Staining Pattern Of Antibody Research Articles

Examination of antinuclear antibody staining patterns and titers in patients with childhood-onset systemic lupus erythematosus.

Antinuclear antibodies (ANA) staining patterns can provide useful information in systemic lupus erythematosus (SLE). In our study, we examined the frequency of ANA staining patterns in disease-related features in childhood-onset SLE patients. ANA and its staining patterns were assessed in childhood-onset SLE patients. Two hundred twenty-three patients were included (F/M = 3/1). Their median age at diagnosis was 14.3 (11.9-16.1) years. The anti-cell (AC)-4/5 (fine or large speckled) pattern was the most common nuclear ANA pattern (75.8%), while the AC-19 (dense fine speckled) pattern was the most frequently detected cytoplasmic ANA pattern (13.1%). The AC-4/5 (fine or large speckled) patterns were notably seen in fever, acute and chronic cutaneous lupus, arthritis, serositis, hematologic involvement, renal involvement, neuropsychiatric involvement, gastrointestinal involvement, and cardiopulmonary involvement (all p < .001). Conversely, the AC-1 (homogeneous) pattern was significantly detected in oral/nasal ulcers and non-scarring alopecia (both p < .001). Regarding the laboratory features, the AC-4/5 (fine or large speckled) patterns exhibited a predominant seen in autoimmune hemolytic anemia, leukopenia, thrombocytopenia, elevated ESR and CRP, hypocomplementemia, direct Coombs, anti-Smith (Sm), anti-SSA and SS-B, anti-ribonucleoprotein (RNP), anti-histone, anti-ribosomal P, lupus anticoagulant, anti-cardiolipin immunoglobulin (Ig)M/IgG, and anti-β2-glycoprotein IgM/IgG positivities (all p < .001). In contrast, the AC-1 (homogeneous) pattern was detected in anti-double-stranded (ds) DNA and anti-histone positivity (both p < .001). Our study showed that AC-4/5 and AC-1 patterns of ANA are frequently detected in many clinical and serological features of childhood-onset SLE patients. However, further studies are needed in larger populations to verify these results.

Antinuclear antibody staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America

Introduction/ObjectiveThis study aimed to describe the frequency of antinuclear antibody (ANA) staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America. Materials and methodsThis retrospective, descriptive, and observational study evaluated data from all patients undergoing antinuclear antibody indirect immunofluorescence assay from a single-tertiary center (University Hospital Fundación Valle del Lili, Cali-Colombia) in 2020. ResultsOne thousand and eight patients met the inclusion criteria. The median patient age was 47 (34–59.2) years, and most were female (769, 75.3%). A positive ANA immunofluorescence assay was observed in approximately two-thirds of patients (664, 65.8%). ANA test results were primarily used to exclude a suspected diagnosis in approximately half of the patients (466, 46.2%). Thirty-seven percent (250/664) of the cohort with ANA-positive titers had a systemic autoimmune rheumatic disease (SARD). The most prevalent SARDs included rheumatoid arthritis (RA) (55, 8.2%) followed by systemic lupus erythematosus (SLE) (37, 5.5%). The vast majority of ANA-positive patients had a reported speckled pattern (anti-cell [AC]-2,4,5; 269; 40.5%) followed by homogenous (AC-1; 266; 40%), nucleolar (AC-8,9,10; 46; 6.9%), and centromere (AC-3; 16; 2.4%). The most frequent patterns observed among SLE patients included homogenous (AC-1) patterns in 17 (45.9%) patients, speckled (AC-2,4,5) nuclear patterns in 11 (29.7%) patients, mixed patterns in 7 (18.9%) patients, and reticular/anti-mitochondrial antibody (AMA, AC-21) cytoplasmic patterns in 2 (5.4%) patients. ConclusionThis study is the first to describe ANA patterns in a Colombian population. Speckled and homogenous patterns were predominant in patients with SARDs.

Staining pattern of specific and cross-reacting Melan-A antibodies: A comparative study on 15,840 samples from 133 human tumor types.

