Search Results (692)

Peptidyl arginine deiminase 4 (PAD4) is a Ca²⁺-dependent enzyme that catalyzes histone citrullination and plays a central role in chromatin decondensation during neutrophil extracellular trap (NET) formation. Dysregulated PAD4-mediated NETosis contributes to the pathogenesis of diverse inflammatory and immune-related diseases, including autoimmune disorders, cancer, and thrombosis. Although several synthetic PAD4 inhibitors have been developed, their therapeutic application has been limited by issues related to selectivity, irreversible covalent reactivity, and suboptimal pharmacokinetic properties, prompting growing interest in natural products as alternative modulators of PAD4 activity and NETosis. This article presents a structural and mechanistic overview of natural products that target PAD4 and regulate NETosis. Based on enzyme kinetics, structural analyses, and functional validation, natural PAD4 modulators are classified into four categories: (i) active-site-directed inhibitors that bind within the U-shaped substrate tunnel, (ii) mixed and active-site-adjacent inhibitors that engage surface pockets flanking the catalytic site, (iii) allosteric and hybrid modulators that bind to regulatory regions distinct from the active site, and (iv) functionally validated PAD4 binders supported by biophysical and cellular evidence. Integration of structural, biochemical, and cellular data highlights that indirect or noncanonical modes of PAD4 regulation represent biologically coherent strategies for controlling pathological NETosis. Full article

Citrulline (CIT), a natural non-protein amino acid and nitric oxide (NO) precursor, plays a vital role in plant physiological regulation. Its use as an eco-friendly biostimulant aligns with global efforts to reduce dependence on synthetic agrochemicals and strengthen sustainable crop production systems. This study represents the first report investigating the effects of exogenous citrulline (0, 0.5, and 1 mM) as a biostimulant/eustressor in two faba bean (Vicia faba) cultivars (Giza 843 and Sakha 1) and the first report to evaluate the variety and dose-dependent responses to foliar CIT application. The morphological, biochemical, and antioxidant responses were assessed. CIT significantly improved several growth and yield attributes in a cultivar-dependent manner, with Giza 843 performing best at 1 mM and Sakha 1 showing optimal shoot performance at 0.5 mM. CIT increased H₂O₂ levels, flavonoids, and catalase activity, which modulate the response mechanisms of treated plants of two varieties of faba bean. In contrast to Giza 843, Sakha 1 increased proline and the activities of peroxidase and ascorbate peroxidase, which is parallel with decreasing soluble sugars and proteins in response to CIT application. These results showed that Sakha 1 had more effective defense mechanisms than Giza 843. These findings demonstrate that CIT at an optimal dose is a promising, eco-friendly biostimulant. It may be suitable to integrate into sustainable crop management programs to enhance crop resilience and productivity. Full article

In inflammatory states, eosinophils are exposed to stimuli leading to activation, increased survival, and/or different cell death subroutines, which have differing effects on tissue inflammation. The mechanisms of signal integration are poorly understood. In this manuscript, we investigated cell death types in response to stimuli mimicking the inflammatory microenvironment. Mouse bone marrow-derived eosinophils (BMDeos) were stimulated with cytokines, cell-cell interaction mimics, pathogen-associated molecular patterns (PAMP), and broad cell activation stimuli. Both PMA and crosslinking of CD95 (cCD95) induced cell death of BMDeos. However, cCD95-induced cell death was consistent with apoptosis, while activation with PMA lead to EETosis. Both stimuli lead to caspase 3 activation and increased total level of histone H3 citrullination, indicating that these outcomes are not able to discriminate between the two cell death types. Flow cytometry for annexinV/7AAD pattern at early time points, and morphologic assessment by immunofluorescence (for DNA, eosinophil granule protein and citH3) were the most reliable outcomes for distinguishing the cell death subtypes. While LPS alone did not decrease BMDeos viability, LPS in the presence of caspase inhibition (zVAD) caused delayed cell death, which did not conform to either of the two cell death types. Finally, LPS and LPS/zVAD led to an increased level of surface expression of CD274 (type 1 activation), while both cCD95 and PMA increased the surface expression of CD101 (type 2 activation). In summary, at least three different activation-associated cell death pathways are seen in BMDeos activated with microenvironment-mimicking stimuli. Crosslinking CD95 leads to type 2 activation and apoptotic cell death. PMA also leads to type 2 activation but EETosis-associated cell death. LPS and LPS/zVAD are associated with type 1 activation, and only LPS/zVAD lead to cell death via a subtype different from both apoptosis and EETosis. Full article

