Following the white rabbit : release and regulation of HMGB1 in inflammation

Dysregulated inflammatory responses are characterized by excessive release of endogenous pro-inflammatory molecules, danger-associated molecular patterns (DAMPs). A prototypical DAMP, High Mobility Group Box 1 protein (HMGB1), is primarily bound to DNA in the nucleus of most eukaryotic cells. However, when HMGB1 exits the cell, it acquires novel functions and can trigger immune activation. The aim of this thesis was to study the release of HMGB1, follow its fate at the site of inflammation and therapeutically block its activity. Multiple studies have reported that cells release HMGB1 in either a passive or an active manner. In order to understand the possible impact of cell death in inflammatory conditions, we performed well-controlled in vitro analyses of HMGB1 release under different cell death modes. Necrotic cells instantly released high levels of HMGB1, while apoptotic cells retained HMGB1 unless they advanced to secondary necrosis. The controlled immunogenic cell death types, such as pyroptosis and necroptosis, secreted HMGB1 along with other pro-inflammatory molecules in comparable or higher levels than cells activated with lipopolysaccharide (LPS) and interferon gamma (IFNγ). Next, we investigated HMGB1 proteolytic regulation at the site of inflammation. Human neutrophil elastase (HNE), cathepsin G (CG) and matrix metalloproteinase 3 (MMP-3), endoproteases present in arthritis-affected joints, cleaved HMGB1 into smaller peptides in vitro. While HNE and MMP-3 truncated the acidic C-terminal tail domain of HMGB1, CG completely degraded the protein. We showed that the C-terminal tail truncation negatively regulated HMGB1 binding to Toll-like receptor 2 (TLR2). Only the protein form devoid the acidic tail domain bound TLR2 in vitro. Thus, truncation of HMGB1 by endoproteases might improve its affinity to receptors. TLR2 and HMGB1 interaction did not result in cytokine induction. However, both full-length and truncated HMGB1 formed complexes with potent TLR2 ligand peptidoglycan (PGN) to potentiate the inflammatory response in peripheral blood mononuclear cells (PBMCs). Lastly, we developed a novel tool that could enable anti-HMGB1 therapy in the clinic by engineering a chimeric, partially humanized, monoclonal antibody (h2G7). The antibody displayed anti-inflammatory effects when tested in a mouse model of paracetamol-induced liver toxicity. Experiments with mutated variants, non-Fc receptor binding and non-complement binding variants, of the novel antibody suggested HMGB1 neutralization as the mode of action. Taken together, results presented in my thesis increased the knowledge about HMGB1 biology regarding its release during immunogenic cell death, proteolytic regulation at the site of inflammation and interaction with its receptor TLR2. In addition, the generation of a partially humanized monoclonal antibody could promote the introduction of anti-HMGB1 therapy in the clinic

LSEC Fenestrae Are Preserved Despite Pro-inflammatory Phenotype of Liver Sinusoidal Endothelial Cells in Mice on High Fat Diet

Healthy liver sinusoidal endothelial cells (LSECs) maintain liver homeostasis, while LSEC dysfunction was suggested to coincide with defenestration. Here, we have revisited the relationship between LSEC pro-inflammatory response, defenestration, and impairment of LSEC bioenergetics in non-alcoholic fatty liver disease (NAFLD) in mice. We characterized inflammatory response, morphology as well as bioenergetics of LSECs in early and late phases of high fat diet (HFD)-induced NAFLD. LSEC phenotype was evaluated at early (2–8 week) and late (15–20 week) stages of NAFLD progression induced by HFD in male C57Bl/6 mice. NAFLD progression was monitored by insulin resistance, liver steatosis and obesity. LSEC phenotype was determined in isolated, primary LSECs by immunocytochemistry, mRNA gene expression (qRT-PCR), secreted prostanoids (LC/MS/MS) and bioenergetics (Seahorse FX Analyzer). LSEC morphology was examined using SEM and AFM techniques. Early phase of NAFLD, characterized by significant liver steatosis and prominent insulin resistance, was related with LSEC pro-inflammatory phenotype as evidenced by elevated ICAM-1, E-selectin and PECAM-1 expression. Transiently impaired mitochondrial phosphorylation in LSECs was compensated by increased glycolysis. Late stage of NAFLD was featured by prominent activation of pro-inflammatory LSEC phenotype (ICAM-1, E-selectin, PECAM-1 expression, increased COX-2, IL-6, and NOX-2 mRNA expression), activation of pro-inflammatory prostaglandins release (PGE2 and PGF2α) and preserved LSEC bioenergetics. Neither in the early nor in the late phase of NAFLD, were LSEC fenestrae compromised. In the early and late phases of NAFLD, despite metabolic and pro-inflammatory burden linked to HFD, LSEC fenestrae and bioenergetics are functionally preserved. These results suggest prominent adaptive capacity of LSECs that might mitigate NAFLD progression

Nine forward–backward translations of the Hopkins symptom checklist-25 with cultural checks

