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The Earth's mean surface temperature is already approximately 1.1°C higher than pre-industrial levels. Exceeding a mean 1.5°C rise by 2050 will make global adaptation to the consequences of climate change less possible. To protect public health, anaesthesia providers need to reduce the contribution their practice makes to global warming. We convened a Working Group of 45 anaesthesia providers with a recognised interest in sustainability, and used a three-stage modified Delphi consensus process to agree on principles of environmentally sustainable anaesthesia that are achievable worldwide. The Working Group agreed on the following three important underlying statements: patient safety should not be compromised by sustainable anaesthetic practices; high-, middle- and low-income countries should support each other appropriately in delivering sustainable healthcare (including anaesthesia); and healthcare systems should be mandated to reduce their contribution to global warming. We set out seven fundamental principles to guide anaesthesia providers in the move to environmentally sustainable practice, including: choice of medications and equipment; minimising waste and overuse of resources; and addressing environmental sustainability in anaesthetists' education, research, quality improvement and local healthcare leadership activities. These changes are achievable with minimal material resource and financial investment, and should undergo re-evaluation and updates as better evidence is published. This paper discusses each principle individually, and directs readers towards further important references.

Background: Adrenocortical tumours (ACT) in children are part of the Li-Fraumeni cancer spectrum and are frequently associated with a germline TP53 pathogenic variant. TP53 p.R337H is highly prevalent in the south and southeast of Brazil and predisposes to ACT with low penetrance. Thus, we aimed to investigate whether genetic variants exist which are associated with an increased risk of developing ACT in TP53 p.R337H carrier children., Methods: A genetic association study was conducted in trios of children (14 girls, 7 boys) from southern Brazil carriers of TP53 p.R337H with ( n  = 18) or without ( n  = 3) ACT and their parents, one of whom also carries this pathogenic variant (discovery cohort). Results were confirmed in a validation cohort of TP53 p.R337H carriers with ( n  = 90; 68 girls, 22 boys) or without ACT ( n  = 302; 165 women, 137 men)., Findings: We analysed genomic data from whole exome sequencing of blood DNA from the trios. Using deep learning algorithms, according to a model where the affected child inherits from the non-carrier parent variant(s) increasing the risk of developing ACT, we found a significantly enriched representation of non-coding variants in genes involved in the cyclic AMP (cAMP) pathway known to be involved in adrenocortical tumorigenesis. One among those variants (rs2278986 in the SCARB1 gene) was confirmed to be significantly enriched in the validation cohort of TP53 p.R337H carriers with ACT compared to carriers without ACT (OR 1.858; 95% CI 1.146, 3.042, p = 0.01)., Interpretation: Profiling of the variant rs2278986 is a candidate for future confirmation and possible use as a tool for ACT risk stratification in TP53 p.R337H carriers., Funding: Centre National de la Recherche Scientifique (CNRS), Behring Foundation, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)., Competing Interests: None of the authors has anything to declare., (© 2024 The Author(s).)

Introduction: In systemic sclerosis (SSc), B-cells are activated and present in the skin and lung of patients where they can interact with fibroblasts. The precise impact and mechanisms of the interaction of B-cells and fibroblasts at the tissular level are poorly studied., Objective: We investigated the impact and mechanisms of B-cell/fibroblast interactions in cocultures between B-cells from patients with SSc and 3-dimensional reconstituted healthy skin model including fibroblasts, keratinocytes and extracellular matrix., Methods: The quantification and description of the B-cell infiltration in 3D cocultures were performed using cells imagery strategy and cytometry. The effect of coculture on the transcriptome of B-cells and fibroblasts was studied with bulk and single-cell RNA sequencing approaches. The mechanisms of this interaction were studied by blocking key cytokines like IL-6 and TNF., Results: We showed a significant infiltration of B-cells in the 3D healthy skin model. The amount but not the depth of infiltration was higher with B-cells from SSc patients and with activated B-cells. B-cell infiltrates were mainly composed of naïve and memory cells, whose frequencies differed depending on B-cells origin and activation state: infiltrated B-cells from patients with SSc showed an activated profile and an overexpression of immunoglobulin genes compared to circulating B-cells before infiltration. Our study has shown for the first time that activated B-cells modified the transcriptomic profile of both healthy and SSc fibroblasts, toward a pro-inflammatory (TNF and IL-17 signaling) and interferon profile, with a key role of the TNF pathway., Conclusion: B-cells and 3D skin cocultures allowed the modelization of B-cells infiltration in tissues observed in SSc, uncovering an influence of the underlying disease and the activation state of B-cells. We showed a pro-inflammatory effect on skin fibroblasts and pro-activation effect on infiltrating B-cells during coculture. This reinforces the role of B-cells in SSc and provide potential targets for future therapeutic approach in this disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Le Maître, Guerrier, Collet, Derhourhi, Meneboo, Toussaint, Bonnefond, Villenet, Sebda, Bongiovanni, Tardivel, Simon, Jendoubi, Daunou, Largy, Figeac, Dubucquoi and Launay.)