Background: Citrulline malate (CM) supplementation has been shown to improve resistance exercise performance. However, there is limited research on the dose–response effects of CM ingestion. The aim of this study was to investigate a moderate (8 g; CM-MOD) and high (12 g; CM-HIGH) dose of CM on resistance exercise performance. Methods: Twelve resistance-trained individuals (7 females, 5 males, age = 24 ± 2 years; body mass = 70 ± 10 kg; height = 172 ± 7 cm) volunteered for this randomised, double-blind, crossover trial. Following a familiarisation trial that consisted of determining one repetition maximum, participants completed barbell bent-over rows and leg presses following acute ingestion of either 8 g CM (CM-MOD), 12 g CM (CM-HIGH), or a placebo 1 h prior to exercise. Each exercise comprised two sets of 10 repetitions (70% one-repetition maximum (RM)) and a third set to exhaustion at 70% 1 RM. Results: The linear mixed-effect model found no significant differences in the completed repetitions between exercise type but did reveal a significant main effect of CM-HIGH on repetitions completed (p = 0.032), which was not found for CM-MOD, and only increases in leg press repetitions were observed (estimated marginal means: placebo = 17; CM-MOD = 19; CM-HIGH = 20). Conclusions: In conclusion, CM-HIGH resulted in small improvements to total repetitions performed during resistance exercise performance and likely only during leg press activity, though the underlying mechanisms remain unclear and further investigation is warranted. Full article

Neutrophils and their extracellular traps (NETs) are pivotal elements of the immune response. This study investigates the dynamics of neutrophil-related markers during the perioperative period of branched endovascular aortic repair (BEVAR) in patients with thoracoabdominal aortic aneurysms (TAAAs) and evaluates their association with one-year clinical outcomes. A prospective, single-center study was conducted on 20 TAAA patients treated with T-branch devices. The analysis focused on surrogate markers associated with NETosis, including double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and citrullinated histone H3 (citH3). Peripheral venous blood was collected 24 h before BEVAR, and on the third and fifth postoperative days. Patients were monitored for one year to evaluate mortality and hospitalization risks, with predictors identified using Cox regression analysis. Increased postoperative levels of inflammatory markers were significantly associated with higher risks of mortality and hospital readmission. On the third postoperative day, key parameters emerged as predictors of adverse outcomes: dsDNA (HR = 1.000; 95% CI 1.000–1.000; p = 0.027), ssDNA (HR = 1.000; 95% CI 1.000–1.000; p = 0.022), and NLR (HR = 1.226; 95% CI 1.043–1.440; p = 0.013). Markers assessed in the early postoperative period (the third postopearive day) demonstrated superior predictive utility compared to those measured on the fifth postoperative day. CitH3 levels did not show statistical significance as a prognostic factor. Early postoperative evaluation of NET-associated markers, particularly dsDNA and ssDNA, offers prognostic value for predicting mortality and hospitalization risks in TAAA patients undergoing BEVAR. These markers may provide superior predictive accuracy compared to conventional post-implantation syndrome criteria. Enhanced postoperative monitoring of these markers could help identify high-risk patients who may benefit from intensified follow-up. Full article

