A Method for Analysis of Oxidized Phospholipids from Biological Samples Using Mass Spectrometry.

Oxidized phospholipids (oxPLs) are generated during innate immunity and inflammation, where they play a variety of biological roles, including regulation of autoimmunity and coagulation. Some are generated by enzymatic reactions, leading to stereo- and regiospecificity, while many others can be formed through nonenzymatic oxidation and truncation and can be used as biomarkers of oxidative stress. Mass spectrometry methods have been developed over many years for oxPL analysis, which can provide robust estimations of molecular species and amounts, where standards are available. Here we present a method used for the analysis of enzymatically-generated oxPL (eoxPL), which allows quantification of mono-hydroxy oxylipin-containing species. We also show profiling of many other partially characterized structures in tissue samples and provide typical chromatograms obtained.

Quantification of Octadecanoids in Human Plasma Using Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry.

Octadecanoids are a subset of oxylipins derived from 18-carbon fatty acids. These compounds have historically been understudied but have more recently attracted attention to their purported biological activity. One obstacle to the study of octadecanoids has been a lack of specific analytical methods for their measurement. A particular limitation has been the need for chiral-based methods that enable separation and quantification of individual stereoisomers. The use of chirality provides an additional dimension for distinguishing analytes produced enzymatically from those formed through autoxidation. In this chapter, we describe a comprehensive method using chiral supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) for the quantification of octadecanoids in human plasma. This method stands as an effective approach for quantifying octadecanoids and is applicable to diverse research applications including clinical research.

Tensile force impairs lip muscle regeneration under the regulation of interleukin-10.

BACKGROUND: Orbicularis oris muscle, the crucial muscle in speaking, facial expression and aesthetics, is considered the driving force for optimal lip repair. Impaired muscle regeneration remains the main culprit for unsatisfactory surgical outcomes. However, there is a lack of study on how different surgical manipulations affect lip muscle regeneration, limiting efforts to seek effective interventions. METHODS: In this study, we established a rat lip surgery model where the orbicularis oris muscle was injured by manipulations including dissection, transection and stretch. The effect of each technique on muscle regeneration was examined by histological analysis of myogenesis and fibrogenesis. The impact of tensile force was further investigated by the in vitro application of mechanical strain on cultured myoblasts. Transcriptome profiling of muscle satellite cells from different surgical groups was performed to figure out the key factors mediating muscle fibrosis, followed by therapeutic intervention to improve muscle regeneration after lip surgeries. RESULTS: Evaluation of lip muscle regeneration till 56 days after injury revealed that the stretch group resulted in the most severe muscle fibrosis (n = 6, fibrotic area 48.9% in the stretch group, P < 0.001, and 25.1% in the dissection group, P < 0.001). There was the lowest number of Pax7-positive nuclei at Days 3 and 7 in the stretch group (n = 6, P < 0.001, P < 0.001), indicating impaired satellite cell expansion. Myogenesis was impaired in both the transection and stretch groups, as evidenced by the delayed peak of centrally nucleated myofibers and embryonic MyHC. Meanwhile, the stretch group had the highest percentage of Pdgfra+ fibro-adipogenic progenitors infiltrated area at Days 3, 7 and 14 (n = 6, P = 0.003, P = 0.006, P = 0.037). Cultured rat lip muscle myoblasts exhibited impaired myotube formation and fusion capacity when exposed to a high magnitude (ε = 2688 µ strain) of mechanical strain (n = 3, P = 0.014, P = 0.023). RNA-seq analysis of satellite cells isolated from different surgical groups demonstrated that interleukin-10 was the key regulator in muscle fibrosis. Administration of recombinant human Wnt7a, which can inhibit the expression of interleukin-10 in cultured satellite cells (n = 3, P = 0.041), exerted an ameliorating effect on orbicularis oris muscle fibrosis after stretching injury in surgical lip repair. CONCLUSIONS: Tensile force proved to be the most detrimental manoeuvre for post-operative lip muscle regeneration, despite its critical role in correcting lip and nose deformities. Adjunctive biotherapies to regulate the interleukin-10-mediated inflammatory process could facilitate lip muscle regeneration under conditions of high surgical tensile force.