The Melan-A (melanocyte antigen) protein, also termed 'melanoma antigen recognized by Tcells 1' (MART-1) is a protein with unknown function whose expression is specific for the melanocyte lineage. Antibodies against Melan-A are thus used for identifying melanocytic tumors, but some Melan-A antibodies show an additional - diagnostically useful - cross-reactivity against an unspecified protein involved in corticosteroid hormone synthesis. To comprehensively compare the staining patterns of a specific and a cross-reactive Melan-A antibody in normal and neoplastic tissues, tissue microarrays containing 15,840 samples from 133 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry. For the Melan-A-specific antibody 'Melan-A specific' (MSVA-900M), Melan-A positivity was seen in 96.0% of 25 benign nevi, 93.0% of 40 primary and 86.7% of 75 metastatic melanomas, 82.4% of 85 renal angiomyolipomas as well as 96.4% of 84 neurofibromas, 2.2% of 46 granular cell tumors, 1.0% of 104 schwannomas, and 1.1% of 87 leiomyosarcomas. The cross-reactive antibody 'Melan-A+' (MSVA-901M+) stained 98.1% of the tumors stained by 'Melan-A specific'. In addition, high positivity rates were seen in sex-cord-stroma tumors of the ovary (35.3%-100%) and the testis (86.7%) as well as for adrenocortical neoplasms (76.3%-83.0%). Only nine further tumor groups showed Melan-A+ staining, including five different categories of urothelial carcinomas. Our data provide a comprehensive overview on the staining patterns of specific and cross-reactive Melan-A antibodies. The data demonstrate that both antibodies are highly useful for their specific purpose. It is important for pathologists to distinguish these two Melan-A antibody subtypes for their daily work.

Immunohistochemical Diagnosis of Amyloid Typing: Utility and Limitations as Determined by Liquid Chromatography-Tandem Mass Spectrometry.

Although immunohistochemical techniques and proteomic analysis are widely used for typing diagnosis of amyloidosis, the diagnostic utility of immunohistochemical evaluation is not well understood. We used immunohistochemical techniques to characterize staining patterns of in-house rabbit polyclonal anti-κ, anti-λ, anti-transthyretin antibodies, and commercial anti-amyloid A and anti-β2-microglobulin antibodies in 40 autopsy cases. In thirty cases (75%), the subtype was determined by using the criterion that amyloid is strongly and diffusely positive for one antibody while negative for other antibodies. We then performed proteomic analysis of all 40 cases. In 39 cases, we identified only one amyloid protein and confirmed the immunohistochemically determined subtypes of the abovementioned 30 cases. In seven other cases, we could retrospectively determine subtypes with immunohistochemistry by using information from proteomic analysis, which increased the immunohistochemistry diagnosis rate to 92.5% (37/40). In one case, we identified double subtypes, both immunohistochemically and with proteomic analysis. In the remaining three cases, proteomic analysis was essential for typing diagnosis. The present findings suggest that combined immunohistochemistry and proteomic analysis is more useful than immunohistochemistry alone. Our findings highlight the importance of carefully interpreting immunohistochemistry for anti-TTR and light chain and offer insights that can guide amyloid typing through immunohistochemistry.

Flow cytometric-based detection of CD80 is a useful diagnostic marker of acute myeloid leukemia in dogs.

