Antibody responses to dietary antigens are accompanied by specific plasma cells in the infant thymus.

BACKGROUND: Human infants develop IgG responses to dietary antigens during the first 2 years of life. Yet, the source of these antibodies is unclear. In previous studies we reported on the thymus as a unique functional niche for plasma cells (PCs) specific to environmental antigens. OBJECTIVE: We sought to examine whether PCs specific to dietary antigens are detected in the infant thymus. METHODS: We tested IgG reactivity to 112 food antigens and allergens in the serum of 20 neonates and infants using microarrays. The presence of PC-secreting IgG specific to the most prominent antigens was then assessed among thymocytes in the same cohort. Using an LC-MS proteomics approach, we looked for traces of these antigens in the thymus. RESULTS: Our studies first confirmed that cow's milk proteins are prevalent targets of serum IgG in early life. Subjects with the highest serum IgG titers to cow's milk proteins also harbored IgG-producing PCs specific to the same antigens in the thymic niche. Furthermore, we detected multiple peptide fragments of cow's milk antigens in the thymus. Lastly, we verified that both serum IgG and IgG secreted by thymic PCs recognized the peptide epitopes found in the thymus. CONCLUSIONS: Our studies reveal the presence of antibody-secreting PCs specific to common dietary antigens in the infant thymus. The presence of these antigens in the thymus suggested that activation and differentiation of specific PCs occurred in this organ. Further studies are now warranted to evaluate the possible implication of these cells in tolerance to dietary antigens.

Automation of Amplicon-Based Library Preparation for Next-Generation Sequencing by Centrifugal Microfluidics.

Next-generation sequencing (NGS) has become a mainstream method in bioanalysis. Improvements in sequencing and bioinformatics turned the complex and cumbersome library preparation to the bottleneck in terms of reproducibility and costs in the complete NGS workflow. Here, we introduce an automated library preparation approach based on a generic centrifugal microfluidic cartridge. Multiplex polymerase chain reaction amplification and subsequent cleanup were performed with all reagents prestored on the disk, including cell-line-based DNA as quality control. Exchange of prestored reagents allows applying the cartridge to different target genes. Sequencing of automatically prepared libraries from T-cell receptor and immunoglobulin gene rearrangements in context of lymphoproliferative disorders demonstrated excellent cleanup performance between 91.9 and 99.9% of target DNA reads and successful amplification of all target regions by up to 15 forward primers combined with 4 reverse primers. The fully automated library preparation by centrifugal microfluidics thus offers attractive automation options in diagnostic settings.

Restricted myeloperoxidase epitopes drive the adaptive immune response in MPO-ANCA vasculitis.

BACKGROUND: Treatment of autoimmune diseases has relied on broad immunosuppression. Knowledge of specific interactions between human leukocyte antigen (HLA), the autoantigen, and effector immune cells, provides the foundation for antigen-specific therapies. These studies investigated the role of HLA, specific myeloperoxidase (MPO) epitopes, CD4+ T cells, and ANCA specificity in shaping the immune response in patients with anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis. METHODS: HLA sequence-based typing identified enriched alleles in our patient population (HLA-DPB1*04:01 and HLA-DRB4*01:01), while in silico and in vitro binding studies confirmed binding between HLA and specific MPO epitopes. Class II tetramers with MPO peptides were utilized to detect autoreactive CD4+ T cells. TCR sequencing was performed to determine the clonality of T cell populations. Longitudinal peptide ELISAs assessed the temporal nature of anti-MPO447-461 antibodies. Solvent accessibility combined with chemical modification determined the buried regions of MPO. RESULTS: We identified a restricted region of MPO that was recognized by both CD4+ T cells and ANCA. The autoreactive T cell population contained CD4+CD25intermediateCD45RO+ memory T cells and secreted IL-17A. T cell receptor (TCR) sequencing demonstrated that autoreactive CD4+ T cells had significantly less TCR diversity when compared to naïve and memory T cells, indicating clonal expansion. The anti-MPO447-461 autoantibody response was detectable at onset of disease in some patients and correlated with disease activity in others. This region of MPO that is targeted by both T cells and antibodies is not accessible to solvent or chemical modification, indicating these epitopes are buried. CONCLUSIONS: These observations reveal interactions between restricted MPO epitopes and the adaptive immune system within ANCA vasculitis that may inform new antigen-specific therapies in autoimmune disease while providing insight into immunopathogenesis.