Comprehensive analysis of aging-related gene expression patterns and identification of potential intervention targets.

PURPOSE: This study aims to understand the molecular mechanisms underlying the aging process and identify potential interventions to mitigate age-related decline and diseases. METHODS: This study utilized the GSE168753 dataset to conduct comprehensive differential gene expression analysis and co-expression module analysis. Machine learning and Mendelian randomization analyses were employed to identify core aging-associated genes and potential drug targets. Molecular docking simulations and mediation analysis were also performed to explore potential compounds and mediators involved in the aging process. RESULTS: The analysis identified 4164 differentially expressed genes, with 1893 upregulated and 2271 downregulated genes. Co-expression analysis revealed 21 modules, including both positively and negatively correlated modules between older age and younger age groups. Further exploration identified 509 aging-related genes with distinct biological functions. Machine learning and Mendelian randomization analyses identified eight core genes associated with aging, including DPP9, GNAZ, and RELL2. Molecular docking simulations suggested resveratrol, folic acid, and ethinyl estradiol as potential compounds capable of attenuating aging through modulation of RELL2 expression. Mediation analysis indicated that eosinophil counts and neutrophil count might act as mediators in the causal relationship between genes and aging-related indicators. CONCLUSION: This comprehensive study provides valuable insights into the molecular mechanisms of aging and offers important implications for the development of anti-aging therapeutics. Key Messages What is already known on this topic - Prior research outlines aging's complexity, necessitating precise molecular targets for intervention. What this study adds - This study identifies novel aging-related genes, potential drug targets, and therapeutic compounds, advancing our understanding of aging mechanisms. How this study might affect research, practice, or policy - Findings may inform targeted therapies for age-related conditions, influencing future research and clinical practices.

Targeting PAR2-mediated inflammation in osteoarthritis: a comprehensive in vitro evaluation of oleocanthal's potential as a functional food intervention for chondrocyte protection and anti-inflammatory effects.

BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention. METHODS: To simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC's therapeutic effects on chondrocytes. RESULTS: The results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1ß, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA. CONCLUSIONS: This study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC's comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC's therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders.

Unwinding the Threads of Mesoporous Silica Nanoparticles as Cutting-Edge for the Management of Inflammation: An Updated Review.

BACKGROUND: Inflammation serves as a protective response to combat cellular and tissue damage. There is currently a wide array of synthetic and traditional therapies available for the treatment of inflammatory diseases. However, it is necessary to create a drug delivery system based on nanotechnology that can improve the solubility, permeability, and bioavailability of current treatments. Mesoporous silica nanoparticles (MSNPs) are inorganic materials known for their organised porous interiors, high pore volumes, substantial surface area, exceptional selectivity, permeability, low refractive index, and customisable pore sizes. OBJECTIVE: This review offers concise insights into the progression of the pathophysiology of inflammation, as well as the inducers, mediators, and effectors that are involved in the inflammatory pathway. This study focuses on the growing significance of MSNPs in the treatment of neuroinflammation, inflammatory bowel disease, arthritic inflammation, lung inflammation, and wound healing applications. This review also presents the latest information on the crucial role of MSNPs in delivering herbal medicines for the treatment of inflammation. METHODS: A comprehensive literature search was conducted for this aim, utilising the Google Scholar, PubMed, and ScienceDirect databases. A systematic review was undertaken utilising scholarly articles published in peer-reviewed journals from 2000 to 2024. RESULTS: The inflammatory mediators involved in the pathophysiology of inflammation include platelet-activating factor, lipoxygenase, cyclooxygenase, Interferon-α, interleukin-6, interleukin- 1ß, matrix metalloproteinases, inducible nitric oxide synthase, nuclear factor-κB, prostaglandins, nitric oxide, and phospholipase A2. MSNPs have the potential to be used in the treatment of neuroinflammation, inflammatory bowel disease, arthritic inflammation, lung inflammation, and wound healing. The investigation of the MSNPs of plant-based compounds such as berberine, tetrahydrocannabinol, curcumin, and resveratrol has shown successful results in recent years for the purpose of managing inflammation. CONCLUSION: This review demonstrates that MSNPs have a strong potential to play a positive role in delivering synthetic and plant-based therapies for the treatment of inflammatory illnesses.

