Anti-histone Antibodies Research Articles

Real-Time, High-Throughput Microscopic Quantification of Human Neutrophil Extracellular Trap Release and Assessing the Pharmacology of Antagonists.

Neutrophils play an important role in innate immune defense by using several strategies, including the release of neutrophil extracellular traps (NETs) in a process referred to as NETosis. However, in the past two decades, it has become clear that the accumulation of NETs in tissues contributes to the pathophysiology of multiple inflammatory and autoimmune diseases. Therefore, interest in the development of NETosis antagonists has risen. Variable and non-standardized methods to detect and analyze NETosis were developed concomitantly, each with its own advantages and limitations. Here, we describe a real-time microscopy method for the quantification of human NET release, allowing to study NETosis as well as NET inhibition in a high-throughput manner. The surface area-based semi-automated analysis recognizes NETs and distinguishes them from non-netting activated neutrophils. We demonstrate that the non-physiological NETosis inducers, calcium ionophore and phorbol-12-myristate-13-acetate (PMA), trigger the release of NETs with different characteristics and kinetics. Furthermore, we show that this approach allows studying NET release in response to disease-relevant stimuli, including immune complexes, N-Formylmethionine-leucyl-phenylalanine (fMLF), monosodium urate crystals, and calcium pyrophosphate crystals. To exemplify the utility of this method to study NETosis antagonists, we used CIT-013, a first-in-class monoclonal antibody inhibitor of NET release. CIT-013 targets citrullinated histone H2A and H4 and efficiently inhibits NET release with an IC50 of 4.6 nM. Other anti-histone antibodies tested lacked this NETosis-inhibitory capacity. Altogether, we demonstrate that this protocol enables specific, reliable, and reproducible high-throughput quantification of NETs, enhancing the study of NET release characteristics, kinetics, and pharmacology of NETosis antagonists.

Emerging therapeutic strategies targeting extracellular histones for critical and inflammatory diseases: an updated narrative review.

Extracellular histones are crucial damage-associated molecular patterns involved in the development and progression of multiple critical and inflammatory diseases, such as sepsis, pancreatitis, trauma, acute liver failure, acute respiratory distress syndrome, vasculitis and arthritis. During the past decade, the physiopathologic mechanisms of histone-mediated hyperinflammation, endothelial dysfunction, coagulation activation, neuroimmune injury and organ dysfunction in diseases have been systematically elucidated. Emerging preclinical evidence further shows that anti-histone strategies with either their neutralizers (heparin, heparinoids, nature plasma proteins, small anion molecules and nanomedicines, etc.) or extracorporeal blood purification techniques can significantly alleviate histone-induced deleterious effects, and thus improve the outcomes of histone-related critical and inflammatory animal models. However, a systemic evaluation of the efficacy and safety of these histone-targeting therapeutic strategies is currently lacking. In this review, we first update our latest understanding of the underlying molecular mechanisms of histone-induced hyperinflammation, endothelial dysfunction, coagulopathy, and organ dysfunction. Then, we summarize the latest advances in histone-targeting therapy strategies with heparin, anti-histone antibodies, histone-binding proteins or molecules, and histone-affinity hemoadsorption in pre-clinical studies. Finally, challenges and future perspectives for improving the clinical translation of histone-targeting therapeutic strategies are also discussed to promote better management of patients with histone-related diseases.

Generalized lymphadenopathy as an initial presentation of systemic lupus erythematosus: case report and review of the literature

BackgroundSystemic lupus erythematosus (SLE) is a chronic multisystemic autoimmune disorder with a wide array of clinical manifestations. Lymphadenopathy is occasionally observed in patients with SLE, and although it is sometimes a feature of active disease, it is rarely the initial presenting feature at the time of diagnosis.Case presentationWe report the case of a 35-year-old woman with clinical features of generalized lymphadenopathy, fatigue, fever, and malaise of approximately 3 months duration with no other constitutional disorders or systemic involvement suggestive of SLE.Despite extensive laboratory, radiological, and histological evaluations for malignancies and infectious causes of generalized lymphadenopathy, all results returned normal. About a month later, she developed inflammatory polyarthritis, with a markedly elevated erythrocyte sedimentation rate (ESR) of 120 mm/h, and a subsequent serologic workup confirmed the diagnosis of SLE.Autoimmune serology testing revealed positive results for various antibodies, including antinuclear antibody (ANA) at a titre of 1:5120, homogenous and speckled pattern, anti-Sm, anti-dsDNA, U1-snRNP, Ku, SSA-AntiRo, SSB-AntiLa, anti-SM/RNP, anti-Histone, ribosomal-p protein, and anti-nucleosome antibodies. The diagnosis of SLE was established using the 2019 European League Against Rheumatism/American College of Rheumatology (EULAR/ ACR) criteria.She was on oral prednisolone, hydroxychloroquine, and calcium/vitamin D3 tablets. The patient improved remarkably, all swollen lymph nodes regressed spontaneously and she had no further need for a hematology review.ConclusionWhile similar cases have been reported in Europe and some parts of Asia, in Africa, a careful search of the literature revealed that such cases have been reported only from Egypt in North Africa and not from other parts of the continent; hence, this case report highlights the importance of recognizing atypical presentations of SLE to improve awareness and diagnosis in different regions.

