Dietary omega-3 fatty acids modulate the production of platelet-derived microvesicles in an in vivo inflammatory arthritis model.

PURPOSE: The aim of this study was to investigate the effects of different ω-3 polyunsaturated fatty acid (PUFA) enriched diets, including a novel renewable plant source of ω-3 fatty acids (Buglossoides arvensis), on the development and progression of rheumatoid arthritis (RA). METHODS: RA was induced in mice consuming experimental diets using the K/BxN model. The experimental diets consisted of either a western control diet (control), diets containing B. arvensis oil or fish oil. The effects of the diets on platelets, platelet microvesicles (PMVs), and inflammatory markers such as clinical index, ankle thickness and cytokine/chemokine release were measured. RESULTS: While ω-3 PUFA-enriched diets did not prevent the development of arthritis in the K/BxN model, a significant decrease in ankle swelling was observed compared to the control group. Platelets isolated from mice consuming either low content of B. arvensis oil or fish oil diets exhibited significantly decreased PMVs production compared to mice consuming the control diet. CONCLUSION: Our study provides insight into the contribution of ω-3 PUFA supplementation in modulating the pro-inflammatory phenotype of platelets in RA pathology. Furthermore, our study suggests that low concentrations of dietary B. arvensis oil may have similar anti-inflammatory potential seen with dietary fish oil supplementation.

Single and multiple inhibitors of the biosynthesis of 5-, 12-, 15-lipoxygenase products derived from cinnamyl-3,4-dihydroxy-α-cyanocinnamate: Synthesis and structure-activity relationship.

The involvement of lipoxygenases in various pathologies, combined with the unavailability of safe and effective inhibitors of the biosynthesis of their products, is a source of inspiration for the development of new inhibitors. Based on a structural analysis of known inhibitors of lipoxygenase products biosynthesis, a comprehensive structure-activity study was carried out, which led to the discovery of several novel compounds (16a-c, 17a) demonstrating promising potency to inhibit the biosynthesis of products of 5-, 12- and 15-LO. Compounds 16b and 16c outperformed zileuton (1), the only FDA-approved 5-LO inhibitor, as well as known inhibitors such as caffeic acid phenethyl ester (CAPE (2)) and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC (4)). However, the introduction of a cyano group at the α-position of the carbonyl abolished the activity. Compounds 16a and 17a also inhibited the biosynthesis of 12- and 15-LO products. Compounds 16a, 17a far surpassed baicalein, a known 12-LO inhibitor, as inhibitors of 12-LO products biosynthesis. Compound 17a and CDC (4) showed equivalent inhibition of LO products, proposing that the double bond in the ester moiety is not necessary for the inhibitory activity. The introduction of the cyano group, as in compound 17a, at the α-position of the carbonyl in compound 16a significantly reduced the inhibitory activity against the biosynthesis of 15-LO products. In addition to the interactions with residues His372 and Phe421 also found with zileuton and CAPE, compounds 16a and 16c each interact with residue His367 as shown by molecular docking. This new interaction may explain their high affinity with the 5-LO active site.

Algerian Propolis from Distinct Geographical Locations: Chemical Profiles, Antioxidant Capacity, Cytotoxicity and Inhibition of 5-Lipoxygenase Product Biosynthesis.

Propolis was collected from honeybee hives in three geographically distinct Algerian climates and extracts were characterized for composition and bioactivity. Bees were identified as native subspecies using an in-silico DraI mtDNA COI-COII test. Over 20 compounds were identified in extracts by LC-MS. Extracts from the Medea region were more enriched in phenolic content (302±28 mg GAE/g of dry extract) than those from Annaba and Ghardaia regions. Annaba extracts had the highest flavonoid content (1870±385 mg QCE/g of dry extract). Medea extracts presented the highest free-radical scavenging activity (IC50=13.5 µg/mL) using the DPPH radical assay while Ghardaia extracts from the desert region were weak (IC50>100 µg/mL). Antioxidant activities measured using AAPH oxidation of linoleic acid were similar in all extracts with IC50 values ranging from 2.9 to 4.9 µg/mL. All extracts were cytotoxic (MTT assay) and proapoptotic (Annexin-V) against human leukemia cell lines in the low µg/mL range, although the Annaba extract was less active against the Reh cell line. Extracts inhibited cellular 5-lipoxygenase product biosynthesis with IC50 values ranging from 0.6 to 3.2 µg/mL. Overall, examined propolis extracts exhibited significant biological activity that warrant further characterization in cellular and in vivo models.