Systemic sclerosis (SSc) is the most severe systemic autoimmune disease with currently no cure. Intravenous immunoglobulins (IVIg) are an attractive candidate in this disease to counteract inflammation and fibrosis but data are scarce and conflicting. This study, assessed the effects of IVIg in a murine HOCl-induced model of SSc. We showed that IVIg prevented skin inflammation and fibrosis, by mitigating the immune cell infiltration (p = 0.04), proinflammatory cytokines gene overexpression (IL1β, p = 0.04; TNFα, p = 0.04; IL6, p = 0.05), skin and dermal thickening (p = 0.003 at d21 and p = 0.0003 at d42), the expression markers of fibrosis, such as αSMA (p = 0.031 for mRNA and p = 0.05 for protein), collagen (p = 0.05 for mRNA and p = 0.04 for protein, p = 0.05 for the hydroxyproline content) and fibronectin (p = 0.033 for mRNA). Moreover, IVIg prevented HOCl-induced alterations in splenic cell homeostasis. When administered in curative mode, despite their ability to reduce skin and dermal thickness (p < 0.0001 and p = 0.0002), IVIg showed partial or more mixed effects on skin inflammation and established fibrosis. These data favor further clinical trials in SSc patients on the potential efficiency of early and/or repeated IVIg administration., (© 2023. Springer Nature Limited.)

Systemic sclerosis (SSc) is an autoimmune, inflammatory and fibrotic disease with limited treatment options. Developing new therapies is therefore crucial to address patient needs. To this end, we focused on galectin-3 (Gal-3), a lectin known to be associated with several pathological processes seen in SSc. Using RNA sequencing of whole-blood samples in a cross-sectional cohort of 249 patients with SSc, Gal-3 and its interactants defined a strong transcriptomic fingerprint associated with disease severity, pulmonary and cardiac malfunctions, neutrophilia and lymphopenia. We developed new Gal-3 neutralizing monoclonal antibodies (mAb), which were then evaluated in a mouse model of hypochlorous acid (HOCl)-induced SSc. We show that two of these antibodies, D11 and E07, reduced pathological skin thickening, lung and skin collagen deposition, pulmonary macrophage content, and plasma interleukin-5 and -6 levels. Moreover, E07 changed the transcriptional profiles of HOCl-treated mice, resulting in a gene expression pattern that resembled that of control mice. Similarly, pathological pathways engaged in patients with SSc were counteracted by E07 in mice. Collectively, these findings demonstrate the translational potential of Gal-3 blockade as a therapeutic option for SSc., (© 2023. Springer Nature Limited.)

