative to controls. Genes up-regulated in WG PBMCs were involved in myeloid differentiation, and these included the WG autoantigen PR3. The coordinated regulation of myeloid differentiation genes was confirmed by GSEA. The median expression values of the 86 up-regulated genes in WG PBMCs were associated with disease activity (P = 1.3 x 10(-4)), and WG patients with low-level expression of the WG signature genes showed expression profiles that were only modestly different from that in healthy controls (P = 0.07). PR3 transcription was significantly up-regulated in WG PBMCs (P = 1.3 x 10(-5), false discovery rate [FDR] 0.002), but not in WG PMNs (P = 0.03, FDR 0.28), and a preliminary longitudinal analysis showed that the fold change in PR3 RNA levels in WG PBMCs corresponded to changes in the BVAS-WG score over time. CONCLUSION: Transcription of PR3 and related myeloid differentiation genes in PBMCs may represent novel markers of disease activity in WG.

Immunopathogenic pathways in canine inflammatory myopathies resemble human myositis.

Progress in the treatment of inflammatory myopathies is impeded by the lack of suitable animal models. Inflammatory myopathies occur spontaneously in the dog, are a heterogeneous group of disorders, and are more common than in humans. Clinical signs of weakness and muscle atrophy are reliably present, and there are histological and immunohistological similarities to forms of human myositis. In this study, microarray technology followed by quantitative real-time PCR and immunohistochemistry on muscle biopsy sections was used to investigate gene expression in cases of canine inflammatory myopathies. Several genes involved with innate and adaptive immunity were highly upregulated including those that participate in macrophage and dendritic cell activation and migration, and antigen processing and presentation. Other genes including those that participate in B cell growth, development, migration and activation, immunoglobulin genes, genes in pro-inflammatory and anti-inflammatory pathways, and genes involved with tissue remodeling were upregulated. In previous reports utilizing microarray technology in human myositis, there was activation of similar pathways involved in the immune response. This study strengthens the argument that forms of canine myositis may be important animal models of human myositis and suggests useful biomarkers for therapeutic response using the dog in pre-clinical trials.

Analysis of environmental contamination resulting from catastrophic incidents: part 1. Building and sustaining capacity in laboratory networks.

Catastrophic incidents, such as natural disasters, terrorist attacks, and industrial accidents, can occur suddenly and have high impact. However, they often occur at such a low frequency and in unpredictable locations that planning for the management of the consequences of a catastrophe can be difficult. For those catastrophes that result in the release of contaminants, the ability to analyze environmental samples is critical and contributes to the resilience of affected communities. Analyses of environmental samples are needed to make appropriate decisions about the course of action to restore the area affected by the contamination. Environmental samples range from soil, water, and air to vegetation, building materials, and debris. In addition, processes used to decontaminate any of these matrices may also generate wastewater and other materials that require analyses to determine the best course for proper disposal. This paper summarizes activities and programs the United States Environmental Protection Agency (USEPA) has implemented to ensure capability and capacity for the analysis of contaminated environmental samples following catastrophic incidents. USEPA's focus has been on building capability for a wide variety of contaminant classes and on ensuring national laboratory capacity for potential surges in the numbers of samples that could quickly exhaust the resources of local communities. USEPA's efforts have been designed to ensure a strong and resilient laboratory infrastructure in the United States to support communities as they respond to contamination incidents of any magnitude. The efforts include not only addressing technical issues related to the best-available methods for chemical, biological, and radiological contaminants, but also include addressing the challenges of coordination and administration of an efficient and effective response. Laboratory networks designed for responding to large scale contamination incidents can be sustained by applying their resources during incidents of lesser significance, for special projects, and for routine surveillance and monitoring as part of ongoing activities of the environmental laboratory community.

Analysis of environmental contamination resulting from catastrophic incidents: part 2. Building laboratory capability by selecting and developing analytical methodologies.

Catastrophic incidents can generate a large number of samples of analytically diverse types, including forensic, clinical, environmental, food, and others. Environmental samples include water, wastewater, soil, air, urban building and infrastructure materials, and surface residue. Such samples may arise not only from contamination from the incident but also from the multitude of activities surrounding the response to the incident, including decontamination. This document summarizes a range of activities to help build laboratory capability in preparation for sample analysis following a catastrophic incident, including selection and development of fit-for-purpose analytical methods for chemical, biological, and radiological contaminants. Fit-for-purpose methods are those which have been selected to meet project specific data quality objectives. For example, methods could be fit for screening contamination in the early phases of investigation of contamination incidents because they are rapid and easily implemented, but those same methods may not be fit for the purpose of remediating the environment to acceptable levels when a more sensitive method is required. While the exact data quality objectives defining fitness-for-purpose can vary with each incident, a governing principle of the method selection and development process for environmental remediation and recovery is based on achieving high throughput while maintaining high quality analytical results. This paper illustrates the result of applying this principle, in the form of a compendium of analytical methods for contaminants of interest. The compendium is based on experience with actual incidents, where appropriate and available. This paper also discusses efforts aimed at adaptation of existing methods to increase fitness-for-purpose and development of innovative methods when necessary. The contaminants of interest are primarily those potentially released through catastrophes resulting from malicious activity. However, the same techniques discussed could also have application to catastrophes resulting from other incidents, such as natural disasters or industrial accidents. Further, the high sample throughput enabled by the techniques discussed could be employed for conventional environmental studies and compliance monitoring, potentially decreasing costs and/or increasing the quantity of data available to decision-makers.

Novel conformation of histidyl-transfer RNA synthetase in the lung: the target tissue in Jo-1 autoantibody-associated myositis.

OBJECTIVE: We previously proposed that novel expression and/or conformation of autoantigens in the target tissue may play a role in generating phenotype-specific immune responses. The strong association of autoantibodies to histidyl-transfer RNA synthetase (HisRS, Jo-1) with interstitial lung disease in patients with myositis led us to study HisRS expression and conformation in the lung. METHODS: Normal human tissue specimens were probed with a novel anti-HisRS antibody recognizing its granzyme B-cleavable conformation by immunoblotting and immunohistochemistry. The HisRS granzyme B site was mapped using site-directed mutagenesis, and its relationship to the antibody recognition domain was evaluated in tandem immunoprecipitation/granzyme B cleavage studies. RESULTS: The HisRS alpha-helical coiled-coil N-terminal domain recognized by autoantibodies is bounded by a granzyme B cleavage site. In immunoprecipitation studies with patient sera, HisRS was found to exist in 2 conformations, defined by sensitivity to cleavage by granzyme B and modification by autoantibody binding. Despite similar global expression of HisRS in different tissue, expression of its granzyme B-cleavable form was enriched in the lung and localized to the alveolar epithelium. CONCLUSION: A proteolytically sensitive conformation of HisRS exists in the lung, the target tissue associated with this autoantibody response. We thus propose that autoimmunity to HisRS is initiated and propagated in the lung.