CD80, a co-stimulatory molecule required for optimal T cell activation, is expressed on antigen-presenting cells, including monocytes and dendritic cells, in dogs and humans. We hypothesized that CD80 would be expressed on tumor cells in dogs from acute myeloid leukemia (AML) but not dogs with lymphoid neoplasms. We first evaluated the cellular staining pattern of a hamster anti-murine CD80 antibody (clone 16-10A1, ThermoFisher Scientific Cat# 17-0801-82, RRID: AB_469417) in blood and bone marrow aspirates from healthy dogs. Using flow cytometric analysis and examination of modified Wright's-stained cytologic smears of unsorted and flow cytometric or immunomagnetic bead-sorted leukocytes, we show that the antibody binds to mature and immature neutrophils and monocytes, but not lymphocytes or eosinophils, in blood and bone marrow. We then added the antibody to routine flow cytometric panels for immunophenotyping hematopoietic neoplasms in dogs. We found that the antibody labeled tumor cells in 72% of 39 dogs with AML and 36% of 11 dogs with acute leukemia expressing lymphoid and myeloid markers ("mixed lineage") but none of the dogs with B (n = 37) or T (n = 35) lymphoid neoplasms. A higher proportion of tumor cells in dogs with AML were labeled with the anti-CD80 antibody vs antibodies against other myeloid-associated antigens, including CD4 (36%, p = 0.003), CD11b (44%), CD11c (46%), CD14 (38%, p = 0.006) and CD18 (59%, clone YFC118). In contrast, antibodies against CD11b and CD11c bound to tumor cells in 8-32% of the lymphoid neoplasms. We show that CD80, as detected by antibody clone 16-10A1, is a sensitive and specific marker for AML and would be useful to include in flow cytometric immunophenotyping panels in dogs.

Fluorescent protein tagging promotes phase separation and alters the aggregation pathway of huntingtin exon-1

Fluorescent protein tags are convenient tools for tracking the aggregation states of amyloidogenic or phase separating proteins, but the effect of the tags is often not well understood. Here, we investigated the impact of a C-terminal red fluorescent protein (RFP) tag on the phase separation of huntingtin exon-1 (Httex1), an N-terminal portion of the huntingtin protein that aggregates in Huntington's disease. We found that the RFP-tagged Httex1 rapidly formed micron-sized, phase separated states in the presence of a crowding agent. The formed structures had a rounded appearance and were highly dynamic according to electron paramagnetic resonance and fluorescence recovery after photobleaching, suggesting that the phase separated state was largely liquid in nature. Remarkably, the untagged protein did not undergo any detectable liquid condensate formation under the same conditions. In addition to strongly promoting liquid-liquid phase separation, the RFP tag also facilitated fibril formation, as the tag-dependent liquid condensates rapidly underwent a liquid-to-solid transition. The rate of fibril formation under these conditions was significantly faster than that of the untagged protein. When expressed in cells, the RFP-tagged Httex1 formed larger aggregates with different antibody staining patterns compared to untagged Httex1. Collectively, these data reveal that the addition of a fluorescent protein tag significantly impacts liquid and solid phase separations of Httex1 invitro and leads to altered aggregation in cells. Considering that the tagged Httex1 is commonly used to study the mechanisms of Httex1 misfolding and toxicity, our findings highlight the importance to validate the conclusions with untagged protein.

Antinuclear antibody staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America

Introduction/ObjectiveThis study aimed to describe the frequency of antinuclear antibody (ANA) staining patterns by indirect immunofluorescence assay observed in patients from a tertiary health center in Latin America. Materials and methodsThis retrospective, descriptive, and observational study evaluated data from all patients undergoing antinuclear antibody indirect immunofluorescence assay from a single-tertiary center (University Hospital Fundación Valle del Lili, Cali-Colombia) in 2020. ResultsOne thousand and eight patients met the inclusion criteria. The median patient age was 47 (34–59.2) years, and most were female (769, 75.3%). A positive ANA immunofluorescence assay was observed in approximately two-thirds of patients (664, 65.8%). ANA test results were primarily used to exclude a suspected diagnosis in approximately half of the patients (466, 46.2%). Thirty-seven percent (250/664) of the cohort with ANA-positive titers had a systemic autoimmune rheumatic disease (SARD). The most prevalent SARDs included rheumatoid arthritis (RA) (55, 8.2%) followed by systemic lupus erythematosus (SLE) (37, 5.5%). The vast majority of ANA-positive patients had a reported speckled pattern (anti-cell [AC]-2,4,5; 269; 40.5%) followed by homogenous (AC-1; 266; 40%), nucleolar (AC-8,9,10; 46; 6.9%), and centromere (AC-3; 16; 2.4%). The most frequent patterns observed among SLE patients included homogenous (AC-1) patterns in 17 (45.9%) patients, speckled (AC-2,4,5) nuclear patterns in 11 (29.7%) patients, mixed patterns in 7 (18.9%) patients, and reticular/anti-mitochondrial antibody (AMA, AC-21) cytoplasmic patterns in 2 (5.4%) patients. ConclusionThis study is the first to describe ANA patterns in a Colombian population. Speckled and homogenous patterns were predominant in patients with SARDs.