Comparison of Clinical and Laboratory Characteristics in Lupus Nephritis vs. Non-Lupus Nephritis Patients-A Comprehensive Retrospective Analysis Based on 921 Patients.

Background: Lupus nephritis (LN) is an inflammation of the kidneys that is related to systemic lupus erythematosus (SLE). This study aimed to evaluate the differences in clinical and laboratory characteristics between LN and non-LN SLE patients. Methods: We conducted a retrospective analysis of medical records collected from SLE patients treated at the University Hospital in Kraków, Poland, from 2012 to 2022. All patients met the 2019 European League Against Rheumatism and the American College of Rheumatology (EULAR/ACR) criteria for SLE. Results: Among 921 SLE patients, LN was documented in 331 (35.94%). LN patients were younger at SLE diagnosis (29 vs. 37 years; p < 0.001) and had a male proportion that was 2.09 times higher than the non-LN group (16.62% vs. 7.97%; p < 0.001). They were more often diagnosed with serositis and hematological or neurological involvement (p < 0.001 for all). Hypertension and hypercholesterolemia occurred more frequently in these patients (p < 0.001 for both). LN patients exhibited a higher frequency of anti-dsDNA, anti-histone, and anti-nucleosome antibodies (p < 0.001 for all). Conversely, the non-LN group had a 1.24-fold (95% CI: 1.03-1.50; p = 0.021) increase in the odds ratio of having positive anti-cardiolipin IgM antibody results. LN patients were more frequently treated with immunosuppressants. The risk factors for experiencing at least three LN flares included female sex, younger age at the onset of LN or SLE, LN occurring later than SLE onset, the presence of anti-nucleosome or anti-dsDNA antibodies, and certain SLE manifestations such as myalgia, arthritis, proteinuria > 3.5 g/day, and pathological urinary casts in the urine sediment. Conclusions: LN patients differ from non-LN patients in the age of SLE diagnosis, treatment modalities, and autoantibody profile and have more frequent, severe manifestations of SLE. However, we still need more prospective studies to understand the diversity of LN and its progression in SLE patients.

Development of Autoimmune Diseases Among Children With Pediatric Acute-Onset Neuropsychiatric Syndrome

Epidemiologic studies indicate a high rate of autoimmune conditions among patients with obsessive-complusive disorder and other psychiatric conditions. Furthering the understanding of the inflammatory diatheses of psychiatric conditions may open doors to new treatment paradigms for psychiatric disorders. To evaluate whether pediatric acute-onset neuropsychiatric syndrome (PANS) is associated with an inflammatory diathesis by assessing signs of immune activation and vasculopathy during a psychiatric symptom exacerbation (flare), estimating the risk of developing arthritis and other autoimmune diseases, and characterizing subtypes of arthritis. This retrospective cohort study used longitudinal clinical data on 193 consecutive patients with PANS followed up within the Stanford Immune Behavioral Health Clinic from September 1, 2012, to December 31, 2021. Medical records were reviewed, and a predefined set of immune markers that were measured during a flare and the features and imaging findings of arthritis and other autoimmune diseases were collected. Immune activation markers included (1) autoimmunity signs (antinuclear antibody, antihistone antibody, antithyroglobulin antibody, C1q binding assay, and complement levels [C3 and C4]); (2) immune dysregulation or inflammation signs (leukopenia, thrombocytosis, C-reactive protein, and erythrocyte sedimentation rate); and (3) vasculopathy signs (livedo reticularis, periungual redness and swelling, abnormally prominent onychodermal band, palatal petechiae, high von Willebrand factor antigen, and high d-dimer). Last, the cumulative risk of developing arthritis and autoimmune diseases was estimated using product limit (Kaplan-Meier) survival probability. The study included data from 193 children (112 boys [58.0%]) who had PANS at a mean (SD) age of 7.5 (3.5) years. They were followed up for a mean (SD) of 4.0 (2.1) years. Among those tested for immune activation markers, 54.2% (97 of 179) had nonspecific markers of autoimmunity, 12.0% (22 of 184) had nonspecific signs of immune dysregulation or inflammation, and 35.8% (69 of 193) had signs of vasculopathy. By 14 years of age, the estimated cumulative incidence of arthritis was 28.3% (95% CI, 20.8%-36.3%), and the estimated cumulative incidence of another autoimmune disease was 7.5% (95% CI, 4.0%-12.4%). Novel findings in the subgroup with arthritis include joint capsule thickening (55.0% [22 of 40]), distal interphalangeal joint tenderness (81.8% [45 of 55]), and spinous process tenderness (80.0% [44 of 55]). Among the 55 patients with arthritis, the most common subtypes of arthritis included enthesitis-related arthritis (37 [67.3%]), spondyloarthritis (27 [49.1%]), and psoriatic arthritis (10 [18.2%]). This study found that patients with PANS show signs of immune activation and vasculopathy during psychiatric symptom flares and have an increased risk of developing arthritis and other autoimmune diseases compared with the general pediatric population. The most common arthritis subtype was enthesitis-related arthritis. These findings suggest that PANS may be part of a multisystem inflammatory condition rather than an isolated psychiatric or neuroinflammatory disorder.

