Erik Sundberg

Evidence of disseminated intravascular coagulation in a hemorrhagic fever with renal syndrome-scoring models and severe illness.

Background Viral hemorrhagic fevers (VHF) are considered to be a serious threat to public health worldwide with up to 100 million cases annually. The general hypothesis is that disseminated intravascular coagulation (DIC) is an important part of the pathogenesis. The study objectives were to study the variability of DIC in consecutive patients with acute hemorrhagic fever with renal syndrome (HFRS), and to evaluate if different established DIC-scores can be used as a prognostic marker for a more severe illness. Method and Findings In a prospective study 2006–2008, data from 106 patients with confirmed HFRS were analyzed and scored for the presence of DIC according to six different templates based on criteria from the International Society on Thrombosis and Haemostasis (ISTH). The DIC-scoring templates with a fibrinogen/CRP-ratio were most predictive, with predictions for moderate/severe illness (p<0.01) and bleeding of moderate/major importance (p<0.05). With these templates, 18.9–28.3% of the patients were diagnosed with DIC. Conclusions DIC was found in about one fourth of the patients and correlated with a more severe disease. This supports that DIC is an important part of the pathogenesis in HFRS. ISTH-scores including fibrinogen/CRP-ratio outperform models without. The high negative predictive value could be a valuable tool for the clinician. We also believe that our findings could be relevant for other VHFs.

Increased Thrombopoiesis and Platelet Activation in Hantavirus-Infected Patients

BACKGROUND Thrombocytopenia is a common finding during viral hemorrhagic fever, which includes hemorrhagic fever with renal syndrome (HFRS). The 2 main causes for thrombocytopenia are impaired thrombopoiesis and/or increased peripheral destruction of platelets. In addition, there is an increased intravascular coagulation risk during HFRS, which could be due to platelet activation. METHODS Thrombopoiesis was determined by quantification of platelet counts, thrombopoietin, immature platelet fraction, and mean platelet volume during HFRS. The in vivo platelet activation was determined by quantification of soluble P-selectin (sP-selectin) and glycoprotein VI (sGPVI). The function of circulating platelets was determined by ex vivo stimulation followed by flow cytometry analysis of platelet surface-bound fibrinogen and P-selectin exposure. Intravascular coagulation during disease was determined by scoring for disseminated intravascular coagulation (DIC) and recording thromboembolic complications. RESULTS The levels of thrombopoietin, immature platelet fraction, and mean platelet volume all indicate increased thrombopoiesis during HFRS. Circulating platelets had reduced ex vivo function during disease compared to follow-up. Most interestingly, we observed significantly increased in vivo platelet activation in HFRS patients with intravascular coagulation (DIC and thromboembolic complications) as shown by sP-selectin and sGPVI levels. CONCLUSIONS HFRS patients have increased thrombopoiesis and platelet activation, which contributes to intravascular coagulation.

HMGB1 levels are increased in patients with juvenile idiopathic arthritis, correlate with early onset of disease, and are independent of disease duration.

Objective. High mobility group box chromosomal protein 1 (HMGB1) has been implicated as a mediator of inflammation in rheumatoid arthritis (RA), while its role in juvenile idiopathic arthritis (JIA) has not been described. To evaluate the role of HMGB1 in the inflammatory process in JIA and its potential as a therapeutic target, we investigated whether extracellular HMGB1 is detectable in JIA and if so, to correlate the levels with established inflammatory markers and clinical measures. Methods. Matching samples of blood and synovial fluid (SF) were collected from 23 patients with JIA. Levels of HMGB1, soluble receptor for advanced glycation endproducts, S100A12, myeloid-related protein 8/14, and other inflammatory mediators were analyzed. Results. Significantly increased HMGB1 levels were recorded in SF compared to blood samples from patients with JIA. The amount of HMGB1 was highest in patients with early disease onset irrespective of disease duration. In contrast, the proinflammatory S100 protein and interleukin 8 were highest in patients in early phases of disease. Matrix metalloproteinase-3, a marker of cartilage destruction, was higher in patients with late disease onset, indicating similarities with RA in that patient subgroup. Conclusion. Levels of extracellular HMGB1 are increased in the inflamed joints of patients with JIA. This warrants further studies of HMGB1 as a mediator of JIA pathogenesis as well as a biomarker for inflammatory activity and as a target for therapy. The variation in levels of HMGB1 and S100 proteins in relation to disease onset indicates a difference in inflammatory phenotype during disease progression.