Introduction: The Hopkins Symptom Checklist-25 (HSCL-25) is an effective, reliable, and ergonomic tool that can be used for depression diagnosis and monitoring in daily practice. To allow its broad use by family practice physicians (FPs), it was translated from English into nine European languages (Greek, Polish, Bulgarian, Croatian, Catalan, Galician, Spanish, Italian, and French) and the translation homogeneity was confirmed. This study describes this process. Methods: First, two translators (an academic translator and an FP researcher) were recruited for the forward translation (FT). A panel of English-speaking FPs that included at least 15 experts (researchers, teachers, and practitioners) was organized in each country to finalize the FT using a Delphi procedure. Results: One or two Delphi procedure rounds were sufficient for each translation. Then, a different translator, who did not know the original version of the HSCL-25, performed a backward translation in English. An expert panel of linguists compared the two English versions. Differences were listed and a multicultural consensus group determined whether they were due to linguistic problems or to cultural differences. All versions underwent cultural check. Conclusion: All nine translations were finalized without altering the original meaning

Say it in Croatian - Croatian translation of the EGPRN definition of Multimorbidity using a Delphi consensus technique

Patients coming to their family physician (FP) usually have more than one condition or problem. Multimorbidity as well as dealing with it, is challenging for FPs even as a mere concept. The World Health Organization (WHO) has simply defined multimorbidity as two or more chronic conditions existing in one patient. However, this definition seems inadequate for a holistic approach to patient care within Family Medicine. Using systematic literature review the European General Practitioners Research Network (EGPRN) developed a comprehensive definition of multimorbidity. For practical and wider use, this definition had to be translated into other languages, including Croatian. Here presented is the Croatian translation of this comprehensive definition using a Delphi consensus procedure for Forward/Backward translation. 23 expert FPs fluent in English were asked to rank the translation from 1 (absolutely disagreeable) to 9 (fully agreeable) and to explain each score under 7. It was previously defined that consensus would be reached when 70 % of the scores are above 6. Finally, a backward translation from Croatian into English was undertaken and approved by the authors of the English definition. Consensus was reached after the first Delphi round with 100% of the scores above 6; therefore the Croatian translation was immediately accepted. The authors of the English definition accepted the backward translation. A comprehensive definition of multimorbidity is now available in English and Croatian, as well as other European languages which will surely make further implications for clinicians, researchers or policy makers

Cross-Cultural Validation of the Definition of Multimorbidity in the Bulgarian Language.

INTRODUCTION: Multimorbidity is a health issue with growing importance. During the last few decades the populations of most countries in the world have been ageing rapidly. Bulgaria is affected by the issue because of the high prevalence of ageing population in the country with multiple chronic conditions. The AIM of the present study was to validate the translated definition of multimorbidity from English into the Bulgarian language. MATERIALS AND METHODS: The present study is part of an international project involving 8 national groups. We performed a forward and backward translation of the original English definition of multimorbidity using a Delphi consensus procedure. RESULTS: The physicians involved accepted the definition with a high percentage of agreement in the first round. The backward translation was accepted by the scientific committee using the Nominal group technique. DISCUSSION: Some of the GPs provided comments on the linguistic expressions which arose in order to improve understanding in Bulgarian. The remarks were not relevant to the content. The conclusion of the discussion, using a meta-ethnographic approach, was that the differences were acceptable and no further changes were required. CONCLUSIONS: A native version of the published English multimorbidity definition has been finalized. This definition is a prerequisite for better management of multimorbidity by clinicians, researchers and policy makers

The linguistic validation of the gut feelings questionnaire in three European languages

BACKGROUND: Physicians' clinical decision-making may be influenced by non-analytical thinking, especially when perceiving uncertainty. Incidental gut feelings in general practice have been described, namely, as "a sense of alarm" and "a sense of reassurance". A Dutch Gut Feelings Questionnaire (GFQ) was developed, validated and afterwards translated into English following a linguistic validation procedure. The aims were to translate the GFQ from English into French, German and Polish; to describe uniform elements as well as differences and difficulties in the linguistic validation processes; to propose a procedural scheme for future GFQ translations into other languages. METHODS: We followed a structured, similar and equivalent procedure. Forward and backward-translations, repeated consensus procedures and cultural validations performed in six steps. Exchanges between the several research teams, the authors of the Dutch GFQ, and the translators involved continued throughout the procedure. RESULTS: 12 translators, 52 GPs and 8 researchers in the field participated to the study in France, Germany, Switzerland and Poland. The collaborating research teams created three versions of the 10-item GFQ. Each research team found and agreed on compromises between comparability and similarity on one hand, and linguistic and cultural specificities on the other. CONCLUSIONS: The gut feeling questionnaire is now available in five European languages: Dutch, English, French, German and Polish. The uniform procedural validation scheme presented, and agreed upon by the teams, can be used for the translation of the GFQ into other languages. Comparing results of research into the predictive value of gut feelings and into the significance of the main determinants in five European countries is now possible