Multiple sclerosis (MS) pathogenesis involves not only immune-mediated myelin injury but also glial responses. We examined how three charge isomers of myelin basic protein (MBP)—native (C1), phosphorylated (C4), and citrullinated (C8)—modulate rat astrocytes. Cytokines were quantified and grouped (pro/anti-inflammatory, chemotactic, neurotrophic, angiogenic, tissue remodeling), and regulatory markers assessed. C1 strongly upregulated the lipid-sensing receptor LXR, and reduced global DNA methylation; C4 moderately enhanced LXR; C8 failed to activate LXR or alter methylation. Functionally, C1 attenuated IL-1β, IL-6 and GM-CSF while increasing IL-10 and certain chemokines. C4 elicited an intermediate pattern, inducing CX3CL1 (fractalkine), CCL20, VEGF-A and TIMP-1 with minor effects on classical cytokines. In contrast, C8 triggered a robust pro-inflammatory phenotype, increasing IL-1α/β, TNF-α and GM-CSF, with higher IL-10, fractalkine, CCL20, VEGF-A and TIMP-1. All isomers suppressed IFN-γ, IL-4 and CNTF. These data indicate that MBP post-translational modifications drive distinct astrocyte phenotypes through integrated cytokine, metabolic and epigenetic pathways: C1 favors immune regulation and repair, C4 blends inflammatory and reparative cues, and C8 amplifies neuroinflammation. Understanding how modified MBP shapes astrocyte behavior provides mechanistic insight into lesion evolution in MS and suggests astrocyte-directed strategies to modulate neuroinflammation and promote remyelination. Full article

A 78-year-old woman with a history of rheumatoid arthritis (treated with methotrexate) developed disturbed consciousness, emesis, and intestinal perforation. Initial labs revealed hyperammonemia (189 μg/dL) and hypertonic dehydration. Despite ammonia normalization, her neurological status improved only slightly, necessitating additional tests. Cerebrospinal fluid analysis showed no pleocytosis but positive oligoclonal bands and markedly elevated myelin basic protein (>500 pg/mL). Serum autoimmune markers were negative, including anti-aquaporin-4 (AQP4), anti-myelin oligodendrocyte glycoprotein (MOG), and anti-glial fibrillary acidic protein (GFAP) antibodies. MRI revealed T2/DWI-hyperintense lesions in the left parietal lobe and cerebellum. Crucially, extensive T2/DWI-hyperintense lesions with diffusion restriction spanned the white matter from the medulla oblongata to the lumbar spinal cord. Axial spinal DWI demonstrated diffuse hyperintensity throughout the entire white matter, accompanied by gray matter atrophy. Subsequent metabolic screening revealed low folate and hypocupremia (34 μg/dL) as well as urinary orotic acid and low serum citrulline, suggesting late-onset ornithine transcarbamylase (OTC) deficiency. Given the clinical context, this was interpreted as a metabolic breakdown rather than an established genetic diagnosis. This case is characterized by a long, diffusion-restricted lesion from the brainstem to the spinal cord that does not correspond to vascular territories. She experienced sudden death. We hypothesize that an underlying metabolic disorder, nutritional deficiencies and drug-induced neurotoxicity contributed to lesion formation. Full article

Background: Sepsis-induced organ dysfunction poses a significant clinical challenge with limited therapeutic options. This study investigated the therapeutic potential of the glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide in sepsis and its underlying mechanisms, focusing on modulation of the gut microbiota-derived metabolome. Methods: Public transcriptomic data analysis identified overlapping targets between liraglutide and sepsis-related genes. In a murine cecal ligation and puncture (CLP) model, liraglutide treatment was evaluated for its effects on survival, systemic inflammation, and organ injury. The gut microbiota composition and fecal metabolome were assessed via 16S rRNA sequencing and UPLC-MS. We also measured plasma GLP-1 in sepsis patients and examined the microbiota-dependency of liraglutide’s effects using antibiotic-depleted mice and fecal microbiota transplantation (FMT) from liraglutide-treated mice. Additionally, citrulline, a key identified metabolite, was functionally validated both in vitro and in a clinical cohort. Results: Liraglutide significantly improved survival, reduced pro-inflammatory cytokines, and alleviated lung, liver, and colon damage in septic mice. It partially restored sepsis-induced gut dysbiosis and modulating associated metabolites, including increasing citrulline. The survival benefit of liraglutide was abolished in microbiota-depleted mice, while FMT from liraglutide-treated mice conferred protection against sepsis, confirming the gut microbiota as a critical mediator. Furthermore, citrulline exhibited direct anti-inflammatory properties in cellular assays, and its plasma levels were negatively correlated with sepsis biomarkers (PCT and CRP) in patients. Conclusions: Taken together, our findings indicate that liraglutide mitigates sepsis by modulating the gut microbiota and regulating associated metabolic pathways. Citrulline may represent a potential microbial mediator or exploratory biomarker within this axis, warranting further mechanistic investigation. Full article