Inhibitory Anti-Peroxidasin Antibodies in Pulmonary-Renal Syndromes.

BACKGROUND: Goodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur via disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP. METHODS: We used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score. RESULTS: We detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production in vitro. The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease. CONCLUSIONS: Anti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients.

Interfacing centrifugal microfluidics with linear-oriented 8-tube strips and multichannel pipettes for increased throughput of digital assays.

We present a centrifugal microfluidic cartridge for the eight-fold parallel generation of monodisperse water-in-oil droplets using standard laboratory equipment. The key element is interfacing centrifugal microfluidics with its design based on polar coordinates to the linear structures of standard high-throughput laboratory automation. Centrifugal step emulsification is used to simultaneously generate droplets from eight samples directly into standard 200 µl PCR 8-tube strips. To ensure minimal manual liquid handling, the design of the inlets allows the user to load the samples and the oil via a standard multichannel pipette. Simulation-based design of the cartridge ensures that the performance is consistent in each droplet generation unit despite the varying radial positions that originate from the interface to the linear oriented PCR 8-tube strip and from the integration of linear oriented inlet holes for the multichannel pipettes. Within 10 minutes, sample volumes of 50 µl per droplet generation unit are emulsified at a fixed rotation speed of 960 rpm into 1.47 × 105 monodisperse droplets with a mean diameter of 86 µm. The overall coefficient of variation (CV) of the droplet diameter was below 4%. Feasibility is demonstrated by an exemplary digital droplet polymerase chain reaction (ddPCR) assay which showed high linearity (R2 ≥ 0.999) across all of the eight tubes of the strip.

Automated library preparation for whole genome sequencing by centrifugal microfluidics.

Next generation sequencing is evolving from a research tool into a method applied in diagnostic routine. The complete sequencing workflow includes sample pre-processing, library preparation, sequencing and bioinformatics. High quality in each of these steps is necessary to obtain excellent sequencing results. The tedious and error-prone library preparation poses a significant challenge for smaller laboratories, where high throughput pipetting robots are not cost-effective. Here we present an automated library preparation for whole genome sequencing using centrifugal microfluidics. Two samples can be run per cartridge. Precise metering of reagents allows the required liquid volumes to be reduced by 40% and the amount of sample used by 60%. The functionality of the cartridge is demonstrated with bacteria and DNA extracted from a human FFPE sample. For the bacterial sample, mean sequencing depths from 140 to 183 reads and a coverage of 99.8% of the reference genome were detected. For the human DNA, mean sequencing depths of 4.4-5.7 reads and a coverage of 78.2% of the effective reference genome were observed.

Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP.

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

Immunoglobulins G from patients with ANCA-associated vasculitis are atypically glycosylated in both the Fc and Fab regions and the relation to disease activity.