Introduction: Soluble markers of B cell activation are interesting diagnostic and prognostic tools in autoimmune diseases. Data in systemic sclerosis (SSc) are scarce and few studies focused on their association with disease characteristics., Methods: 1. Serum levels of 14 B cell biomarkers (β2-microglobulin, rheumatoid factor (RF), immunoglobulins (Ig) G, IgA, IgM, BAFF, APRIL, soluble (s)TACI, sBCMA sCD21, sCD23, sCD25, sCD27, CXCL13) were measured in SSc patients and healthy controls (HC). 2. Associations between these biomarkers and SSc characteristics were assessed. 3. The pathophysiological relevance of identified associations was explored by studying protein production in B cell culture supernatant., Results: In a discovery panel of 80 SSc patients encompassing the broad spectrum of disease manifestations, we observed a higher frequency of RF positivity, and increased levels of β2-microglobulin, IgG and CXCL13 compared with HC. We found significant associations between several biomarkers and SSc characteristics related to disease phenotype, activity and severity. Especially, serum IgG levels were associated with pulmonary hypertension (PH); β2-microglobulin with Nt-pro-BNP and DLCO; and BAFF with peak tricuspid regurgitation velocity (TRV). In a validation cohort of limited cutaneous SSc patients without extensive ILD, we observed lower serum IgG levels, and higher β2-microglobulin, sBCMA, sCD23 and sCD27 levels in patients with pulmonary arterial hypertension (PAH). BAFF levels strongly correlated with Nt-pro-BNP levels, FVC/DLCO ratio and peak TRV in SSc-PAH patients. Cultured SSc B cells showed increased production of various angiogenic factors (angiogenin, angiopoietin-1, VEGFR-1, PDGF-AA, MMP-8, TIMP-1, L-selectin) and decreased production of angiopoietin-2 compared to HC., Conclusion: Soluble markers of B cell activation could be relevant tools to assess organ involvements, activity and severity in SSc. Their associations with PAH could plead for a role of B cell activation in the pathogenesis of pulmonary microangiopathy. B cells may contribute to SSc vasculopathy through production of angiogenic mediators., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sanges, Guerrier, Duhamel, Guilbert, Hauspie, Largy, Balden, Podevin, Lefèvre, Jendoubi, Speca, Hachulla, Sobanski, Dubucquoi and Launay.)

We provide an original multi-stage approach identifying a gene signature to assess murine fibroblast polarization. Prototypic polarizations (inflammatory/fibrotic) were induced by seeded mouse embryonic fibroblasts (MEFs) with TNFα or TGFß1, respectively. The transcriptomic and proteomic profiles were obtained by RNA microarray and LC-MS/MS. Gene Ontology and pathways analysis were performed among the differentially expressed genes (DEGs) and proteins (DEPs). Balb/c mice underwent daily intradermal injections of HOCl (or PBS) as an experimental murine model of inflammation-mediated fibrosis in a time-dependent manner. As results, 1456 and 2215 DEGs, and 289 and 233 DEPs were respectively found in MEFs in response to TNFα or TGFß1, respectively. Among the most significant pathways, we combined 26 representative genes to encompass the proinflammatory and profibrotic polarizations of fibroblasts. Based on principal component analysis, this signature deciphered baseline state, proinflammatory polarization, and profibrotic polarization as accurately as RNA microarray and LC-MS/MS did. Then, we assessed the gene signature on dermal fibroblasts isolated from the experimental murine model. We observed a proinflammatory polarization at day 7, and a mixture of a proinflammatory and profibrotic polarizations at day 42 in line with histological findings. Our approach provides a small-size and convenient gene signature to assess murine fibroblast polarization., (© 2022. The Author(s).)

Autoantibodies (Aabs) are frequent in systemic sclerosis (SSc). Although recognized as potent biomarkers, their pathogenic role is debated. This study explored the effect of purified immunoglobulin G (IgG) from SSc patients on protein and mRNA expression of dermal fibroblasts (FBs) using an innovative multi-omics approach. Dermal FBs were cultured in the presence of sera or purified IgG from patients with diffuse cutaneous SSc (dcSSc), limited cutaneous SSc or healthy controls (HCs). The FB proteome and transcriptome were explored using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and microarray assays, respectively. Proteomic analysis identified 3,310 proteins. SSc sera and purified IgG induced singular protein profile patterns. These FB proteome changes depended on the Aab serotype, with a singular effect observed with purified IgG from anti-topoisomerase-I autoantibody (ATA) positive patients compared to HC or other SSc serotypes. IgG from ATA positive SSc patients induced enrichment in proteins involved in focal adhesion, cadherin binding, cytosolic part, or lytic vacuole. Multi-omics analysis was performed in two ways: first by restricting the analysis of the transcriptomic data to differentially expressed proteins; and secondly, by performing a global statistical analysis integrating proteomics and transcriptomics. Transcriptomic analysis distinguished 764 differentially expressed genes and revealed that IgG from dcSSc can induce extracellular matrix (ECM) remodeling changes in gene expression profiles in FB. Global statistical analysis integrating proteomics and transcriptomics confirmed that IgG from SSc can induce ECM remodeling and activate FB profiles. This effect depended on the serotype of the patient, suggesting that SSc Aab might play a pathogenic role in some SSc subsets., Competing Interests: DL reports grants from: GSK, Actelion, Boehringer Ingelheim, Takeda, CSL Behring, Biocryst. VS reports consultancies and speaking fees from Boehringer Ingelheim and Grifols (less than $10 000) and research support from Actelion, Grifols, GSK, Octapharma, Pfizer, Shire, outside the submitted work. GM works with former students, now employed by Diagrams Technologies and Genes Diffusion, outside of this present work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest, (Copyright © 2022 Chepy, Vivier, Bray, Ternynck, Meneboo, Figeac, Filiot, Guilbert, Jendoubi, Rolando, Launay, Dubucquoi, Marot and Sobanski.)