ARF1 prevents aberrant type I interferon induction by regulating STING activation and recycling.

Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling.

pSTAT5 is associated with improved survival in patients with thick or ulcerated primary cutaneous melanoma.

Identifying prognostic biomarkers to predict clinical outcomes in stage I and II cutaneous melanomas could guide the clinical application of adjuvant and neoadjuvant therapies. We aimed to investigate the prognostic value of phosphorylated signal transducer and activator of transcription 5 (pSTAT5) as a biomarker in early-stage melanoma. This study evaluated all initially staged Ib and II melanoma patients undergoing sentinel node biopsy at a tertiary centre in Brisbane, Australia between 1994 and 2007, with survival data collected from the Queensland Cancer Registry. Primary melanoma tissue from 189 patients was analysed for pSTAT5 level through immunohistochemistry. Cox regression modelling, with adjustment for sex, age, ulceration, anatomical location, and Breslow depth, was applied to determine the association between pSTAT5 detection and melanoma-specific survival. Median duration of follow-up was 7.4 years. High pSTAT5 detection was associated with ulceration and increased tumour thickness. However, multivariate analysis indicated that high pSTAT5 detection was associated with improved melanoma-specific survival (hazard ratio: 0.15, 95% confidence interval: 0.03-0.67) as compared to low pSTAT5 detection. This association persisted when pSTAT5 detection was limited to immune infiltrate or the vasculature, as well as when sentinel node positivity was accounted for. In this cohort, staining for high-pSTAT5 tumours identified a subset of melanoma patients with increased survival outcomes as compared to low-pSTAT5 tumours, despite the former having higher-risk clinicopathological characteristics at diagnosis. pSTAT5 is likely an indicator of local immune activation, and its detection could represent a useful tool to stratify the risk of melanoma progression.

Targeting the chemokine receptor CXCR4 with histamine analog to reduce inflammation in juvenile arthritis.

Introduction: Among immune cells, activated monocytes play a detrimental role in chronic and viral-induced inflammatory pathologies, particularly in Juvenile Idiopathic Arthritis (JIA), a childhood rheumatoid arthritis (RA) disease. The uncontrolled activation of monocytes and excessive production of inflammatory factors contribute to the damage of bone-cartilage joints. Despite the moderate beneficial effect of current therapies and clinical trials, there is still a need for alternative strategies targeting monocytes to treat RA. Methods: To explore such an alternative strategy, we investigated the effects of targeting the CXCR4 receptor using the histamine analog clobenpropit (CB). Monocytes were isolated from the blood and synovial fluids of JIA patients to assess CB's impact on their production of key inflammatory cytokines. Additionally, we administered daily intraperitoneal CB treatment to arthritic mice to evaluate its effects on circulating inflammatory cytokine levels, immune cell infiltrates, joints erosion, and bone resorption, as indicators of disease progression. Results: Our findings demonstrated that CXCR4 targeting with CB significantly inhibited the spontaneous and induced-production of key inflammatory cytokines by monocytes isolated from JIA patients. Furthermore, CB treatment in a mouse model of collagen-induce arthritis resulted in a significant decrease in circulating inflammatory cytokine levels, immune cell infiltrates, joints erosion, and bone resorption, leading to a reduction in disease progression. Discussion: In conclusion, targeting CXCR4 with the small amino compound CB shows promise as a therapeutic option for chronic and viral-induced inflammatory diseases, including RA. CB effectively regulated inflammatory cytokine production of monocytes, presenting a potential targeted approach with potential advantages over current therapies. These results warrant further research and clinical trials to explore the full therapeutic potential of targeting CXCR4 with CB-like molecules in the management of various inflammatory diseases.