P24 A case of limited cutaneous systemic sclerosis evolving into an overlap syndrome with systemic lupus erythematosus and pyoderma gangrenosum

P24 A case of limited cutaneous systemic sclerosis evolving into an overlap syndrome with systemic lupus erythematosus and pyoderma gangrenosum

Anti-Nuclear Antibodies Patterns in Patients With Systemic Lupus Erythematosus and Their Correlation With Other Diagnostic Immunological Parameters.

BackgroundAntinuclear antibodies (ANA) are major immunodiagnostic tools in systemic lupus erythematosus (SLE); however, their clinical and pathogenic roles are not yet elucidated and are a subject of controversy.ObjectivesThe aim of the study is to explore the pathogenic significance of ANA patterns among SLE patients, by analyzing their association with ANA titers, complement levels and other pathogenic immune markers, namely, anti-double-stranded DNA (anti-dsDNA), complements C3 and C4, rheumatoid factor (RF), anticardiolipin antibodies IgG (ACL IgG) and IgM (ACL IgM), Beta-2 Glycoprotein 1 Antibodies (β2-GP) IgG (β2-IgM) and IgM (β2-IgM), and lupus anticoagulant (LA).MethodA comparative cross-sectional study was conducted among 495 SLE patients, who were diagnosed and classified by consultant rheumatologists according to the new European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) 2019 criteria. SLE immunodiagnostic profiles were analyzed including the following parameters: ANA antibody titers and staining patterns, anti-dsDNA, C3 and C4 levels, aCL, and anti-β2-GP and LA.ResultThe most frequently observed ANA patterns were the speckled (52.1%) and homogeneous (35.2%) patterns, while other patterns were rare representing less than 7% of the patients each. ANA titers were highest in patients with mixed pattern followed by the speckled pattern. Of all the investigated patterns, the peripheral pattern showed the most pathogenic immune profile, namely, highest levels of anti-dsDNA, lowest levels of C4, and highest levels of aCL and β2-GP IgG and IgM.ConclusionThis retrospective study showed that speckled followed by homogeneous ANA patterns were predominant accounting for 52.1 and 35.2% of the patients. The ANA pattern showed several associations with other immune markers that are documented to have significant clinical implications in SLE. Peripheral, mixed, and speckled patterns were associated with higher profiles of immune markers indicative of a potential prognostic value of these patterns in SLE.

Abstract P2-07-09: Characterization of the tumor microenvironment in patients with hormone receptor positive, HER2 negative early breast cancer