Background: A differential diagnosis between angiotensin-converting enzyme inhibitor (ACEi) angioedema (AE) and histaminergic AE (hAE) might be challenging. Follow-up data may help discriminate these conditions but are scarcely reported. Objective: To report on the follow-up of patients with suspected ACEi-AE and to describe the baseline characteristics of AE attacks in patients with a diagnosis of ACEi-AE after follow-up. Methods: Sixty-four patients with suspected ACEi-AE ( i.e. , with exposure to ACEi before the first attack, no urticaria associated, and normal C1-inhibitor levels) and at least one follow-up visit were included. Data were retrospectively collected at baseline and during the follow-up. Results: After the follow-up, the diagnosis of ACEi-AE was probable in only 30 patients. The remaining patients were reclassified as having probable hAE (21 patients) or undetermined-mechanism AE (13 patients). Patients with ACEi-AE were mostly men (61%), with a median age of 64 years (interquartile range [IQR] ±17 years), with a highly variable delay from ACEi introduction (median: 23 months; interquartile range: 103 months). Attacks preferentially involved lips (50%), tongue (47%), and throat (30%). Interestingly, patients with probable ACEi-AE after a follow-up also frequently presented with a history of allergy and atopic conditions (20%), attacks with preferential evening onset (25%), and spontaneous resolution in < 24 hours (26%), which are usually considered as suggestive of hAE. ACEi-AE attacks responded to icatibant in 79% of the patients. Conclusion: Patients with probable ACEi-AE were mostly men with facial involvement. A third of the patients with an initial suspected diagnosis of ACEi-AE had a final diagnosis of probable hAE. Although a follow-up of all patients should be a standard of care, it is critical to the correct diagnosis in the case of suspected bradykinin-associated AE, which may actually be due to histamine.

Fibroblasts (Fb) are key effector cells in systemic sclerosis (SSc). Fb stimulation with transforming growth factor beta 1 (TGF-β1) is considered as a positive control in studies assessing fibrogenesis. The lack of standardization of TGF-β1 stimulation might be responsible for discrepancies in experiments performed in different conditions. Using quantitative proteomics analysis, we evaluated the impact of changes in experimental conditions on proteomic profiles of primary Fb. Principal component analysis (PCA) identified several groups of differentially expressed proteins influenced by cell passage, culture medium, and both concentration and duration of exposure to TGF-β1 stimulation. Bioinformatics analysis revealed that late passages expressed proteins involved in senescence. TGF-β1 concentration and time of stimulation were correlated with the expression of proteins involved in the fibrogenesis and inflammatory processes. These data underline the need for standardization of culture conditions to allow inter-data comparisons in future in vitro studies, especially when using "omics" approaches., (© 2021 Wiley-VCH GmbH.)