EGCG inhibits the inflammation and senescence inducing properties of MDA-MB-231 triple-negative breast cancer (TNBC) cells-derived extracellular vesicles in human adipose-derived mesenchymal stem cells.

BACKGROUND: Triple-negative breast cancer (TNBC) cells' secretome can induce a pro-inflammatory phenotype in human adipose-derived mesenchymal stem cells (hADMSC). This can be prevented by the green tea polyphenol epigallocatechin-3-gallate (EGCG). The impact of EGCG on the paracrine regulation that the extracellular vesicles (EVs) specifically exert within the TNBC secretome remains unknown. METHODS: EVs were obtained from a TNBC-derived serum-starved MDA-MB-231 cell model treated or not with EGCG under normoxic or hypoxic (< 1% O2) culture conditions. RNA-Seq analysis was used to assess the EVs' genetic content. The modulation of inflammatory and senescence markers in hADMSC was evaluated by RT-qPCR using cDNA arrays and validated by immunoblotting. A protein profiler phospho-kinase array was used to explore signaling pathways. RESULTS: While hypoxic culture conditions did not significantly alter the genetic content of MDA-MB-231-secreted EVs, the addition of EGCG significantly modified EVs genetic material at low oxygen tension. Gene expression of cancer-associated adipocyte pro-inflammatory markers CXCL8, CCL2 and IL-1ß was increased in hADMSC treated with EVs. Concomitantly, EVs isolated from MDA-MB-231 treated with EGCG (EGCG-EVs) downregulated CCL2 and IL-1ß, while inducing higher expression of CXCL8 and IL-6 levels. EVs activated CHK-2, c-Jun, AKT and GSK-3ß signaling pathways in hADMSC, whereas EGCG-EVs specifically reduced the latter two as well as the serum starvation-induced senescence markers p21 and ß-galactosidase. Finally, the mitochondrial content within the TNBC cells-derived EVs was found reduced upon EGCG treatment. CONCLUSION: This proof of concept study demonstrates that the chemopreventive properties of diet-derived polyphenols may efficiently target the paracrine regulation that TNBC cells could exert upon their surrounding adipose tissue microenvironment.

Pathogenic variants in the NLRP3 LRR domain at position 861 are responsible for a boost-dependent atypical CAPS phenotype.

BACKGROUND: Cryopyrin-associated periodic syndrome (CAPS) is associated with NLRP3 pathogenic variants, mostly located in the NACHT (neuronal apoptosis inhibitor protein, MHC class 2 transcription activator, incompatibility locus protein from Podospora anserina, telomerase-associated protein) domain. Cold-induced urticarial rash is among the main clinical features. However, this study identified a series of 14 patients with pathogenic variants of the Y861 residue (p.Tyr861) of the LRR domain of NLRP3 and minimal prevalence of cold-induced urticarial rash. OBJECTIVES: This study aimed to address a possible genotype/phenotype correlation for patients with CAPS and to investigate at the cellular levels the impact of the Y861C substitution (p.Tyr861Cys) on NLRP3 activation. METHODS: Clinical features of 14 patients with CAPS and heterozygous substitution at position 861 in the LRR domain of NLRP3 were compared to clinical features of 48 patients with CAPS and pathogenic variants outside the LRR domain of NLRP3. IL-1ß secretion by PBMCs and purified monocytes from patients and healthy donors was evaluated following LPS and monosodium urate crystal stimulation. RESULTS: Patients with substitution at position 861 of NLRP3 demonstrated a higher prevalence of sensorineural hearing loss while being less prone to skin urticarial. In contrast to patients with classical CAPS, cells from patients with a pathogenic variant at position 861 required an activation signal to secrete IL-1ß but produced more IL-1ß during the early and late phase of secretion than cells from healthy donors. CONCLUSIONS: Pathogenic variants of Y861 of NLRP3 drive a boost-dependent oversecretion of IL-1ß associated with an atypical CAPS phenotype.