Drug-induced lupus erythematosus in childhood: Case-based review.

Drug-induced lupus erythematosus (DILE) is the development of lupus-like syndrome following a drug exposure. DILE has been reported less frequently among children than adults. In this study, we present four children with DILE and similar published cases through a systematic literature review. We report four children (three girls and one boy) who developed DILE associated with the use of topiramate, doxycycline, etanercept, and ethosuximide. Three of them were positive for anti-histone antibodies. In all patients, the drug was discontinued and symptoms resolved completely. The literature review revealed 48 articles describing 61 children with DILE. In the evaluation of 65 patients (our 4 patients and 61 patients from the literature), the most frequently reported drugs associated with DILE were ethosuximide (n = 13) and minocycline (n = 12). Fever (n = 33), arthralgia (n = 31), rash (n = 30), and arthritis (n = 29) were the most common clinical manifestations. Antinuclear antibody (ANA) was positive in 93.5% of patients and anti-histone antibodies were detected in 72.2% of the patients. As for treatment, the responsible drug was discontinued in all patients, and corticosteroids were initiated in 53.3%. Improvement was achieved in 92.0% of patients. For children presenting with SLE features, proper drug history is crucial since DILE may be more frequent than anticipated. An association of the relevant drug with the symptoms, and resolution of symptoms on drug withdrawal provides evidence for the diagnosis of DILE.

Associations of Digital Ulcers in Patients with Systemic Sclerosis: An 8-Year Retrospective Study

Introduction: This study aimed to investigate the associations of digital ulcers (DUs) in patients with systemic sclerosis (SSc). Methods: This retrospective study investigated the demographic characteristics, specific autoantibodies, organ involvement, and laboratory tests in patients with SSc from our hospital. Results: This study enrolled 144 patients with SSc. The DU+ group consisted of 15 (10.4%) patients. Patients with SSc having DUs have longer disease duration, higher fibrinogen, higher fibrin degradation product, and lower cholesterol. None of the patients used cholesterol-lowering drugs before onset of DUs. The study also demonstrated a higher prevalence of anti-dsDNA and anti-histone antibodies in patients with SSc with DUs. Anti-dsDNA antibody is a specific antibody for SLE with a specificity of 96–99%. A total of 86.1% (124/144) of patients suffered from diffuse cutaneous SSc, and 28.5% (41/144) of patients suffered from overlap syndrome. Conclusion: Our study indicated that patients with SSc with fibrinogen of >2.895 g/L (p = 0.043) and cholesterol of <3.340 mmol/L (p = 0.036), which is equal to 129.258 mg/dL, are at high risk of developing DUs.

Anti-histone and anti-nucleosome rather than anti-dsDNA antibodies associate with IFN-induced biomarkers in Sudanese and Swedish Systemic Lupus Erythematosus patients.