Mitigative role of cysteamine against unilateral renal reperfusion injury in Wistar rats.

Background: Ischemia-reperfusion injury (IRI) is unavoidable during kidney transplant and it is responsible for delayed or non-function after kidney transplantation. Cysteamine is the standard drug in the management of nephropathic cystinosis and its extra-renal complications. Thus, we designed this study to investigate its potential against renal reperfusion injury. Results: Significant elevation of H2O2, MDA, and nitrite and reduced GPx, GSH, and protein thiol in the Ischemia-reperfusion injury rats was reversed by cysteamine (50 and 100 mg/kg). Serum MPO, TNF-α, IL-1ß, creatinine, and AOPP were significantly elevated in IRI while rats treated with cysteamine revealed a significant decrease (p < 0.05) in the activities of these pro-inflammatory and renal injury markers. Conclusion: Based on its activity against inflammation, apoptosis, and free radical-induced stress, cysteamine has great potential to be used as a kidney transplant pre-operative drug to prevent renal reperfusion injury.

Pharmacological effects of specialized pro-resolving mediators in sepsis-induced organ dysfunction: a narrative review.

Sepsis is a life-threatening syndrome of organ dysfunction, characterized by uncontrolled inflammatory response and immune dysregulation, often leading to multiple organ failure and even death. Specialized pro-resolving mediators (SPMs), which are typically thought to be formed via consecutive steps of oxidation of polyenoic fatty acids, have been shown to suppress inflammation and promote timely resolution of inflammation. They are mainly divided into four categories: lipoxins, resolvins, protectins, and maresins. The SPMs may improve the prognosis of sepsis by modulating the immune and inflammatory balance, thereby holding promise for clinical applications. However, their biosynthetic and pharmacological properties are very complex. Through a literature review, we aim to comprehensively elucidate the protective mechanisms of different SPMs in sepsis and its organ damage, in order to provide sufficient theoretical basis for the future clinical translation of SPMs.

Comparision of interleukin-1ß concentrations in posttreatment endodontic disease and other pulpal and periapical conditions - A clinical study.

Aim: To evaluate interleukin (IL)-1ß concentrations in periapical tissue fluid (PTF) in persistent apical periodontitis requiring endodontic retreatment and to compare the levels of IL-1ß with chronic apical periodontitis, symptomatic irreversible pulpitis (SIP), normal pulpal, and periapical tissues. Materials and Methods: The patients were selected based on inclusion and exclusion criteria and divided into 4 groups based on the pulpal and periapical status: Posttreatment endodontic diseases (PTED): Teeth with PTED due to failed primary root canal treatment having periapical radiolucency. PNAP: Teeth requiring root canal treatment due to pulpal necrosis having periapical radiolucency. SIP: Teeth with symptoms of SIP with healthy periapical tissues). Intentional root canal treatment (IRCT): Teeth requiring IRCT (healthy pulp and periapical tissues). The access cavity was redefined and the preexisting filling was removed using H-files. The root canals were minimally enlarged followed by collection of PTF using paper points, in the case of group PTED. For groups PNAP, SIP, and IRCT, conventional access cavity preparation was done followed by enlargement of canals till 20, 0.02. PTF was collected using 15, 0.02 size absorbent points 2 mm beyond the apex. Levels of IL-1ß was assessed by enzyme-linked immunosorbent assay. Results: A statistically significant difference was seen in levels of IL-1ß in all the groups. The highest concentration was seen in group PTED (85.07 ± 11.57 pg/mL) followed by group PNAP (37.60 ± 10.94 pg/mL), group SIP (8.40 ± 1.99 pg/mL), and the least was seen in group IRCT (3.47 ± 1.36 pg/mL). Conclusion: The levels of IL-1ß were highest in PETD cases followed by PNAP, SIP, and IRCT. This indicates the severity of inflammation in PETD cases as compared to other endodontic diseases.