Chronic arthritis following arthritogenic alphavirus infections presents symptoms resembling autoimmune rheumatic diseases, raising questions about the underlying mechanisms, including molecular mimicry and autoantibody production. This systematic review evaluated evidence supporting molecular mimicry and the potential role of autoantibodies as predictive biomarkers in alphavirus-induced chronic arthritis. A comprehensive search of PubMed, Scopus and Web of Science was conducted following PRISMA 2020 guidelines and PECO framework. Thirteen studies met the inclusion criteria: four computational studies assessing peptide homology between viral and human proteins, and nine clinical studies evaluating autoantibodies in chronic post-alphavirus arthritis. Computational analyses identified conserved alphavirus peptides with sequence and structural similarity to human proteins implicated in autoimmunity, supporting the hypothesis of molecular mimicry. However, most lacked experimental validation. Clinical studies showed variable detection of autoantibodies, rheumatoid factors, anti-cyclic citrullinated peptide, and antinuclear antibodies in chronic patients, though seropositivity rates were inconsistent and generally low. Only one study reported a significant association between autoantibody levels and disease chronicity. The findings suggest a potential autoimmune component in post-alphavirus arthritis driven by molecular mimicry, though current evidence remains inconclusive due to methodological heterogeneity and limited validation. Autoantibodies may contribute to pathogenesis but are not reliable predictors of chronicity. Future longitudinal studies with standardized assays and validation of computational findings in human models are needed. Full article

Air-dried pork coppa is highly favored for its unique organoleptic and flavor characteristics. However, the traditional long processing cycle and uncontrollable environmental conditions lead to unstable product quality. Staphylococcus mediates the reduction in nitrite to nitric oxide via nitrite reductase; the resulting nitric oxide then binds to myoglobin, forming nitrosylmyoglobin that endows meat products with a characteristic bright red. It could also improve the activity of lipase and protease, promoting the flavor. In this study, Staphylococcus carnosus and Staphylococcus xylosus as starter cultures have been applied to air-dried coppa. After Staphylococcus inoculation, the water activity and pH value of coppa significantly decreased compared with those of naturally fermented coppa (p < 0.05). Meanwhile, it improved the color and increased the hardness and chewiness, which in turn enhanced the overall taste of organoleptic acceptability. The ¹H NMR spectra showed that the main taste metabolites were free amino acids and organic acids. Citrulline, formic acid, isobutyric acid, and isovaleric acid might be the key metabolites distinguishing between those with or without Staphylococcus inoculation. This study suggested that inoculation with Staphylococcus xylosus and Staphylococcus carnosus played an important role in improving the physicochemical properties and taste development of coppa. Full article