In SLE, anti-dsDNA can co-occur with autoantibodies against other chromatin components, like histones and nucleosomes. These antibodies induce type-1 interferon production, a hallmark of SLE. We measured antinuclear antibody (ANA) sub-specificities and investigated their associations to inflammatory biomarkers including interferon-regulated chemokines. We included 93 Sudanese and 480 Swedish SLE patients and matched controls (N = 104 + 192). Autoantibodies targeting ANA-subspecificites: dsDNA, Sm, Sm/U1RNPcomplex, U1RNP, SSA/Ro52, SSA/Ro60, SSB/La, ribosomal P, PCNA and histones were quantified in all subjects, anti-nucleosome only in the Swedish patients, with a bead-based multiplex immunoassay. Levels of 72 plasma biomarkers were determined with Proximity Extension Assay technique or ELISA. Among Sudanese patients, the investigated antibodies significantly associated with 9/72 biomarkers. Anti-histone antibodies showed the strongest positive correlations with MCP-3 and S100A12 as well as with interferon I-inducible factors MCP-1 and CXCL10. Anti-dsDNA antibodies associated with CXCL10 and S100A12, but in multivariate analyses, unlike anti-histone, associations lost significance.Among Swedish patients, MCP-1, CXCL10, SA100A12 also demonstrated stronger associations to anti-histone and anti-nucleosome antibodies, compared with anti-dsDNA and other ANA sub-specificities. In multiple regression models, anti-histone/nucleosome retained the strongest associations. When excluding anti-histone or anti-nucleosome positive patients, the associations between MCP-1/CXCL10 and anti-dsDNA were lost. In contrast, when excluding anti-dsDNA positive patients, associations with anti-histone and anti-nucleosome remained significant. In two cohorts of different ethnical origin, autoantibodies targeting chromatin correlate stronger with IFN-induced inflammatory biomarkers than anti-dsDNA or other ANA sub-specificities. Our results suggest that anti-histone/nucleosome autoantibodies may be main drivers of type-1 interferon activity in SLE.

Abstract 13315: Recurrent Cardiac Tamponade Due to Minoxidil Associated Drug Induced Lupus Erythematosus

Case description A 59 year old man with history of MGUS, CKD stage 2, DM, HTN, and OSA presented with gradual worsening of dyspnea and intermittent dizziness over the past few days. Echocardiography showed diastolic collapse of the RV, RA collapse, IVC plethora, marked tricuspid in-flow velocities, and pericardial effusion suggestive of cardiac tamponade (CT). Subxiphoid pericardial window was emergently performed. Serosanguineous fluid 900 mL was drained, and fluid analysis was negative for malignancy or infection. Pericardial biopsy revealed fibro-connective tissue with mild peri-vascular chronic inflammation. He significantly improved and was discharged after 5 days. One month later, he presented again with 1 day symptom of severe dyspnea due to recurrent pericardial effusion with CT physiology. Emergent pericardiocentesis was performed to remove 1,800 ml of serous fluid. Serologic workup for autoimmune disease was strongly positive for anti-histone antibody. ESR (124 mm/hr) and c-reactive protein (5.8 mg/dL) were elevated. Upon review of prescription list, he had been treated for HTN in the past 5 years with minoxidil, amlodipine, lisinopril, and carvedilol. Discussion The use of topical minoxidil, a potent peripheral arterial vasodilator, has been approved for androgenetic alopecia in both men and women for decades, whereas oral minoxidil is limited to refractory hypertension due to side effects such as reflex tachycardia, fluid retention, hypertrichosis, and, rarely, idiosyncratic pericardial effusion. Pericardial effusion was reported in approximately 3-5% of patients receiving minoxidil therapy, which could be attributed to volume overload associated with underlying renal insufficiency. Besides hydralazine, another direct arterial vasodilator commonly implicated in drug-induced lupus erythematosus (DILE), minoxidil also carries a trivial risk of DILE, as suggested by a positive anti-histone antibody and biopsy-proven serositis. Although the association between topical minoxidil and pericardial effusion has never been reported, receding hairline has become a major cosmetic concern and the clinicians should be aware of this rare, but serious, complication associated with minoxidil due to its widespread use.

Clinical value of chemiluminescence method for detection of antinuclear antibody profiles.