SPMs exert anti-inflammatory and pro-resolving effects through positive allosteric modulation of the prostaglandin EP4 receptor.

Inflammation is a protective response to pathogens and injury. To be effective it needs to be resolved by endogenous mechanisms in order to avoid prolonged and excessive inflammation, which can become chronic. Specialized pro-resolving mediators (SPMs) are a group of lipids derived from omega-3 fatty acids, which can induce the resolution of inflammation. How SPMs exert their anti-inflammatory and pro-resolving effects is, however, not clear. Here, we show that SPMs such as protectins, maresins, and D-series resolvins function as biased positive allosteric modulators (PAM) of the prostaglandin E2 (PGE2) receptor EP4 through an intracellular binding site. They increase PGE2-induced Gs-mediated formation of cAMP and thereby promote anti-inflammatory signaling of EP4. In addition, SPMs endow the endogenous EP4 receptor on macrophages with the ability to couple to Gi-type G-proteins, which converts the EP4 receptor on macrophages from an anti-phagocytotic receptor to one increasing phagocytosis, a central mechanism of the pro-resolving activity of synthetic SPMs. In the absence of the EP4 receptor, SPMs lose their anti-inflammatory and pro-resolving activity in vitro and in vivo. Our findings reveal an unusual mechanism of allosteric receptor modulation by lipids and provide a mechanism by which synthetic SPMs exert pro-resolving and anti-inflammatory effects, which may facilitate approaches to treat inflammation.

The dual role of lipids in chronic kidney disease: Pathogenic culprits and therapeutic allies.

Chronic kidney disease (CKD) is a significant health burden, with rising incidence and prevalence, attributed in part to increasing obesity and diabetes rates. Lipid accumulation in the kidney parenchyma and chronic, low-grade inflammation are believed to significantly contribute to the development and progression of CKD. The effect of dysregulated kidney lipid metabolism in CKD progression, including altered cholesterol and fatty acid metabolism contribute to glomerular and tubular cell injury through the activation of oxidative stress and inflammatory signalling cascades. In contrast, classes of endogenous specialized pro-resolving lipid mediators (SPMs) have been described that act to limit the inflammatory response and promote the resolution of inflammation. This review highlights our current understanding of how lipids can cause damage within the kidney, and classes of protective lipid metabolites that offer therapeutic benefits.

Impact of the local renin-angiotensin system in perivascular adipose tissue on vascular health and disease.

Perivascular adipose tissue (PVAT) is found locally around blood vessels. It has the ability to release vasoactive chemicals, such as factors that relax and contract blood vessels. PVAT is now recognized as an endocrine organ with metabolic activity and as a "protagonist" for maintaining vascular homeostasis. Angiotensin II, a powerful vasoconstrictor of the renin-angiotensin system (RAS) that can increase blood pressure and vascular tone, is produced locally by PVAT. To mitigate the multiple vascular effects of angiotensin II, PVAT also generates molecules such as angiotensin (1-7), adiponectin, and nitric oxide. Reactive oxygen species and proinflammatory cytokines are produced in greater amounts when PVAT-mediated angiotensin II is present, resulting in endothelial dysfunction, inflammation, atherosclerosis, and other vascular disorders. The anticontractile and procontractile nature of PVAT is frequently disrupted in obese individuals, which increases the production of angiotensin II and decreases the production of anti-inflammatory and vasodilatory factors. These changes in turn exacerbate vascular inflammation, hypertension, and atherosclerosis. PVAT, which is crucial for maintaining vascular homeostasis, loses its anticontractile effect in obesity due to adipocyte hypertrophy, inflammation, and oxidative stress, exacerbating endothelial dysfunction. Overactive RAS in PVAT facilitates the migration and proliferation of vascular smooth muscle cells and atherosclerotic plaques, both of which accelerate the development of atherosclerosis. Targeting PVAT and the local RAS can offer therapeutic benefits in treating hypertension, atherosclerosis, and other vascular diseases. This review highlights the scientific underpinnings of the function of PVAT in regulating the autocrine and paracrine activities of vascular RAS constituents.