Abstract Background: Patients (pts) with early stage HER2+ breast cancer (HER2+ EBC) are often treated with neoadjuvant chemotherapy (NACT). Recent studies have demonstrated that increased densities of tumor infiltrating lymphocytes (TIL) are associated with increased rates of pathologic complete response (pCR) in HER2+ EBC. However, outside of standard TIL analysis, a more comprehensive description of the tumor microenvironment (TME) and its impact on clinical outcomes in HER+ EBC is not well defined. Using novel methods, we sought to characterize the TME of patients with hormone receptor positive (HR+), HER2+ EBC treated with NACT. In addition to standard H&amp;E based TIL analysis, we utilized quantitative multiplex immunofluorescence (qmIF), spatial analysis and analysis of RNA-based gene signatures to assess for pretreatment TME characteristics associated with pCR to NACT in HER2+/HR+ EBC. Methods: We identified 29 pts with HER2+/HR+ EBC treated with NACT between 2005 and 2015 for which pretreatment biopsy tissue was available. 13 pts (45%) were treated with an anthracycline, 26 (90%) were treated with trastuzumab in addition to chemotherapy and 14 (48%) were treated with trastuzumab and pertuzumab. At surgery, 13 pts (45%) achieved a pCR. A breast cancer pathologist performed TIL analysis according to international guidelines on pretreatment H&amp;E slides. qmIF was performed on 28 pretreatment specimens using an antibody panel targeting CD3, CD8, CD68, FOXP3, and pancytokeratin. We analyzed multispectral images using HALO software to perform tissue segmentation (tumor vs stroma) and phenotypic analysis based on antibody staining patterns. Upon spatially resolving these components, we then performed proximity analysis. We obtained mRNA-based gene signatures via the Nanostring nCounter Breast Cancer 360 Panel for 28 patients. T-tests were performed to evaluate for associations of TME features with pCR. Results: We evaluated the association of pCR with: manual H&amp;E-based TIL assessments, lymphocyte densities and interactions (assessed by spatial proximity) from qmIF, and RNA signatures assessed from the NanoString BC360 panel. Univariate analysis revealed strong associations of inflammation signatures derived from qmIF with pCR, including higher stromal densities of CD3+ (p&amp;lt;0.01), CD8+ (p=0.02) and CD3+CD8-FOXP3- (p=0.02) cells. pCR was also associated with higher ratios of CD3+:CD68+ (p&amp;lt;0.01). This finding remained significant in T-cell subsets CD8+:CD68+ (p=0.04) and was driven by CD3+CD8-FOXP3-:CD68+ (p=0.02). High numbers of lymphocyte interactions, assessed by counting pairs of cells within 30 microns of each other, were also associated with pCR, including CD3+:FOXP3+ (p&amp;lt;0.01) and CD3+CD8-FOXP3-:FOXP3+ (p&amp;lt;0.01). We found no significant associations between pCR and manual H&amp;E based TIL assessments or RNA signatures. Conclusion: In this population of HER2+/HR+ EBC treated with NACT, stromal and intratumoral TIL assessment by standard H&amp;E was not associated with pCR. Higher stromal densities of CD3+, CD8+, and CD3+CD8-FOXP3- TIL subsets were associated with pCR by qmIF and further analysis revealed the presence and proximity of lymphocyte subpopulations correlated with pCR, suggesting that spatial relationships between these populations within the TME contributes to sensitivity to NACT. The lack of association between TILs and pCR in our cohort by standard H&amp;E is likely due to small numbers; however, significance was seen by qmIF, indicating this method as a potentially more sensitive approach to this analysis. Analysis of RNA signatures by NanoString BC360 panel did not reveal signatures associated with pCR . While our study demonstrates the feasibility of this approach to TME evaluation, further studies of larger cohorts are needed to validate markers predictive of response to NACT. Citation Format: Matthew Ryan Kearney, Rami Vanguri, Qi Wang, Kathleen Fenn, Hua Guo, Douglas Marks, Hanina Hibshoosh, Kevin M Kalinsky, Eileen Connolly. Characterization of the tumor microenvironment in patients with hormone receptor positive, HER2 negative early breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-07-09.

Staining of antinuclear antibodies and antibodies against removable nuclear antigens in connective tissue diseases

Introduction and objectivesConnective tissue diseases are inflammatory, autoimmune diseases and threaten quality of life. To determine the relationship between staining patterns of antinuclear antibodies and antibodies against extractable nuclear antigens in patients with connective tissue disease. Materials and methodsObservational, basic, analytical and transversal study. Study conducted in the Immunology Service of the Arzobispo Loayza National Hospital between January 2017 and June 2017. We analyzed 291 samples of patients with CTD and for the detection of anti-nuclear antibody staining patterns, the immunological kit and observation with microscope of at 40X Immunofluorescence and for the detection of the antibodies against extractable nuclear antigens. The Immunoblot method was employed. Statistical analyses were carried out with the statistical package SPSS version 21 for Windows. We used the Pearson Chi-square test for the categorical variables, a value of p<0.05 was considered significant. ResultsThere was a significant relationship p<0.05 of the homogeneous pattern, the mottled pattern with Anti-histones (p=0.000), Anti-nucleosomes (p=0.000), Anti-Ro 52 (p=0.000), Anti-SSA (p=0.001), Anti-SSB (p=0.003), Anti-dsDNA (p=0.000) with the Pearson Chi-square test. There was a significant relationship of p<0.05 of the centromeric pattern with Anti-Cenp B (p=0.000) with Fisher⿿s exact statistic. ConclusionsThere was a significant relationship between the anti-nuclear antibody staining patterns and the antibodies to the core extractable antigens in patients with systemic lupus erythematosus, Sjögren⿿s syndrome, Calcinosis, Raynaud⿿s phenomenon, esophageal Dysmotility, sclerodactyly and Telangiectasia (CREST), Scleroderma and Polymyositis.