High Systemic Levels of the Cytokine-Inducing HMGB1 Isoform Secreted in Severe Macrophage Activation Syndrome

Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. High mobility group box 1 (HMGB1) is a nuclear protein extensively leaked extracellularly during necrotic cell death or actively secreted by natural killer (NK) cells, macrophages and additional cells during infection or sterile injury. Extracellular HMGB1 orchestrates key events in inflammation as a prototypic alarmin. The redox states of its three cysteines render the molecule mutually exclusive functions: fully reduced “all-thiol HMGB1” exerts chemotactic activity; “disulfide HMGB1” has cytokine-inducing, toll-like receptor 4 (TLR4)-mediated effects—while terminally oxidized “sulfonyl HMGB1” lacks inflammatory activity. This study examines the kinetic pattern of systemic HMGB1 isoform expression during therapy in four children with severe MAS. Three of the four patients with underlying systemic rheumatic diseases were treated with biologics and two suffered from triggering herpes virus infections at the onset of MAS. All patients required intensive care unit therapy due to life-threatening illness. Tandem mass-spectrometric analysis revealed dramatically increased systemic levels of the cytokine-inducing HMGB1 isoform during early MAS. Disease control coincided with supplementary etoposide therapy initiated to boost apoptotic cell death, when systemic HMGB1 levels drastically declined and the molecule emerged mainly in its oxidized, noninflammatory isoform. Systemic interferon (IFN)-γ and ferritin peaked concomitantly with HMGB1, whereas interleukin (IL)-18 and monocyte chemotactic protein (MCP)-1 levels developed differently. In conclusion, this work provides new insights in HMGB1 biology, suggesting that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of disulfide HMGB1 antagonists to improve outcome of MAS.

Sensitization Prediction and Validation for Al 5xxx Alloys Exposed to Long-Term Cyclical and Constant Heating at Low Temperatures

A number of Al 5xxx alloys were aged at constant temperatures (40, 50, 60, 70°C) and cyclic temperatures (40 to 45, 30 to 70, 50 to 70°C) for as long as 57.5 months. ASTM G67 nitric acid mass loss test (NAMLT) results of Al 5050-H32, 5052-H32, 5154-H32, 5083-H116, 5083-H131, and 5456-H116 alloys were obtained to evaluate the degree of sensitization. An intergranular β-phase growth model that considers nucleation, growth, and coarsening processes was developed to predict the formation and continuity of the β phase that leads to sensitization. Results obtained from scanning transmission electron microscopy were used to verify the model. A linear relationship between continuity and mass loss was adopted to predict the mass loss of Al 5083-H116 and -H131 aged at constant and cyclical temperature, and the modeling results agree well with experimental mass loss data.

Characterization of Al-Mg Alloy Aged at Low Temperatures

Long-term aged [343 K (70 °C) for 30 months and natural exposure for over 10 years] Al 5456 H116 samples were characterized using electron backscatter diffraction (EBSD), scanning transmission electron microscopy (STEM), state-of-the-art energy-dispersive X-ray spectroscopy (EDS) systems, and small-angle neutron scattering (SANS). ASTM G-67 mass loss tests of the sensitized Al 5456 alloy samples were conducted. Intragranular Mg-rich precipitates, such as Guinier–Preston (GP) zones, were confirmed in Al 5456 H116 aged at 343 K (70 °C) for 30 months, and the volume of these precipitates is 1.39 pct. β′ phase is identified at the grain boundary of a navy ship sample, while high-resolution STEM results reveal no intragranular precipitates. Intergranular corrosion (IGC) of Al 5456 was found to be related to the continuity of intergranular precipitates.