The assessment of usefulness of cytokines and other soluble mediators as the predictors of sequalae development in various forms of tick-borne encephalitis (TBE).

AIM: The aim of the study was to assess the usefulness of cytokines and other soluble mediators in differentiation between severe and mild course of tick-borne encephalitis (TBE) as well as the predictor of sequalae development. MATERIAL AND METHODS: 122 patients (mean age 47.66 ± 14.77 years, 43 females, 79 males) with TBE were included in the study. Concentrations of 82 cytokines, growth factors, selectins, matrix metalloproteinases and other soluble mediators were measured in serum and CSF samples according to the manufacturer's instruction on a Bio-Plex 200 System using the custom made Luminex assays. Enzyme-linked immunosorbent assays for the quantitative detection of human IL-26, IL-29 IL-22, CXCL12 were performed. RESULTS: No significant differences between serum concentrations of examined factors between group with sequelae and group with complete recovery were observed. In the CSF the concentrations of GM-CSF, Il-1α, Il-2, Il-4, Il-6, Il-12p70, Il-17A, CXCL1, CXCL6, Il-8, CCL4, CCL20, TRAIL, CD40L, MMP8 were significantly higher in patients who developed sequelae than in patients with complete recovery. For TRAIL concentration over 26.65 pg/ml in CSF the probability of sequalae development was 10.5 higher. In case of CCL20 - the concentration over 21.38 pg/ml in CSF the odds ratio was 6.429 times. For MMP-8 over 4210.54 pg/ml, the odds ratio was 11.222 times. CONCLUSIONS: TRAIL, CCL-20 and MMP-8 are promising biomarkers of prediction of the sequalae development of TBE. Pro-inflammatory cytokines IL-8, IL-1, IL-2, IL-12, IL-17A also associate well with the risk of sequelae and could be further evaluated as prognostic markers in TBE, individually or as elements of a larger model.

Levels of matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, osteopontin, pentraxin-3, and thymic stromal lymphopoietin in crevicular fluid samples from peri-implantitis, periodontitis, and healthy sites.

AIM: Periodontitis and peri-implantitis are chronic inflammatory diseases characterized by the destruction of supporting tissues. Despite some similarities, it is essential to understand the differences in how these diseases elicit unique host responses within the oral tissues, including the production of selected matrix metalloproteinases (MMPs) and inflammatory mediators involved in tissue remodelling. The aim of this study was to evaluate the levels of proteolytic enzymes MMP-1, MMP-2, MMP-3, as well as the inflammatory mediators osteopontin (OPN), pentraxin-3 (PTX3), and thymic stromal lymphopoietin (TSLP) in crevicular fluid samples collected from healthy, periodontitis-affected, and peri-implantitis sites. METHODS: Gingival crevicular fluid (GCF) and peri-implant crevicular fluid (PICF) samples were collected from healthy and diseased teeth and implant sites of 163 patients. The MMP-1, MMP-2, MMP-3, OPN, PTX3, and TSLP levels were determined using commercially available immunoassays. A linear mixed model procedure was adopted for multilevel analyses, using biomarker levels as the outcome variable to compare two types of sites. The diagnostic accuracy of the biomarkers was evaluated by Youden's index to estimate the sensitivity, specificity and the area under curve (AUC). RESULTS: The levels of MMP-1, MMP-2, MMP-3, OPN, and TSLP were higher at sites with periodontitis and peri-implantitis compared to the levels at sites with healthy teeth and healthy implants. No significant differences were observed in the levels of the measured markers between the sites diagnosed with periodontitis and those diagnosed with peri-implantitis. The highest diagnostic potential at implant sites was found for MMP-2 (AUC = 0.74) and TSLP (AUC = 0.72). The highest AUC (0.82) at tooth sites was found for OPN. CONCLUSIONS: The findings indicate that the proteolytic enzyme MMP-2 and the cytokine TSLP might be potential biomarkers for both periodontitis and peri-implantitis, whereas the proinflammatory cytokine OPN may serve as a biomarker for periodontitis. Further studies are required to confirm the utility of these biomarkers and explore their potential clinical applications.