Identification of adenylate kinase 5 antibodies during routine diagnostics in a tissue-based assay: Three new cases and a review of the literature

Antibodies against adenylate kinase 5 (AK5) have been described in patients with non-paraneoplastic limbic encephalitis, mainly in men around 70 years of age. Routine testing with specific cell-based assays is not yet available. Three patients with episodic anterograde memory problems and depression had extensive limbic lesions and developed severe atrophy, mainly of the medial temporal lobes. The antibodies were identified in serum and CSF based on the typical staining pattern of AK5 antibodies on a tissue-based assay (here, unfixed mouse brain). Subsequently, they were confirmed by a research laboratory through a cell-based assay.

The highly GABARAP specific rat monoclonal antibody 8H5 visualizes GABARAP in immunofluorescence imaging at endogenous levels

The determination of unique functions of GABARAP (gamma-aminobutyric acid type A receptor-associated protein), a member of the highly conserved protein family of mammalian autophagy-related 8 protein (mATG8), within diverse cellular processes remains challenging. Because available anti-GABARAP antibodies perform inadequate, especially within various microscopy-based applications, we aimed to develop an antibody that targets GABARAP but not its close orthologs. Following the latest recommendations for antibody validation including fluorescence protein tagging, genetic and orthogonal strategies, we characterized the resulting anti-GABARAP (8H5) antibody during confocal immunofluorescence imaging in-depth. We compared the antibody staining pattern with that obtained for fluorescence protein tagged GABARAP, GABARAPL1 or GABARAPL2 each ectopically expressed in GABARAP knockout cells. Furthermore, we imaged cells expressing all mATG8 family members at endogenous levels and checked GABARAP knockout cells for unspecific staining under fed or macroautophagy-inducing conditions. Finally, we simultaneously stained cells for endogenous GABARAP and the common autophagosomal marker LC3B. Summarized, the presented antibody shows high specificity for GABARAP without cross-reactivity to other mATG8 family members in immunofluorescence imaging making it a valuable tool for the identification of unique GABARAP functions.

The distribution and the origin of unmyelinated fibers in the central nervous system

Background: The distribution and the origin of unmyelinated fibers in the central nervous system(CNS) are not well known. This is because the positive markers of unmyelinated fibers have not been known or electron micrograph(EM) studies were used for the identification of unmyelinated fibers. Well-known unmyelinated fibers in CNS are hippocampal mossy fibers and cerebellar parallel fibers. Recently we have reported the striatal projection fibers are unmyelinated based on the diffuse staining pattern of antibodies to sodium channel beta4 subunit, which stain Ranvier nodes on myelinated fibers as dot pattern(Miyazaki et al. Nat Commun 2014). We also found anti-Nav1.2 antibodies stain diffusely striatal unmyelinated fibers.

Size-based detection of sarcoma circulating tumor cells and cell clusters.