Antinuclear antibodies (ANAs) are crucial in diagnosing autoimmune diseases, mainly systemic lupus erythematosus (SLE). This study aimed to compare the performance of chemiluminescence assay (CLIA) and line immunoassay (LIA) in detecting ANAs in patients with autoimmune diseases, evaluate their diagnostic accuracy for SLE, and develop a novel diagnostic model using CLIA-detected antibodies for SLE. Specimens from patients with autoimmune diseases and physical examination specimens were collected to parallel detect specific antibodies. Individual antibodies' diagnostic performance and a model combining multiple antibodies were assessed. The findings provide valuable insights into improving the diagnosis of SLE through innovative approaches. To compare the performance of CLIA and LIA in detecting ANAs in patients with autoimmune diseases, assess their accuracy for SLE, and develop a novel diagnostic model using CLIA-detected antibodies for SLE. Specimens have been obtained from 270 patients with clinically diagnosed autoimmune disorders, as well as 130 physical examination specimens. After that, parallel detection of anti-double-stranded DNA (dsDNA) antibody, anti-histone (Histone) antibody, anti-nucleosome (Nuc) antibody, anti-Smith (Sm) antibody, anti-ribosomal P protein (Rib-P) antibody, anti-sicca syndrome A (Ro60) antibody, anti-sicca syndrome A (Ro52) antibody, anti-sicca syndrome (SSB) antibody, anti-centromere protein B (Cenp-B) antibody, anti-DNA topoisomerase 1 (Scl-70) antibody, anti-histidyl tRNA synthetase (Jo-1) antibody, and anti-mitochondrial M2 (AMA-M2) antibody was performed using CLIA and LIA. The detection rates, compliance rates, and diagnostic performance for SLE were compared between the two methodologies, followed by developing a novel diagnostic model for SLE. CLIA and LIA exhibited essentially comparable detection rates for anti-dsDNA antibody, anti-Histone antibody, anti-Nuc antibody, anti-Sm antibody, anti-Rib-P antibody, anti-Ro60 antibody, anti-Ro52 antibody, anti-SSB antibody, anti-Cenp-B antibody, anti-DNAScl-70 antibody, anti-Jo-1 antibody and anti-AMA-M2 antibody (P > 0.05). The two methods displayed identical results for the detection of anti-dsDNA antibody, anti-Histone antibody, anti-Nuc antibody, anti-Sm antibody, anti-Ro60 antibody, anti-Ro52 antibody, anti-SSB antibody, anti-Cenp-B antibody, anti-Scl-70 antibody, and anti-AMA-M2 antibody (Kappa > 0.7, P < 0.05), but showed a moderate agreement for the detection of anti-Rib-P antibody and anti-Jo-1 antibody (Kappa = 0.671 and 0.665; P < 0.05). In addition, the diagnostic performance of these antibodies detected by both methods was similar for SLE. The diagnostic model's area under the curve values, sensitivity, and specificity, including an anti-dsDNA antibody and an anti-Ro60 antibody detected by CLIA, were 0.997, 0.962, and 0.978, respectively. These values were higher than the diagnostic performance of individual antibodies. CLIA and LIA demonstrated excellent overall consistency in detecting ANA profiles. A diagnostic model based on CLIA-detected antibodies can successfully contribute to developing a novel technique for detecting SLE.

Externalized histones fuel pulmonary fibrosis via a platelet-macrophage circuit of TGFβ1 and IL-27

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFβ and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFβ1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFβ1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFβ1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.

Multiple intra-articular injections with adipose-derived stem cells for knee osteoarthritis cause severe arthritis with anti-histone H2B antibody production

IntroductionOsteoarthritis (OA) is the most common form of arthritis. OA results from the breakdown of cartilage, which leads to deterioration of the entire joint and the connective tissue that holds the joint together, and gradually and irreversibly worsens over time. Adipose-derived stem/stromal cells (ADSCs) have been used in the treatment of knee OA. However, the safety and efficacy of ADSC treatment of OA remain unclear. In this study, we investigated the pathophysiology of severe knee arthritis that occurred after ADSC treatment by screening for autoantibodies in synovial fluid from patients who received ADSC treatment. MethodsAdult Japanese patients with OA who received ADSC treatment at Saitama Cooperative Hospital between June 2018 and October 2021 were enrolled. Antibodies (Abs) were screened using immunoprecipitation (IPP) with [35S]-methionine-labeled HeLa cell extracts. The detected protein was identified by liquid chromatography coupled with time-of-flight mass spectrometry (MS) and ion trap MS, and the corresponding proteins were confirmed as autoantigens using immunoblotting. Ab titers were measured using an enzyme-linked immunosorbent assay. ResultsA total of 113 patients received ADSC treatment, and 75% (85/113) received ADSC injection at least twice with a 6-month interval between. No obvious abnormalities were observed in any patient after their first treatment; by contrast, 53% (45/85) of patients who received their second or third ADSC injection showed severe knee arthritis. IPP detected a common anti-15 kDa Ab in synovial fluid of 62% (8/13) of the samples analyzed from patients who showed severe arthritis. This Ab was not detected in synovial fluid obtained from the same joints before treatment. The corresponding autoantigen was identified as histone H2B. All available synovial samples from patients who tested positive for anti-histone H2B Ab were newly positive after the treatment; that is, none had been positive for anti-histone H2B Ab before treatment. ConclusionsMultiple ADSC injections for OA induced severe arthritis in a high percentage of patients, particularly after the second injection. Synovial fluid from some patients with knee arthritis contained Ab to histone H2B that appeared only after ADSC treatment. These findings provide new insights into the pathogenesis of ADSC treatment-induced severe arthritis.