BACKGROUND: Anti-neutrophil cytoplasmic autoantibodies (ANCA) directed against myeloperoxidase (MPO) and proteinase 3 (PR3) are pathogenic in ANCA-associated vasculitis (AAV). The respective role of IgG Fc and Fab glycosylation in mediating ANCA pathogenicity is incompletely understood. Herein we investigate in detail the changes in Fc and Fab glycosylation in MPO-ANCA and Pr3-ANCA and examine the association of glycosylation aberrancies with disease activity. METHODOLOGY: Total IgG was isolated from serum or plasma of a cohort of 30 patients with AAV (14 MPO-ANCA; 16 PR3-ANCA), and 19 healthy control subjects. Anti-MPO specific IgG was affinity-purified from plasma of an additional cohort of 18 MPO-ANCA patients undergoing plasmapheresis. We used lectin binding assays, liquid chromatography, and mass spectrometry-based methods to analyze Fc and Fab glycosylation, the degree of sialylation of Fc and Fab fragments and to determine the exact localization of N-glycans on Fc and Fab fragments. PRINCIPAL FINDINGS: IgG1 Fc glycosylation of total IgG was significantly reduced in patients with active AAV compared to controls. Clinical remission was associated with complete glycan normalization for PR3-ANCA patients but not for MPO-ANCA patients. Fc-glycosylation of anti-MPO specific IgG was similar to total IgG purified from plasma. A major fraction of anti-MPO specific IgG harbor extensive glycosylation within the variable domain on the Fab portion. CONCLUSIONS/SIGNIFICANCE: Significant differences exist between MPO and PR3-ANCA regarding the changes in amounts and types of glycans on Fc fragment and the association with disease activity. These differences may contribute to significant clinical difference in the disease course observed between the two diseases.

Rhenium fac tricarbonyl bisimine complexes: biologically useful fluorochromes for cell imaging applications.

A series of lipophilic and hydrophilic fac tricarbonyl rhenium bisimine complexes have been prepared, their membrane-permeabilities explored in liposomes and their potential for application in fluorescence microscopy cell imaging demonstrated in the first application of MLCT-fluorescent rhenium complexes in cell imaging.

Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis.

Anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.

A qualitative study of the activities and outcomes of domestic violence coordinating councils.

Efforts to promote systems change frequently involve the creation of councils, coalitions, and other collaborative settings. However, research, to date, reports limited empirical evidence that they achieve desired outcomes (Roussos and Fawcett, Annu Rev Public Health 21:369-402, 2000). The precise nature of this evidence base has received less attention. In particular, formal investigations into council effectiveness (a) rarely highlight the specific nature of collaborative efforts; (b) emphasize fairly distal markers as the "gold standard" for effectiveness; (c) focus largely on formative "outcomes" (e.g., action plan quality); and (d) utilize primarily quantitative research approaches. The current study extends previous research by employing a qualitative approach to investigate the particular activities and proximal outcomes of 41 domestic violence coordinating councils. Study findings suggest that councils engage in six primary activities: discussing issues, sharing information, identifying weaknesses in the system's response, providing training for key stakeholders, engaging in public/community education, and lobbying key stakeholders who are not council members. Three proximal outcomes were consistently identified in council efforts: the promotion of knowledge, relationships, and institutionalized change. Attending more directly to proximal outcomes and concrete activities in our research has important implications for conceptualizing and researching the effectiveness of councils and collaborative settings.

Scientists in the swamp: narrowing the language-practice gap in community psychology.

As a confluence of unique values and activities, the collective practice of community psychology is difficult to characterize in a simple way. Increasingly, however, professional contexts are laden with pressure to define any practice--from library work to medical interventions--in the orderly, compact language of traditional science. This trend has historically been resisted in the field by those sensing a fundamental lack of fit between the fluid, emergent aspects of community psychological practice and the fixed, precise language of classic science. In response to this "language-practice gap," some have attempted to adapt the traditional language of science to better fit the field's practice, while others have explored alternative languages of practice seemingly more indigenous to the messy "swamp" of actual communities. While the former effort leaves some theoretical contradictions intact, the latter tends to discount scientific identity entirely. This paper proposes a potential step forward by resituating questions of disciplinary language and identity within a current philosophical discourse where the nature of social science itself remains sharply contested. This suggests shifting attention away from "should we be a science?" to "what kind of science might we be after all?"; in turn, alternative languages may be re-cast as legitimate contributors to a kind of science more authentic to human communities--even a viable "science in the swamp." One such language-philosophical hermeneutics--is presented as a particularly valuable supplement to traditional science. Illustrations highlight ways that hermeneutics may advance the formal language of the field towards a closer fit of what actually happens in practice, while preserving and even bolstering the empirical rigor and scientific identity of the field.