Diverse effects on vaccine-specific serum IgG titres and memory B cells upon methotrexate and anti-TNF-α therapy in children with rheumatic diseases: A cross-sectional study

OBJECTIVES We aimed at a comprehensive evaluation of how anti-TNF-α therapy and methotrexate treatment interferes with B cell memory in children with Paediatric Rheumatic Disease (PRD), by evaluating existing B cell phenotypes, and preserved vaccine-specific memory B cells and IgG titres generated prior to disease and treatment. METHODS In a cross-sectional study on children with PRD on various treatments, we measured titre levels and avidity strength of serum IgG specific against measles, rubella and tetanus. We also quantified transitional B cells and resting, atypical, and activated memory B cells with flow cytometry, and enumerated antigen-specific memory B cells with ELISpot. RESULTS For children who had received a tetanus booster, patients treated with any disease-modifying anti-rheumatic drug (DMARD) had lower tetanus serum IgG compared to healthy controls and NSAID-treated patients. Patients without a measles booster had lower levels of measles-specific memory B cells, but all vaccine-specific memory B cells were preserved in patients with booster. We furthermore found that the mature B cell compartment was phenotypically similar between patients and healthy controls. CONCLUSIONS We concluded that the general and vaccine-specific memory B cell compartment is well preserved in children with PRD and DMARD treatment, but that they might have lower serum tetanus IgG. We emphasize the importance for these children to follow the full vaccination schedule, and suggest to re-measure tetanus titres as they reach adulthood.

Evaluation of the danger signal HMGB1 as a potential biomarker in juvenile idiopathic arthritis (JIA): a preliminary study using the novel biobank jabba

Backgroundand objectives The endogenous danger signal High Mobility Group Box protein 1 (HMGB1) promotes inflammation. HMGB1 has been implicated as a mediator of RA while there are no reports describing its presence in JIA patient samples. HMGB1 is aberrantly expressed in the synovitis of RA patients, intraarticular injection of HMGB1 induces arthritis in mice and HMGB1 blockade has beneficial effects in several different experimental disease models, including arthritis. In this study, the aim was to determine whether HMGB1 is extracellularly increased during JIA and, if so, to correlate the HMGB1 levels both with other more well-described laboratory parameters and with clinical parameters such as age at onset and disease duration. This type of descriptive study forms the basis for the evaluation of HMGB1 as a biomarker of inflammatory activity during JIA and as a potential therapeutic target. The newly established JIA biobank JABBA coupled to a clinical register gives us a unique opportunity to study JIA pathogenesis. Materials and methods Plasma and synovial fluid (SF) was collected from 23 patients with JIA (median age 12 (2–18)) at Astrid Lindgren Childrens hospital, Stockholm, Sweden and at Tartu University Childrens Hospital, Estonia. Samples were analysed by ELISA and Cytometric bead array (CBA) to measure levels of HMGB1, MMP-3, sRAGE, IL-12p70, TNF, IL-10, IL-6, IL-1β, IL-8, MCP-1, IP-10, RANTES, IFNγ, IFNα and IL-17A. Results Increased HMGB1 levels were recorded in SF as compared to plasma from JIA patients. Highest levels of HMGB1 were recorded in patients with a disease onset in early age (before age 10), while no correlation between the HMGB1 levels and disease duration was evident. In contrast, both S100 and IL-8 levels correlated with disease duration being highest during early stages of disease. MMP-3, a marker of cartilage destruction, was higher in patients with late disease onset which indicates similarities with RA. Conclusion The increased levels of HMGB1 in inflamed joints of JIA patients warrants further studies of HMGB1 as a biomarker for inflammatory activity and as a target for therapy. The correlation of HMGB1 levels with age at disease onset and continuously high levels irrespective of disease duration indicates, together with the decreasing levels of S100 and IL-8 during the disease course, that the inflammatory process in JIA evolves over time. Different mediators might thus be of varying importance during the disease progression. The authors conclude that the role of HMGB1 in the pathogenesis of JIA deserves further investigation.