MASH: MEDIATION ANALYSIS OF SURVIVAL OUTCOME AND HIGH-DIMENSIONAL OMICS MEDIATORS WITH APPLICATION TO COMPLEX DISEASES.

Environmental exposures such as cigarette smoking influence health outcomes through intermediate molecular phenotypes, such as the methylome, transcriptome, and metabolome. Mediation analysis is a useful tool for investigating the role of potentially high-dimensional intermediate phenotypes in the relationship between environmental exposures and health outcomes. However, little work has been done on mediation analysis when the mediators are high-dimensional and the outcome is a survival endpoint, and none of it has provided a robust measure of total mediation effect. To this end, we propose an estimation procedure for Mediation Analysis of Survival outcome and High-dimensional omics mediators (MASH) based on sure independence screening for putative mediator variable selection and a second-moment-based measure of total mediation effect for survival data analogous to the R 2 measure in a linear model. Extensive simulations showed good performance of MASH in estimating the total mediation effect and identifying true mediators. By applying MASH to the metabolomics data of 1919 subjects in the Framingham Heart Study, we identified five metabolites as mediators of the effect of cigarette smoking on coronary heart disease risk (total mediation effect, 51.1%) and two metabolites as mediators between smoking and risk of cancer (total mediation effect, 50.7%). Application of MASH to a diffuse large B-cell lymphoma genomics data set identified copy-number variations for eight genes as mediators between the baseline International Prognostic Index score and overall survival.

COVID-19 Inflammatory Syndrome: Lessons from TNFRI and CRP about the Risk of Death in Severe Disease.

Background/Objectives: Cytokine storm in severe COVID-19 is responsible for irreversible tissue damage and death. Soluble mediators from the TNF superfamily, their correlation with clinical outcome, and the use of TNF receptors as a potent predictor for clinical outcome were evaluated. Methods: Severe COVID-19 patients had the levels of soluble mediators from the TNF superfamily quantified and categorized according to the clinical outcome (death versus survival). Statistical modeling was performed to predict clinical outcomes. Results: COVID-19 patients have elevated serum levels from the TNF superfamily. Regardless of sex and age, the sTNFRI levels were observed to be significantly higher in deceased patients from the first weeks following the onset of symptoms. We analyzed hematological parameters and inflammatory markers, and there was a difference between the groups for the following factors: erythrocytes, hemoglobin, hematocrit, leukocytes, neutrophils, band cells, lymphocytes, monocytes, CRP, IL-8, IFN-γ, IL-10, IL-6, IL-4, IL-2, leptin MIF sCD40L, and sTNFRI (p < 0.05). A post hoc analysis showed an inferential capacity over 70% for some hematological markers, CRP, and inflammatory mediators in deceased patients. sTNFRI was strongly associated with death, and the sTNFRI/sTNFRII ratio differed between outcomes (p < 0.001; power above 90%), highlighting the impact of these proteins on clinical results. The final logistic model, including sTNFRI/sTNFRII and CRP, indicated high sensitivity, specificity, accuracy, and an eight-fold higher odds ratio for an unfavorable outcome. Conclusions: The joint use of the sTNFRI/sTNFRII ratio with CRP proves to be a promising tool to assist in the clinical management of patients hospitalized for COVID-19.