Metastatic disease is the most important factor in determining the survival of sarcoma patients. Since sarcoma metastasis is predominantly hematogenous, we hypothesized that detection and quantification of circulating tumor cells (CTCs) could reflect response to therapy and risk of metastatic relapse. We evaluated the presence of CTCs using a novel animal model and in the blood of patients with high grade sarcomas utilizing the CellSieve™ size-based low pressure microfiltration system. Sarcoma CTCs were identified based on antibody staining patterns and nuclear morphology. Additionally, RNA was extracted from the CTCs for molecular analysis including demonstration of an EWS-FLI1 translocation, identification of a previously unrecognized p53 mutation in a patient with Ewing sarcoma, and single cell RNA sequencing of CTC from a child with alveolar rhabdomyosarcoma. In mouse xenograft models, the presence of CTC correlates with disease burden and with clinically silent metastases. In human patients, CTCs were readily detected at diagnosis, decreased with successful treatment, and were detectable in the blood of patients with no radiographic evidence of disease prior to the development of overt metastasis. Although evaluation of CTC is established in the care of patients with carcinomas, this technology has yet to be effectively applied to the evaluation and treatment of sarcoma patients. Our work demonstrates that the CellSieve™ microfiltration system can be used to study the biology of CTC in both mouse models and human sarcoma patients, with the potential for application to the monitoring of disease response and prediction of metastatic relapse.

Anti-centromere antibody exhibits specific distribution levels among anti-nuclear antibodies and may characterize a distinct subset in rheumatoid arthritis

Anti-centromere antibody (ACA) is one of the classical anti-nuclear antibody (ANA) staining patterns. However, characteristics of ACA in comparison with the other ANA patterns and clinical features of ACA-positive subjects have not been elucidated. Here, we examined all ANA patterns by indirect immunofluorescence for 859 rheumatoid arthritis (RA) patients. Together with the ANA data of 9,575 healthy volunteers, we compared distributions of the ANA levels. ACA was the only ANA that demonstrated a definite bimodal distribution of levels. ACA showed significantly higher levels than the other ANA staining patterns in both RA and healthy population (p < 0.0001). ACA-positivity was associated with old age and was observed more in females. We further recruited another cohort of 3,353 RA patients and confirmed the findings. ACA was also associated with Raynaud’s phenomenon (p = 6.8 × 10−11) in RA. As a conclusion, ACA displays a specific ANA staining pattern with a bimodal distribution, and ACA-positive RA may constitute a distinct subset with specific clinical features.

Abstract WP430: Endothelial Cell Collection from Ipsilateral Middle Cerebral Artery in Acute Ischemic Stroke

Introduction: The cerebrovascular endothelium plays a critical role in the pathogenesis of and response to acute ischemic stroke (AIS). To date, techniques to study its function have relied on animal and in vitro models. A robust method of endothelial cell (EC) capture in patients with AIS at early time points, from within the ischemic region, could greatly advance our understanding of cerebrovascular injury. Method: Patients undergoing thrombectomy for middle cerebral artery occlusion (MCA) within 8 hours of onset were offered enrollment if the pass of their stent-retriever device occurred directly into a distal access catheter in the proximal M1 segment, limiting exposure of the device to only the MCA. After retrieval, ECs adherent to the devices were retrieved and stained for EC (CD31) and leukocyte (CD45) markers. EC identity and yield were confirmed by flow cytometry with simultaneous immuno-fluorescence microscopy. Cultured human ECs were used as positive controls. The EC fraction was defined as CD31 + CD45 - with size and morphological features consistent with the positive controls. Results: ECs from stent-retriever devices (n=3) were collected and pooled. Approximately 8% of the collected cells represented ECs. EC collected from the stent-retrievers demonstrated highly similar shape, morphology and antibody staining patterns compared to the positive controls (Figure). Conclusions: Here we provide the first demonstration of a rapid post-thrombectomy method for reliable harvesting of cerebral ECs in humans. The ability to capture these cells in patients with AIS within hours of symptom onset opens many avenues of exploration for determining the role of ECs in AIS.