Colyn Crane-Robinson (20 October 1935–5 March 2023)

Colyn Crane-Robinson, Professor of Biochemistry at the University of Portsmouth, was born on 20 October 1935 and died on 5 March 2023. His death marks the end of a career as a world-leading researcher in the field of chromatin structure and epigenetics.Colyn was born in the Sale area of Greater Manchester to Robin and Marion. After evacuation at the start of World War 2, he returned to Manchester in 1947 and joined Ackworth Secondary School in south-west Yorkshire. At school, he enjoyed cricket, becoming a noted bowler, obtaining the nickname ‘colyncranecricketball’, and later the captain of the first team. He cited his teacher at the time, Mr Harris, as giving him a lasting passion for classical music and secondly, and more critical to his future career, chemistry.In 1954, Colyn joined Christ Church College, Oxford, where his love for research was fostered. After obtaining a first-class degree, he began his DPhil in the laboratory of H.W. Thompson, a renowned infra-red spectroscopist, working on vibrational–rotational spectra studies of gas-phase molecules. This time was noted by having to construct one’s own gas line, meaning a glass-blowing course had to be undertaken, something Colyn managed but, by his own admission, his creations contained many strange shapes and bubbles!After his PhD, Colyn took the unusual step of using a newly arranged treaty between the Royal Society and the USSR, to commence a post-doctoral position in Leningrad at the Soviet Academy of Sciences. Here he became fluent in Russian and studied physico-chemical aspects of polymer synthesis. His time was cut short after he met and proposed to a young woman. Marriages to westerners being unfavourable, he was asked to leave the USSR abruptly. He did successfully get her out of the USSR and they went on to have a family together. Upon his return to the UK, Colyn obtained a job at the polymer firm Courtaulds, but when research there was closed abruptly he looked elsewhere for employment, which brought him to the University of Portsmouth.Colyn arrived in Portsmouth to work in the newly established biophysics laboratory founded by Morton Bradbury. Together they worked on high-resolution NMR studies to investigate conformational changes in biopolymers, producing a series of high-impact Nature publications and leading to an interest in chromatin. Their research moved into detailed studies on the structure of individual histones and then progressed into proposed models of the subunit structure of a eukaryotic chromosome. The group ultimately focused on investigations of histone modifications and their interactions with DNA within the nucleosome, in particular acetylation of the N-terminal tail of histone H4 and its consequential DNA interactions.In 1988, the discovery of the link between histone H4 acetylation and transcriptionally active chromatin was cemented after the group successfully raised an anti-histone acetyl-specific antibody, alongside their development of the ChIP (chromatin immunoprecipitation) assay. These tools were used to show a strong enrichment in active gene sequences versus inactive in antibody-bound fractions, compared to the input material. In Colyn’s own words, he was “the principal midwife at the birth of ChIPs, using antibodies to modified histones”. The publication from this research became highly cited and remains as a critical statement of this fundamental epigenetic control mechanism.I joined Colyn’s research laboratory as a Wellcome Research Fellow in 1995. During my time in his laboratory, the work focused on the role of histone H3 K4 methylation in the control of gene expression, work conducted in collaboration with the Kouzarides laboratory at Cambridge. Indeed, Colyn had collaborative projects across the globe; two of the most prolific and long-standing were with Peter Privalov (Johns Hopkins University, USA) and Doncho Staynov (Imperial College London, UK). The first collaboration was on the principles of the thermodynamics of proteins–DNA interactions; the second focused on interactions of the linker histones with the nucleosome structure.Colyn became a Professor of Biochemistry, taking emeritus status in 2000. His enthusiasm was infectious, and he particularly enjoyed enthusing our younger researchers (PhD, MRes and undergraduates) with his joy for all things scientific. His willingness to communicate his knowledge was continued right to the end; the image shown is from a group research talk given by Colyn only a few months before his passing.

Antibodies to histone in the pediatric population: a retrospective chart review

BackgroundAntibodies to histone have been associated in the adult literature with systemic lupus erythematosus(SLE) and drug induced lupus(DILE). Little data is available regarding the spectrum of pathology that antibodies to histone encompass in the pediatric population. Prior studies suggest an association with SLE, juvenile idiopathic arthritis(JIA), uveitis and linear scleroderma.MethodsPatient charts were reviewed that contained positive anti-histone antibody testing during a consecutive three year period. Patient diagnosis along with the presence of: anti-histone antibody titer, ANA, and the presence of other autoantibodies to SSA, SSB, Sm, RNP, dsDNA and chromatin were obtained. The frequency of SLE, JIA and DILE was further investigated in specific subsets.Results139 individual charts were reviewed containing 41 different diagnoses. The most common diagnosis was hypermobility arthralgia with 22 patients. The most frequent rheumatologic diagnosis was JIA(nonsystemic) with 19. 13 patients in this study were diagnosed with SLE and 2 with DILE. 18 patients had other autoantibody production, of these, 11 had SLE or DILE. Only one of 62 patients with a weak antihistone antibody titer(1.0-1.5) was diagnosed with SLE. When strong titers are present(> 2.5), the antihistone antibody test was associated with a greater than 50% incidence of an underlying rheumatologic disease and ten times higher incidence of SLE than a weak titer. In regards to the frequency of SLE, there was a statistically significant difference between weak and moderate titers and between weak and strong titers.ConclusionThe presence of anti-histone antibody was observed in a variety of diagnoses in the pediatric population. Overall, the presence of anti-histone antibodies appears to have poor diagnostic utility for any specific condition. However, diagnostic utility for SLE does appear to improve with higher titers, when combined with other autoantibody positivity. Strength of titer did not appear to be a factor for JIA, but was the most frequently observed rheumatologic disease in this study.