Background/Objectives: Vitamin D levels tend to be lower in patients with inflammatory rheumatic diseases (IRDs), including rheumatoid arthritis (RA), but there are minimal data on vitamin D levels in rheumatology patients with inflammatory vs. non-inflammatory diagnoses. Methods: In this retrospective, observational study, we used electronic health record data from patients presenting for their first visit at a large rheumatology clinic to assess vitamin D levels and deficiency based on diagnosis, and to evaluate the association between vitamin D and inflammatory markers (including C-reactive protein [CRP]) or autoimmune markers (including rheumatoid factor [RF], anti-citrullinated peptide antibody, and anti-nuclear antibodies). Logistic regression analysis with 13 clinical variables was used to evaluate the association between vitamin D levels and IRD diagnosis, and linear regression was used to evaluate the association between vitamin D levels and CRP or RF. Results: The patient cohort included 4979 patients; 1385 (27.8%) had an IRD. Vitamin D levels were significantly lower in the IRD vs. non-inflammatory subgroup (mean [SD] of 26.6 [13.3] vs. 27.7 [14.3]; p = 0.009), but the difference was not clinically relevant given the small effect size. Vitamin D deficiency rates (<20 ng/mL) were not significantly different between the subgroups, and vitamin D was not associated with an IRD diagnosis in logistic regression analysis. In linear regression analysis, vitamin D was not associated with CRP or RF in the full patient cohort or in the subgroup with RA (n = 539). Conclusions: We conclude that vitamin D levels do not differ substantially based on IRD versus non-inflammatory diagnosis, CRP levels, or RF levels in this clinical cohort. Full article

The biomarker landscape in rheumatoid arthritis (RA) is evolving from reliance on traditional markers toward integrated, multimodal strategies enabling precision medicine approaches. To critically evaluate emerging biomarkers across serological, cellular, genetic, imaging, and multi-omic domains, distinguishing those approaching clinical readiness from those requiring further development. In this study, a narrative review of the literature published between 2000 and 2024 relevant to clinical decision-making in RA was conducted. Among novel serological markers, 14-3-3η protein and anti-carbamylated protein antibodies show the strongest validation for seronegative disease and prognostic stratification. Calprotectin demonstrates utility for disease activity monitoring and de-escalation decisions. Multi-biomarker disease activity scores provide an objective assessment but lack outcome trial validation. Musculoskeletal ultrasound offers accessible imaging biomarker capability, while MRI bone marrow edema remains the strongest structural progression predictor. Synovial tissue pathotyping has demonstrated proof-of-concept for treatment stratification. Genetic, epigenetic, and metabolomic approaches remain investigational. Key clinical implications include using 14-3-3η and calprotectin to inform seronegative diagnosis and de-escalation decisions, integrating ultrasound for remission verification, and recognizing that emerging biomarkers for extra-articular complications, including cardiovascular risk and venous thromboembolism, represent important unmet needs. Full article

Macrophages are key innate immune cells in the host defense against pathogens. Ionizing radiation can impair macrophage functions such as phagocytosis and activate them, potentially exacerbating tissue injury. Macrophage extracellular traps (METs) are formed upon stimulation of macrophages with PAMPs or DAMPs. We hypothesized that macrophages exposed to ionizing radiation can release extracellular traps. Peritoneal macrophages were collected from C57BL/6 mice and subjected to 5 Gy radiation. We performed assays to detect METs, including the immunofluorescence of citrullination of histone H3 and cell-free DNA measurement in cell culture medium as well as cell death. The exposure of ionizing radiation killed a significant number of mouse peritoneal macrophages through pyroptosis, which was mediated by Gasdermin D (GSDMD). The onset of pyroptosis eventually caused METs by suicidal METosis via pyroptosis and vital METosis occurring in the cells surviving after exposure to radiation. We found that exposure of peritoneal macrophages to 5 Gy radiation significantly increased METosis, as revealed by increased levels of citrullinated histone H3 and an increased surface area of extracellular DNA surrounding the cells. We discovered that peptidyl arginine deiminase (PAD) 2 and 4 are required for peritoneal macrophages to generate extracellular traps in response to radiation exposure. Our data demonstrate that the ionizing radiation induces METs via the activation of GSDMD, and we confirmed the requirement of PADs for METosis after exposure to the ionizing radiation. Targeting METs may direct a new therapeutic strategy for mitigating radiation-induced tissue injury. Full article