A 56-year-old woman with breathlessness

Clinical introductionA 56-year-old female with adult-onset asthma was admitted to the cardiology service with intermittent left-sided chest pain and progressive dyspnoea. Twelve months prior to this admission, she had received a course of prednisolone for bilateral anterior uveitis. Physical examination was unremarkable with blood sampling revealing a marked eosinophilia (eosinophil count 17.3×109/L) and a perinuclear antineutrophil cytoplasmic antibody staining pattern on indirect immunofluorescence microscopy (myeloperoxidase antibodies 83 IU/mL). ECG demonstrated anterolateral T-wave inversion (see online supplementary figure S1). High-sensitivity troponin T was elevated at 100 ng/L. Invasive coronary angiography showed unobstructed coronary arteries. Echocardiography and cardiac magnetic resonance (CMR) were performed (figure 1).QuestionWhat is the most appropriate therapy? Beta-blockade and ACE inhibitionMethylprednisoloneIntravenous antibioticsEndocardiectomyImplantable cardioverter defibrillator (ICD)

Cross-reactivity of Antibodies Directed to the Gram-Negative Bacterium Neisseria gonorrhoeae With Heat Shock Protein 60 and ATP-Binding Protein Correlates to Reduced Mitochondrial Activity in HIBCPP Choroid Plexus Papilloma Cells

Antibacterial antibodies can cause neurologic side-effects by cross-reactivity with cellular antigens. Here we investigated interactions of antibodies to Neisseria gonorrhoeae (α-NG) - maternal infections by which increases the offspring's risk for later psychosis-with HIBCPP cells, a cell culture model of choroid plexus epithelium. Immunocytochemistry and Western blotting with α-NG, revealed organelle-like intracellular staining in HIBCPP cells, and labelling of several immunoreactive bands in cellular protein. Two-dimensional Western blotting revealed several immunopositive spots, most prominent of which were identified by mass spectrometry as mitochondrially localized proteins heat shock protein 60 (Hsp60) and ATP-binding protein β-subunit (ATPB). Similarly α-NG interacted with commercial samples of these proteins as revealed by Western blotting. Three alternative methods (JC-1, Janus green and MTT staining) revealed α-NG to cause in HIBCPP cells a significant decrease in mitochondrial activity, which could be reverted by neuroleptic drugs. Immunoreactivity of α-NG with choroid plexus epithelium in human post mortem samples suggests in vivo relevance of these findings. Finally, distinctly different staining patterns of antibodies against Neisseria meningitidis (α-NM), confirmed antibody specificity. To our knowledge this is the first report that α-NG cross-reactivity with Hsp60 and ATPB impairs mitochondrial activity in choroid plexus epithelial cells, pathogenetic relevance of which needs further clarification.

Age and genetic selection affect auto-immune profiles of chickens

Specificity, antibody isotype distribution and levels, of natural autoantibodies (NAAb) may be potential informative parameters for immune mediated natural disease resistance, immune modulation, and maintenance of physiological homeostasis. In a previous study we detected IgM and IgG antibodies to liver antigens in plasma from 1 year old chickens. Auto-immune profiles directed towards liver antigens differed between chicken lines divergently selected for specific antibody responses to sheep red blood cells. In the present study we measured the presence and typed levels and antibody isotypes (IgG and IgM) of NAAb binding the ‘auto-antigen’ complex chicken liver cell lysate (CLL) in plasma samples obtained from chickens at 5 weeks and at 1-year of age, respectively, by quantitative western blotting.Extensive staining patterns of plasma antibodies binding CLL were found for both isotypes and at both ages in all birds. At both ages, IgM and IgG bound similar numbers of CLL antigens, which remained almost constant for IgM, whereas the number of IgG stained bands in time was enhanced. Significant differences of binding patterns of NAAb (stained antigen fragments of CLL and staining intensity) were detected between the three different chicken lines at both ages and between both ages, and lines could be clustered on the basis of their auto-antibody profile. The present results indicate that analysis of the plasma NAAb repertoire of poultry like in mammals could provide a way of distinguishing differences of immune competence (as reflected by the selection criterion of antibody responses) between individuals and lines, and could provide tools to select individual birds for health and other traits. The age-dependency of the auto-immune profile suggest that such profiles may also reflect immune maturation, which should be taken into account when relating an auto-immune profile with other traits.