Comparative Analysis of Molecular Functions and Biological Role of Proteins from Cell-Free DNA-Protein Complexes Circulating in Plasma of Healthy Females and Breast Cancer Patients.

Cell-free DNA (cfDNA) circulates in the bloodstream packed in membrane-coated structures (such as apoptotic bodies) or bound to proteins. To identify proteins involved in the formation of deoxyribonucleoprotein complexes circulating in the blood, native complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies from plasma of healthy females (HFs) and breast cancer patients (BCPs). It was found that the nucleoprotein complexes (NPCs) from HF plasma samples contained shorter DNA fragments (~180 bp) than BCP NPCs. However, the share of DNA in the NPCs from cfDNA in blood plasma in HFs and BCPs did not differ significantly, as well as the share of NPC protein from blood plasma total protein. Proteins were separated by SDS-PAGE and identified by MALDI-TOF mass spectrometry. Bioinformatic analysis showed that in the presence of a malignant tumor, the proportion of proteins involved in ion channels, protein binding, transport, and signal transduction increased in the composition of blood-circulating NPCs. Moreover, 58 (35%) proteins are differentially expressed in a number of malignant neoplasms in the NPCs of BCPs. Identified NPC proteins from BCP blood can be recommended for further testing as breast cancer diagnostic/prognostic biomarkers or as being useful in developing gene-targeted therapy approaches.

Neutrophil extracellular trap formation in anti-neutrophil cytoplasmic antibody-associated and large-vessel vasculitis.

Levels of neutrophil extracellular traps (NETs) were measured in plasma of healthy controls (HC, n=30) and patients with granulomatosis with polyangiitis (GPA, n=123), microscopic polyangiitis (MPA, n=61), Takayasu's arteritis (TAK, n=58), and giant cell arteritis (GCA, n=68), at times of remission or activity and correlated with levels of the platelet-derived thrombospondin-1 (TSP-1). Levels of NETs were elevated during active disease in patients with GPA (p<0.0001), MPA (p=0.0038), TAK (p<0.0001), and GCA (p<0.0001), and in remission for GPA, p<0.0001, MPA, p=0.005, TAK, p=0.03, and GCA, p=0.0009. All cohorts demonstrated impaired NET degradation. Patients with GPA (p=0.0045) and MPA (p=0.005) had anti-NET IgG antibodies. Patients with TAK had anti-histone antibodies (p<0.01), correlating with presence of NETs. Levels of TSP-1 were increased in all patients with vasculitis, and associated with NET formation. NET formation is a common process in vasculitides. Targeting NET formation or degradation could be potential therapeutic approaches for vasculitides.

Removal of chromatin by salt-tolerant endonucleases for production of recombinant measles virus.

Host cell DNA is a critical impurity in downstream processing of enveloped viruses. Especially, DNA in the form of chromatin is often neglected. Endonuclease treatment is an almost mandatory step in manufacturing of viral vaccines. In order to find the optimal performer, four different endonucleases, two of them salt tolerant, were evaluated in downstream processing of recombinant measles virus. Endonuclease treatment was performed under optimal temperature conditions after clarification and before the purification by flow-through chromatography with a core shell chromatography medium: Capto™ Core 700. Virus infectivity was measured by TCID50. DNA and histone presence in process and purified samples was determined using PicoGreen™ assay and Western blot analysis using an anti-histone antibody, respectively. All tested endonucleases allowed the reduction of DNA content improving product purity. The salt-tolerant endonucleases SAN and M-SAN were more efficient in the removal of chromatin compared with the non-salt-tolerant endonucleases Benzonase® and DENARASE®. Removal of chromatin using M-SAN was also possible without the addition of extra salt to the cell culture supernatant. The combination of the endonuclease treatment, using salt-tolerant endonucleases with flow-through chromatography, using core-shell particles, resulted in high purity and purification efficiency. This strategy has all features for a platform downstream process of recombinant measles virus and beyond.