Introduction: During systemic sclerosis (SSc), peripheral B cells display alterations in subset homeostasis and functional properties and are a promising therapeutic target. However, there is only few data regarding whether these anomalies are accurately reproduced in animal models of SSc., Objective: In this work, we assessed the B cell homeostasis modifications in an experimental model of SSc [hypochlorous acid (HOCl)-induced mouse], both at a phenotypic and functional level, during the course of the disease., Methods: Balb/c mice underwent daily intradermal injections of HOCl (or phosphate-buffered saline) and were then sacrificed at day 21 (early inflammatory stage) or day 42 (late fibrotic stage). For phenotypic studies, the distribution of the main spleen cell subsets (B cells, T CD4 and CD8 cells, NK cells, macrophages) and splenic B cell subsets (immature, mature naïve, germinal center, antibody-secreting, memory, B1) was assessed by flow cytometry. For functional studies, splenic B cells were immediately MACS-sorted. Production of interleukin (IL)-6, CCL3, IL-10, and transforming growth factor (TGF)-β was assessed ex vivo by RT-PCR and after 48 h of culture by ELISA. Regulatory B cell (Breg) counts were quantified by flow cytometry., Results: Phenotypic analyses showed an early expansion of transitional B cells, followed by a late expansion of the mature naive subset and decrease in plasmablasts and memory B cells. These anomalies are similar to those encountered in SSc patients. Functional analyses revealed a B-cell overproduction of pro-inflammatory cytokines (IL-6 and CCL3) and an impairment of their anti-inflammatory capacities (decreased production of IL-10 and TGF-β, reduced levels of Bregs) at the early inflammatory stage; and an overproduction of pro-fibrotic cytokines (TGF-β and IL-6) at the late fibrotic stage. These results approximate the anomalies observed in human SSc., Conclusion: This work reports the existence of anomalies in B cell homeostasis and functional properties in an animal model of SSc that approximate those displayed by SSc patients. These anomalies vary over the course of the disease, which pleads for their participation in inflammatory and fibrotic events. This makes the HOCl mouse a relevant experimental model for the study of B cells, and therefore, B-cell-targeted therapies in SSc.

Lamellarin D (LamD) is a marine alkaloid with broad spectrum antitumor activities. Multiple intracellular targets of LamD, which affect cancer cell growth and induce apoptosis, have been identified. These include nuclear topoisomerase I, relevant kinases (such as cyclin-dependent kinase 2) and the mitochondrial electron transport chain. While we have previously demonstrated that LamD at micromolar range deploys strong cytotoxicity by inducing mitochondrial apoptosis, mechanisms of its cytostatic effect have not yet been characterized. Here, we demonstrated that induction of cellular senescence (depicted by cell cycle arrest in G2 associated with β-galactosidase activity) is a common response to subtoxic concentrations of LamD. Cellular senescence is observed in a large panel of cancer cells following in vitro or in vivo exposure to LamD. The onset of cellular senescence is dependent on the presence of intact topoisomerase I since topoisomerase I-mutated cells are resistant to senescence induced by LamD. LamD-induced senescence occurs without important loss of telomere integrity. Instead, incubation with LamD results in the production of intracellular reactive oxygen species (ROS), which are critical for senescence as demonstrated by the inhibitory effect of antioxidants. In addition, cancer cells lacking mitochondrial DNA also exhibit cellular senescence upon LamD exposure indicating that LamD can trigger senescence, unlike apoptosis, in the absence of functional mitochondria. Overall, our results identify senescence-associated growth arrest as a powerful effect of LamD and add compelling evidence for the pharmacological interest of lamellarins as potential anticancer agents.

Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy. Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses. Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α. However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown. We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients. Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α. Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib. Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol. Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition.

Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease., (©2012 AACR.)

F14512, an epipodophyllotoxin derivative equipped with a spermine moiety, is selectively taken up by the polyamine transport system over-active in tumor cells. F14512 was identified as a selective anticancer agent with a broad spectrum of antitumor activities and is currently undergoing phase I clinical trial in onco-hematology. However, the mechanism by which F14512 exerts its selective effects on neoplastic cells remains poorly understood. In this study, using mainly P388 leukemia cells, we showed that activation of the DNA damage response by F14512 did not induce immediate apoptosis but resulted in an early growth arrest. F14512-induced G2 arrest was accompanied by the appearance of a senescence-like phenotype (characterized by an increased β-galactosidase staining) with up-regulation of the cyclin-dependent kinase inhibitor p16, and cyclin D1. The early senescence-based cell cycle block was characterized by a marked increase of the level of the IAP protein survivin, but not cIAP2, in P388 cells as well as in three other leukemia and melanoma cell types. The Thr(34)-phosphorylated form of survivin was observed within 4 h after F14512 exposure. Inhibition of survivin by siRNA resulted in a switch from senescence-like growth arrest to apoptosis. Compared with the parental drug etoposide, F14512-induced DNA damage signaling pathway resulted in greater senescence like-growth arrest and delayed apoptosis. Collectively, our data show that senescence arrest and subsequent apoptosis are powerful mechanisms mediating the chemotherapeutic effects of F14512 and identify survivin as the molecular determinant responsible for a qualitative shift in cell fate from senescence to apoptosis upon treatment with F14512.