Successful treatment of JAK1-associated inflammatory disease.

BACKGROUND: Gain-of-function variants of JAK1 drive a rare immune dysregulation syndrome associated with atopic dermatitis, allergy, and eosinophilia. OBJECTIVES: This study sought to describe the clinical and immunological characteristics associated with a new gain-of-function variant of JAK1 and report the therapeutic efficacy of Janus kinase (JAK) inhibition. METHODS: The investigators identified a family affected by JAK1-associated autoinflammatory disease and performed clinical assessment and immunological monitoring on 9 patients. JAK1 signaling was studied by flow and mass cytometry in patients' cells at basal state or after immune stimulation. A molecular disease signature in the blood was studied at the transcriptomic level. Patients were treated with 1 of 2 JAK inhibitors: either baricitinib or upadacitinib. Clinical, cellular, and molecular response were evaluated over a 2-year period. RESULTS: Affected individuals displayed a syndromic disease with prominent allergy including atopic dermatitis, ichthyosis, arthralgia, chronic diarrhea, disseminated calcifying fibrous tumors, and elevated whole blood histamine levels. A variant of JAK1 localized in the pseudokinase domain was identified in all 9 affected, tested patients. Hyper-phosphorylation of STAT3 was found in 5 of 6 patients tested. Treatment of patients' cells with baricitinib controlled most of the atypical hyper-phosphorylation of STAT3. Administration of baricitinib to patients led to rapid improvement of the disease in all adults and was associated with reduction of systemic inflammation. CONCLUSIONS: Patients with this new JAK1 gain-of-function pathogenic variant displayed very high levels of blood histamine and showed a variable combination of atopy with articular and gastrointestinal manifestations as well as calcifying fibrous tumors. The disease, which appears to be linked to STAT3 hyperactivation, was well controlled under treatment by JAK inhibitors in adult patients.

Caffeic acid phenethyl ester analogues as selective inhibitors of 12-lipoxygenase product biosynthesis in human platelets.

The inflammatory response is an essential process for the host defence against pathogens. Lipid mediators are important in coordinating the pro-inflammatory and pro-resolution phases of the inflammatory process. However, unregulated production of these mediators has been associated with chronic inflammatory diseases such as arthritis, asthma, cardiovascular diseases, and several types of cancer. Therefore, it is not surprising that enzymes implicated in the production of these lipid mediators have been targeted for potential therapeutic approaches. Amongst these inflammatory molecules, the 12-hydroxyeicosatetraenoic acid (12(S)-HETE) is abundantly produced in several diseases and is primarily biosynthesized via the platelet's 12-lipoxygenase (12-LO) pathway. To this day, very few compounds selectively inhibit the 12-LO pathway, and most importantly, none are currently used in the clinical settings. In this study, we investigated a series of polyphenol analogues of natural polyphenols that inhibit the 12-LO pathway in human platelets without affecting other normal functions of the cell. Using an ex vivo approach, we found one compound that selectively inhibited the 12-LO pathway, with IC50 values as low as 0.11 µM, with minimal inhibition of other lipoxygenase or cyclooxygenase pathways. More importantly, our data show that none of the compounds tested induced significant off-target effects on either the platelet's activation or its viability. In the continuous search for specific and better inhibitors targeting the regulation of inflammation, we characterized two novel inhibitors of the 12-LO pathway that could be promising for subsequent in vivo studies.

Heterogeneity and functions of the 13 IFN-α subtypes - lucky for some?

Type I IFNs are critical for host responses to viral infection and are also implicated in the pathogenesis of multiple autoimmune diseases. Multiple subtypes exist within the type I IFN family, in particular 13 distinct IFN-α genes, which signal through the same heterodimer receptor that is ubiquitously expressed by mammalian cells. Both evolutionary genetic studies and functional antiviral assays strongly suggest differential functions and activity between the 13 IFN-α subtypes, yet we still lack a clear understanding of these different roles. This review summarizes the evidence from studies describing differential functions of IFN-α subtypes and highlights potential reasons for discrepancies between the reports. We examine both acute and chronic viral infection, as well as autoimmunity, and integrate a more recent awareness of the importance of anti-IFN-α autoantibodies in shaping the type I IFN responses in these different conditions.