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovitis. The prevalence of RA is estimated to be 0.5–1% worldwide. Methods: This work investigated the therapeutic effects and underlying mechanisms of blue mussel (Mytilus galloprovincialis) oil (BMO) on RA in rats, using green-lipped mussel oil (GMO) and Antarctic krill oil (KO) as controls. Results: The results suggested that BMO, GMO, and KO all alleviated paw swelling in rats and reduced serum levels of rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibody, and pro-inflammatory cytokines such as TNF-$\alpha$ and IL-17. Histopathological assessment further revealed that BMO, GMO, and KO prevented synovial fibroplasia, mitigated inflammatory cell infiltration, and improved cartilage damage in ankle joints. Overall, BMO exhibited slightly superior alleviating effects compared with GMO and KO. Plasma lipidomics analysis revealed that the lipid metabolites altered by BMO showed significant correlations with RA-related indicators, particularly pro-inflammatory cytokines. Functional enrichment analysis suggested the involvement of inflammation-related pathways, particularly the NF-$\kappa$B signaling pathway. Further validation demonstrated that BMO effectively suppressed the production of inflammatory cytokines (TNF-$\alpha$, IL-17) and the expression of NF-$\kappa$B p65, JAK2, and STAT3 proteins in synovial tissue. And IL-17 production in footpad tissues is closely associated with CD3-positive T cells. Similar effects were also observed for GMO and KO. Conclusions: Collectively, BMO might ameliorate RA by inhibiting NF-$\kappa$B and JAK2/STAT3 signaling pathways. Full article

Psoriasis is a common inflammatory skin disease with chronic manifestation in which the role of neutrophil extracellular traps (NETs) and alarmins are increasingly recognized as contributors to systemic and cutaneous inflammation. However, the interaction between alarmins and NET-driven immune responses remains poorly defined. The main aim of this study is to define the role of target alarmins (i.e., IL-33 and TSLP) in NETs induction and its subsequent impact on oxidative stress and inflammation in the peripheral blood. In the present study, we recruited active psoriasis patients (n = 56) and control (n = 56) subjects. The frequency of circulating neutrophils, the levels of NET-associated markers (MPO (myeloperoxidase)–DNA complex, CitH3 (citrullinated histone H3), PAD4 (peptidyl arginine deiminase4), NADPH oxidase, and NE (neutrophil elastase)), and alarmin transcripts (IL (interleukin)-33, TSLP (thymic stromal lymphopoietin), S100A7, S100B, HSP (heat shock protein) 60/70 were quantified using flow cytometry, ELISA (Enzyme-linked immunosorbent assay), and qPCR (quantitative polymerase chain reaction), respectively, in each group. The NET formation potential of isolated neutrophils was assessed in the presence or absence of rhIL-33 and rhTSLP by immunocytofluorescence. The effect of rhIL-33- and rhTSLP-primed NETs in augmenting oxidative stress and inflammation was evaluated on peripheral blood mononuclear cells (PBMCs) by ELISA. Significantly higher circulating neutrophils (p < 0.001) and levels of NET-associated markers (i.e., MPO–DNA complex, CitH3, PAD4, NADPH oxidase, and NE) were observed in active psoriasis patients compared to controls. Lesional skin exhibited strong expression of MPO (p < 0.001) compared to normal skin. The alarmins, IL-33 and TSLP, were markedly upregulated in the blood and skin (p < 0.05). The rhIL-33 and rhTSLP treated neutrophils demonstrated enhanced NETosis in patients (p < 0.001). Increased expression of inflammatory cytokines and oxidative stress markers were reported in PBMCs when incubated with rhIL-33- and rhTSLP-primed NETs. Taken together, our investigation demonstrated the novel mechanism wherein the alarmins IL-33 and TSLP exacerbate NET formation that may drive enhanced inflammation and oxidative stress in psoriasis. Full article