High mobility group box protein 1—A prognostic marker for structural joint damage in 10-year follow-up of patients with juvenile idiopathic arthritis

OBJECTIVE High mobility group box protein 1 (HMGB1) is an important pro-inflammatory mediator in adult rheumatoid arthritis. The diagnostic utility of HMGB1 in Juvenile Idiopathic Arthritis (JIA) is still unclear. The aim was to examine whether serum HMGB1 levels are associated with inflammation, radiological disease progression, and long-term prognosis in JIA. METHODS We included 131 children with JIA from a population-based prevalence study; 38 of them were prospectively followed up for 10 years. Clinical and laboratory disease characteristics at study entry and after 10 years as well as radiological progression over 10 years were recorded. HMGB1 levels were analyzed by an ELISA. RESULTS The HMGB1 levels were similar in children with different JIA subgroups and in children with established (53%) or newly diagnosed (47%) disease. HMGB1 levels did not differ between groups at entry into the study or at 10 years, by sex, or by the presence or absence of RF or ANA antibodies. HMGB1 levels at the study entry correlated with HMGB1 levels at 10 years and with blood neutrophil count. Most importantly, children with destructive arthritis at 10 years had a tendency toward higher HMGB1 levels at study entry (median 1.2 vs 0.6ng/ml, ns) and displayed 4-fold higher circulating HMGB1 levels (median 3.4 vs 0.8ng/ml, p = 0.0014) than children without radiological destructions. CONCLUSIONS Our results suggest that HMGB1 is a marker of inflammatory activity in children with JIA. Higher serum HMGB1 levels are related to more destructive JIA and could be used as a negative prognostic marker at the disease start. TRIAL REGISTRATION Clinicaltrials.gov NCT01905319. Registered July 16, 2013.

A2.28 Characterisation and potential function of HMGB1 in juvenile idiopathic arthritis

Background and objectives High mobility group box protein 1 (HMGB1) is a prototypic alarmin being released from activated, stressed or dying cells. Extracellular HMGB1 has the ability to induce cell migration as well as cytokine production. Pathogenic effects of HMGB1 have been described in several inflammatory diseases, including arthritis, and HMGB1-specific blockade is beneficial in multiple experimental disease models. Recent reports have underlined the importance of post-translational modifications (PTMs) in determination of HMGB1 function and release mechanisms. We investigated the occurrence of PTMs of HMGB1 obtained from synovial fluid (SF) of juvenile idiopathic arthritis patients (JIA). Materials and methods Synovial fluid was obtained from 17 JIA patients. Total Levels of HMGB1 were determined by ELISA. PTMs of HMGB1 were determined HMGB1-specific immunoprecipitation followed by Liquid chromatography tandem mass-spectrometry (LC-MS/MS). Results Analyses of 17 JIA patients confirmed high HMGB1 levels in SF. Liquid chromatography tandem mass-spectrometry (LC-MS/MS) analyses of PTMs revealed that total HMGB1 levels did not associate with increased LDH activity but strongly correlated with nuclear location sequence 2 (NLS2) hyperacetylation, indicating active release of HMGB1. The correlation between total HMGB1 levels and NLS2 hypoacetylation suggests additional, acetylation-independent release mechanisms. Monomethylation of lysine 43 (K43), a proposed neutrophil-specific PTM, strongly associated with high HMGB1 levels, implying that neutrophils are a source of released HMGB1. Analysis of cysteine redox isoforms: fully reduced HMGB1, disulfide HMGB1 and oxidised HMGB1 revealed that HMGB1 acts as both a chemotactic and a cytokine-inducing mediator. These properties were associated with actively released HMGB1. Conclusions This is the first report that characterises HMGB1-specific PTMs during a chronic inflammatory condition. HMGB1 in SF from JIA patients is actively released through both acetylation- and non-acetylation-dependent manners. The presence of various functional HMGB1 redox isoforms confirms the complexity of its pathogenic role during chronic inflammation. Defining HMGB1 release pathways and redox isoforms is critical for the understanding of the contribution of HMGB1 during inflammatory processes.