Comprehensive time-course gene expression evaluation of high-risk beef cattle to establish immunological characteristics associated with undifferentiated bovine respiratory disease.

Bovine respiratory disease (BRD) remains the leading infectious disease in beef cattle production systems. Host gene expression upon facility arrival may indicate risk of BRD development and severity. However, a time-course approach would better define how BRD development influences immunological and inflammatory responses after disease occurrences. Here, we evaluated whole blood transcriptomes of high-risk beef cattle at three time points to elucidate BRD-associated host response. Sequenced jugular whole blood mRNA from 36 cattle (2015: n = 9; 2017: n = 27) across three time points (n = 100 samples; days [D]0, D28, and D63) were processed through ARS-UCD1.2 reference-guided assembly (HISAT2/Stringtie2). Samples were categorized into BRD-severity cohorts (Healthy, n = 14; Treated 1, n = 11; Treated 2+, n = 11) via frequency of antimicrobial clinical treatment. Assessment of gene expression patterns over time within each BRD cohort was modeled through an autoregressive hidden Markov model (EBSeq-HMM; posterior probability ≥ 0.5, FDR < 0.01). Mixed-effects negative binomial models (glmmSeq; FDR < 0.05) and edgeR (FDR < 0.10) identified differentially expressed genes between and across cohorts overtime. A total of 2,580, 2,216, and 2,381 genes were dynamically expressed across time in Healthy, Treated 1, and Treated 2+ cattle, respectively. Genes involved in the production of specialized resolving mediators (SPMs) decreased at D28 and then increased by D63 across all three cohorts. Accordingly, SPM production and alternative complement were differentially expressed between Healthy and Treated 2+ at D0, but not statistically different between the three groups by D63. Magnitude, but not directionality, of gene expression related to SPM production, alternative complement, and innate immune response signified Healthy and Treated 2+ cattle. Differences in gene expression at D63 across the three groups were related to oxygen binding and carrier activity, natural killer cell-mediated cytotoxicity, cathelicidin production, and neutrophil degranulation, possibly indicating prolonged airway pathology and inflammation weeks after clinical treatment for BRD. These findings indicate genomic mechanisms indicative of BRD development and severity over time.

Effects of Omega-3 Polyunsaturated Fatty Acids on the Formation of Adipokines, Cytokines, and Oxylipins in Retroperitoneal Adi-Pose Tissue of Mice.

Oxylipins and specialized pro-resolving lipid mediators (SPMs) derived from polyunsaturated fatty acids (PUFAs) are mediators that coordinate an active process of inflammation resolution. While these mediators have potential as circulating biomarkers for several disease states with inflammatory components, the source of plasma oxylipins/SPMs remains a matter of debate but may involve white adipose tissue (WAT). Here, we aimed to investigate to what extent high or low omega (n)-3 PUFA enrichment affects the production of cytokines and adipokines (RT-PCR), as well as oxylipins/SPMs (liquid chromatography-tandem mass spectrometry) in the WAT of mice during lipopolysaccharide (LPS)-induced systemic inflammation (intraperitoneal injection, 2.5 mg/kg, 24 h). For this purpose, n-3 PUFA genetically enriched mice (FAT-1), which endogenously synthesize n-3 PUFAs, were compared to wild-type mice (WT) and combined with n-3 PUFA-sufficient or deficient diets. LPS-induced systemic inflammation resulted in the decreased expression of most adipokines and interleukin-6 in WAT, whereas the n-3-sufficient diet increased them compared to the deficient diet. The n-6 PUFA arachidonic acid was decreased in WAT of FAT-1 mice, while n-3 derived PUFAs (eicosapentaenoic acid, docosahexaenoic acid) and their metabolites (oxylipins/SPMs) were increased in WAT by genetic and nutritional n-3 enrichment. Several oxylipins/SPMs were increased by LPS treatment in WAT compared to PBS-treated controls in genetically n-3 enriched FAT-1 mice. Overall, we show that WAT may significantly contribute to circulating oxylipin production. Moreover, n-3-sufficient or n-3-deficient diets alter adipokine production. The precise interplay between cytokines, adipokines, and oxylipins remains to be further investigated.