Ligation of free HMGB1 to TLR2 in the absence of ligand is negatively regulated by the C-terminal tail domain

Abstract Background High mobility group box 1 (HMGB1) protein is a central endogenous inflammatory mediator contributing to the pathogenesis of several inflammatory disorders. HMGB1 interacts with toll-like receptors (TLRs) but contradictory evidence regarding its identity as a TLR2 ligand persists. The aim of this study was to investigate if highly purified HMGB1 interacts with TLR2 and if so, to determine the functional outcome. Methods Full length or C-terminal truncated (Δ30) HMGB1 was purified from E.coli. Binding to TLR2-Fc was investigated by direct-ELISA. For the functional studies, proteins alone or in complex with peptidoglycan (PGN) were added to human embryonic kidney (HEK) cells transfected with functional TLR2, TLR 1/2 or TLR 2/6 dimers, macrophages, whole blood or peripheral blood mononuclear cells (PBMCs). Cytokine levels were determined by ELISA. Results In vitro binding experiments revealed that Δ30 HMGB1, lacking the acidic tail domain, but not full length HMGB1 binds dose dependently to TLR2. Control experiments confirmed that the interaction was specific to TLR2 and could be inhibited by enzymatic digestion. Δ30 HMGB1 alone was unable to induce cytokine production via TLR2. However, full length HMGB1 and Δ30 HMGB1 formed complexes with PGN, a known TLR2 ligand, and synergistically potentiated the inflammatory response in PBMCs. Conclusions We have demonstrated that TLR2 is a receptor for HMGB1 and this binding is negatively regulated by the C-terminal tail. HMGB1 did not induce functional activation of TLR2 while both full length HMGB1 and Δ30 HMGB1 potentiated the inflammatory activities of the TLR2 ligand PGN. We hypothesize that Δ30 HMGB1 generated in vivo by enzymatic cleavage could act as an enhancer of TLR2-mediated inflammatory activities

Reshaping interaction in higher/tertiary education (HTE) under the challenges of inclusion

The panel addresses IPrA’s special theme (The shape of interaction: the pragmatics of (a)typicality) by asking how academic communication is or can be (re)shaped as an interaction that strives to be inclusive. Inclusive interaction in higher/tertiary education (HTE) entails but is not restricted to e.g. subtitling, audio description, sign language translation, simplified language (“Easy Languages”) and Universal Design for learning. Students experiencing barriers bring with them their own resources and sensitivity for interacting, communicating and acquiring knowledge. The panel brings together scholars whose research deals with communicative vulnerability and accommodation strategies, experiencing minority status, the pragmatics of asymmetric, inclusive, accessible, and bias-free communication in academic interaction and related research on Universal Design. Methods include, among others, multimodal analyses of interaction data, analyses of institutional talk/text/discourse/media situated in HTE/universities, and research comparing international advances in academic inclusion. The panel also focuses on methodological and theoretical reflections, bridging gaps between linguistic pragmatics and disability studies in HTE. New contradictory developments in interaction in higher education, such as aspiration for more participation and increasing language anxiety will also be explored. The panel is going to answer the following research questions: • Which adaptations in actual academic interaction can accommodate students who experience communicative barriers, e.g., auditory or visual, for various reasons? What can we learn from their experience of “minority status”? • Which types of interactions in HTE bear which risks of exclusion and hindered participation, and how can these situations and settings be described from interaction data? • As interactions between people with and without impairments are often described as “asymmetrical” or “atypical”: In what way are those interactions in HTE contexts actually different from “typical” interactions? How do categories like the ones mentioned shape the research on interactions? • How do terms such as “inclusive”, “barrier-free”, “accessible”, and “(dis-)ability”, among others, shape institutional communication? And how can inter- and transdisciplinary approaches reach more clarity on the terms and help reshape interaction in HTE? • In which ways can issues of diversity – e.g., culture, multilingual societies, the individual students’ linguistic competencies in L1 (first language) and L2 (foreign or second language) and their intersectional needs – be integrated into an approach that has a broad understanding of inclusion and relates inclusive communication to accessible, intercultural and bias-free communication? • What kind of competences are acquired on the part of translators, mobility trainers and others with intermediary roles? • How can knowledge on socio-cultural, linguistic and pragmatic aspects of inclusive communication be explored, shared and transferred internationally in HTE, given the great divides that exist, depending on historical and institutional (pre-)conditions, more or less advanced inclusive institutions and education systems, and provisions in recognition of the UN Convention on the Rights of Persons with Disabilities? The panel is organised by the International Network on Inclusion and Inclusive Communication in Tertiary Education (NIICTE), established at the 2021 IPrA conference. Its purpose is to strengthen interdisciplinary collaboration on inclusion, with a view to how linguistic pragmatics can inform practices of inclusion and inclusive communication in HTE

Additional file 1: of Ligation of free HMGB1 to TLR2 in the absence of ligand is negatively regulated by the C-terminal tail domain

Supplementary information. (PDF 447 kb