Autoantibodies in lupus nephritis useful in distinguishing proliferative nephritis from membranous nephritis.

This study was conducted to determine autoantibodies associated with lupus nephritis, especially those useful in diagnosing proliferative and membranous nephritis. 106 patients with lupus nephritis and 63 patients with SLE but no nephritis were enrolled. 55 patients were selected from the lupus nephritis group and were divided into two groups: proliferative nephritis patients (n=36) and membranous nephritis patients (n=19). The autoantibody profiles of patients' sera were evaluated using the EUROLINE ANA profile 3 (IgG) kit. A higher positivity rate of anti-dsDNA antibody and anti-histone antibody were seen in lupus nephritis patients compared to non-renal SLE patients. In comparing between proliferative and membranous nephritis, positivity of anti-nucleosome antibody was higher in proliferative nephritis compared to membranous nephritis, although it was not statistically significant. However, anti-nucleosome antibody-positive patients with lupus nephritis had higher prevalence of hematuria and pyuria, which are strong indications of proliferative nephritis. Also, a significantly higher positivity rate of anti-RNP70 antibody was seen in membranous nephritis compared to proliferative nephritis. Our results showed anti-nucleosome antibody and anti-RNP70 antibodies may be predictive non-histological factors for discriminating between proliferative and membranous lupus nephritis.

PSUN62 A Rare Case of Atorvastatin Induced Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS) Syndrome - A Case Report.

Abstract Clinical Case A 77 year-old female presented to emergency department with progressively worsening rash that started one week ago. On physical examination blanching erythema was noted in lower back, buttocks and extremities. There was no desquamation or involvement of oral mucosa. No lymphadenopathy or hepatosplenomegaly was noted. Laboratory investigations showed white blood cell count of 21.2×10^3/uL, with 33.1% eosinophils. Creatinine was elevated at 2.8mg/dL from baseline of 0.8mg/dL. Further lab work revealed alkaline phosphate 2314 IU/L, aspartate transaminase 587 IU/L, and alanine transaminase 620 U/L, total bilirubin 7.3mg/dL with direct bilirubin of 5.1mg/dL, Immunoglobulin E level of 2000 IU/ml and normal Tryptase levels. Serology for viral hepatitis, Epstein-Barr virus, Cytomegalovirus and Human immunodeficiency virus was negative. Autoimmune workup including antinuclear Antibody (ANA), anti-neutrophil cytoplasmic antibody (ANCA), mitochondrial antibody, smooth muscle antibody, double stranded DNA, anti-histone antibody, rheumatoid factor, Sjogren's antibody and centromere antibody were negative. Urinalysis, urine protein electrophoresis and serum protein electrophoresis were also normal. Computed tomography (CT) scan of chest showed mild supraclavicular and mediastinal lymphadenopathy. Review of medical records revealed that patient was recently discharged from hospital with a prescription of Atorvastatin following admission for transient ischemic attack 6 weeks ago. Skin biopsy showed superficial perivascular dermatitis with extravasation of red blood cells with no diagnostic evidence of vasculitis, granuloma or malignancy. Based on biopsy findings, recent drug administration followed by appearance of rash, hematological abnormality, lymphadenopathy and multi-system involvement and after exclusion of other possible causes, diagnosis of DRESS syndrome was made. Atorvastatin was held and patient was given Methylprednisolone for three days followed by Prednisone 1mg/kg with taper. Rash as well as kidney and liver functions improved drastically within two days. Discussion Our patient presented with one week of rash associated with deteriorating kidney and liver functions. Atorvastatin was initiated five weeks prior to development of rash. All the possible infectious etiologies and malignancy was ruled out based on the laboratory work mentioned above. Work up for vasculitis was also negative. DRESS syndrome is a late onset drug induced hypersensitivity reaction which usually manifests 3 weeks to months after the offending drug is started. It is very rare with an incidence of 0.9-2 per 100,000 patients per year and accounts for 10-20% of all cutaneous adverse drug reactions. DRESS syndrome if untreated carries significant risk of mortality, between 10-20%. RegiSCAR (European Registry of Severe Cutaneous Adverse Reaction) scoring system can be used to help diagnose DRESS syndrome. It takes into account multiple factors including fever, lymphadenopathy, eosinophilia, rash, number of organs involved, disease duration, skin biopsy findings and negative results of investigations to rule out alternate causes of patient's symptoms. Patient responded well to systemic steroids followed by oral steroids with taper. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

TUBERCULOUS PLEURAL EFFUSION MASQUERADING AS DRUG-INDUCED LUPUS

TUBERCULOUS PLEURAL EFFUSION MASQUERADING AS DRUG-INDUCED LUPUS