The Effect of Ionizing Irradiation on the Autotaxin-Lysophasphatidic Acid Axis and Interleukin-6/8 Secretion in Different Breast Cancer Cell Lines.

BACKGROUND: The Autotaxin (ATX)-lysophosphatidic acid (LPA) axis is involved in decreasing radiation sensitivity of breast tumor cells. This study aims to further elucidate the effect of irradiation on the ATX-LPA axis and cytokine secretion in different breast cancer cell lines to identify suitable breast cancer subtypes for targeted therapies. METHODS: Different breast cancer cell lines (MCF-7 (luminal A), BT-474 (luminal B), SKBR-3 (HER2-positive), MDA-MB-231 and MDA-MB-468 (triple-negative)) and the breast epithelial cell line MCF-10A were irradiated. The influence of irradiation on LPA receptor (LPAR) expression, ATX expression, and Interleukin (IL)-6 and IL-8 secretion was analyzed. Further, the effect of IL-6 and IL-8 on ATX expression of adipose-derived stem cells (ADSC) was investigated. RESULTS: Irradiation increased ATX and LPAR2 expression in MDA-MB-231 cells. Additionally, IL-6 secretion was enhanced in MDA-MB-231, and IL-8 secretion in MDA-MB-231 and MDA-MB-468. Stimulation of ADSC with IL-6 and IL-8 increased ATX expression in ADSC. CONCLUSIONS: Targeting ATX or its downstream signaling pathways might enhance the sensitivity of triple-negative breast cancer cells to radiation. Further exploration of the interplay between irradiation, the ATX-LPA axis, and inflammatory cytokines may elucidate novel pathways for overcoming radioresistance and improving individual treatment outcomes.

A Patent-Pending Ointment Containing Extracts of Five Different Plants Showed Antinociceptive and Anti-Inflammatory Mechanisms in Preclinical Studies.

Background/Objectives: The antinociceptive and anti-inflammatory effects of a patent-pending ointment containing plant extracts from Eucalyptus globulus, Curcuma longa, Hamamelis virginiana, Echinacea purpurea, and Zingiber officinale were evaluated. Methods: Plant extracts were chemically characterized by gas chromatography-mass spectroscopy. The antinociceptive activity of the ointment was assessed using the hot plate, tail flick, and formalin tests, whereas the anti-inflammatory activity was measured using the acute and chronic TPA-induced ear edema tests. Mechanisms of action were evaluated using inhibitors from signaling pathways related to pain response and by using histological analysis and assessing the expression and activity of pro-inflammatory mediators. Results: The ointment showed antinociceptive and anti-inflammatory effects like those observed with diclofenac gel (1.16% v/v) and ketoprofen gel (2.5% v/v). The antinociceptive actions of the ointment are mediated by the possible participation of the opiodergic system and the nitric oxide pathway. The anti-inflammatory response was characterized by a decrease in myeloperoxidase (MPO) activity and by a reduction in ear swelling and monocyte infiltration in the acute inflammation model. In the chronic model, the mechanism of action relied on a decrease in pro-inflammatory mediators such as COX-2, IL-1ß, TNF-α, and MPO. An in-silico study with myristic acid, one of the compounds identified in the ointment's plant mixture, corroborated the in vivo results. Conclusions: The ointment showed antinociceptive activities mediated by the decrease in COX-2 and NO levels, and anti-inflammatory activity due to the reduction in IL-1ß and TNFα levels, a reduction in MPO activity, and a decrease in NF-κB and COX-2 expression.