Bifunctional Europium for Operando Catalyst Thermometry in an Exothermic Chemical Reaction.

Often the reactor or the reaction medium temperature is reported in the field of heterogeneous catalysis, even though it could vary significantly from the reactive catalyst temperature. The influence of the catalyst temperature on the catalytic performance and vice versa is therefore not always accurately known. We here apply EuOCl as both solid catalyst and thermometer, allowing for operando temperature determination. The interplay between reaction conditions and the catalyst temperature dynamics is studied. A maximum temperature difference between the catalyst and oven of +16 °C was observed due to the exothermicity of the methane oxychlorination reaction. Heat dissipation by radiation appears dominating compared to convection in this set-up, explaining the observed uniform catalyst bed temperature. Application of operando catalyst thermometry could provide a deeper mechanistic understanding of catalyst performances and allow for safer process operation in chemical industries.

Efficacy and tolerance of corticosteroids and methotrexate in patients with juvenile dermatomyositis: a retrospective cohort study.

OBJECTIVES: To assess the efficacy and tolerance of the conventional first-line treatment by MTX and CS in patients with JDM regardless of severity. METHODS: We conducted a monocentric retrospective study of patients with newly diagnosed JDM treated with MTX and CS from 2012 to 2020. The proportion of clinically inactive disease (CID) within 6 months of MTX initiation was evaluated using both Paediatric Rheumatology International Trials Organisation (PRINTO) criteria (evaluating muscle inactive disease) and DAS (evaluating skin inactive disease). We compared responders and non-responders using univariate analyses. RESULTS: Forty-five patients with JDM, out of which 30 (67%) severe JDM, were included. After 6 months of treatment with MTX and CS, complete CID, muscle CID and skin CID were achieved in 14/45 (31%), 19/45 (42%) and 15/45 (33%) patients, respectively. The absence of myositis-specific (MSA) or myositis-associated autoantibodies (MAA) at diagnosis was associated with a better overall, cutaneous and muscular therapeutic response, compared with antibody-positive forms (P < 0.01). Requirement for ICU (P = 0.029) and cutaneous ulcerations (P = 0.018) were associated with a less favourable muscle response. MTX was stopped due to intolerance in six patients (13%) before month 6. CONCLUSIONS: Conventional first-line treatment with MTX was not efficient in a large subset of JDM patients, especially in patients with MSA-positive forms, and in patients with severe JDM. Larger, multicentre cohorts are required to confirm these data and to identify new predictive biomarkers of MTX response, in order to treat patients with JDM as early as possible with appropriate targeted drugs.

Native extracellular matrix orientation determines multipotent vascular stem cell proliferation in response to cyclic uniaxial tensile strain and simulated stent indentation.

Cardiovascular disease is the leading cause of death worldwide, with multipotent vascular stem cells (MVSC) implicated in contributing to diseased vessels. MVSC are mechanosensitive cells which align perpendicular to cyclic uniaxial tensile strain. Within the blood vessel wall, collagen fibers constrain cells so that they are forced to align circumferentially, in the primary direction of tensile strain. In these experiments, MVSC were seeded onto the medial layer of decellularized porcine carotid arteries, then exposed to 10%, 1 Hz cyclic tensile strain for 10 days with the collagen fiber direction either parallel or perpendicular to the direction of strain. Cells aligned with the direction of the collagen fibers regardless of the orientation to strain. Cells aligned with the direction of strain showed an increased number of proliferative Ki67 positive cells, while those strained perpendicular to the direction of cell alignment showed no change in cell proliferation. A bioreactor system was designed to simulate the indentation of a single, wire stent strut. After 10 days of cyclic loading to 10% strain, MVSC showed regions of densely packed, highly proliferative cells. Therefore, MVSC may play a significant role in in-stent restenosis, and this proliferative response could potentially be controlled by controlling MVSC orientation relative to applied strain.

Compromised mitochondrial quality control triggers lipin1-related rhabdomyolysis.

LPIN1 mutations are responsible for inherited recurrent rhabdomyolysis, a life-threatening condition with no efficient therapeutic intervention. Here, we conduct a bedside-to-bench-and-back investigation to study the pathophysiology of lipin1 deficiency. We find that lipin1-deficient myoblasts exhibit a reduction in phosphatidylinositol-3-phosphate close to autophagosomes and late endosomes that prevents the recruitment of the GTPase Armus, locks Rab7 in the active state, inhibits vesicle clearance by fusion with lysosomes, and alters their positioning and function. Oxidized mitochondrial DNA accumulates in late endosomes, where it activates Toll-like receptor 9 (TLR9) and triggers inflammatory signaling and caspase-dependent myolysis. Hydroxychloroquine blocks TLR9 activation by mitochondrial DNA in vitro and may attenuate flares of rhabdomyolysis in 6 patients treated. We suggest a critical role for defective clearance of oxidized mitochondrial DNA that activates TLR9-restricted inflammation in lipin1-related rhabdomyolysis. Interventions blocking TLR9 activation or inflammation can improve patient care in vivo.

Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A.

Mitochondrial DNA (mtDNA) has been suggested to drive immune system activation, but the induction of interferon signaling by mtDNA has not been demonstrated in a Mendelian mitochondrial disease. We initially ascertained two patients, one with a purely neurological phenotype and one with features suggestive of systemic sclerosis in a syndromic context, and found them both to demonstrate enhanced interferon-stimulated gene (ISG) expression in blood. We determined each to harbor a previously described de novo dominant-negative heterozygous mutation in ATAD3A, encoding ATPase family AAA domain-containing protein 3A (ATAD3A). We identified five further patients with mutations in ATAD3A and recorded up-regulated ISG expression and interferon α protein in four of them. Knockdown of ATAD3A in THP-1 cells resulted in increased interferon signaling, mediated by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Enhanced interferon signaling was abrogated in THP-1 cells and patient fibroblasts depleted of mtDNA. Thus, mutations in the mitochondrial membrane protein ATAD3A define a novel type I interferonopathy.

Molecular characterization of the anticancer properties associated with bee venom and its components in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a frequent form of malignant glioma. Strategic therapeutic approaches to treat this type of brain tumor currently involves a combination of surgery, radiotherapy and chemotherapy. Nevertheless, survival of GBM patients remains in the 12-15 months range following diagnosis. Development of novel therapeutic approaches for this malignancy is therefore of utmost importance. Interestingly, bee venom and its components have shown promising anti-cancer activities in various types of cancer even though information pertaining to GBMs have been limited. The current work was thus undertaken to better characterize the anti-cancer properties of bee venom and its components in Hs683, T98G and U373 human glioma cells. MTT-based cell viability assays revealed IC50 values of 7.12, 15.35 and 7.60 µg/mL for cell lines Hs683, T98G and U373 treated with bee venom, respectively. Furthermore, melittin treatment of these cell lines resulted in IC50 values of 7.77, 31.53 and 12.34 µg/mL, respectively. Cell viability assessment by flow cytometry analysis confirmed signs of late apoptosis and necrosis after only 1 h of treatment with either bee venom or melittin in all three cell lines. Immunoblotting-based quantification of apoptotic markers demonstrated increased expression of Bak and Bax, while Caspsase-3 levels were significantly lower when compared to control cells. Quantification by qRT-PCR showed increased expression levels of long non-coding RNAs RP11-838N2.4 and XIST in glioma cells treated with either bee venom or melittin. Overall, this study provides preliminary insight on molecular mechanisms via which bee venom and its main components can impact viability of glioma cells and warrants further investigation of its anticancer potential in gliomas.