Background Osteosarcoma progression and treatment resistance are strongly influenced by the tumour microenvironment. One of the key players in tumour microenvironments is cancer-associated fibroblast. Much of the conventional cancer-associated fibroblast-centred research paradigm focuses on the secretory and matrix-remodelling aspects of fibroblasts. However, less is known on how fibroblasts influence osteosarcoma cell behaviour, particular under stress. Methods Human dermal fibroblasts and osteosarcoma cells were studied using direct coculture and size-restricted transwell systems. Fluorescent labelling, confocal microscopy, live-cell imaging, and single-cell tracking were used to assess intercellular transfer, migratory behaviour, proliferation, and survival under chemotherapy and ischemic challenge. Results Osteosarcoma cells acquired fibroblast-derived, mitochondria cargo-enriched material in both direct and indirect coculture systems, with transfer of material enhanced under carboplatin and ischemic stress. Fibroblast coculture increased osteosarcoma cell migration and promoted survival under stress but did not substantially increase basal proliferation. Conclusions These findings identify direct fibroblast-to-cancer cell transfer of mitochondria-enriched cargo as an underappreciated mechanism of tumour–stroma communication in osteosarcoma. Stress-amplified transfer may support tumour adaptation, chemoresistance, and metastatic behaviour, highlighting stromal intercellular communication as a potential therapeutic target.

Osteosarcoma progression and treatment resistance are strongly influenced by the tumour microenvironment. One of the key players in tumour microenvironments is cancer-associated fibroblast. Much of the conventional cancer-associated fibroblast-centred research paradigm focuses on the secretory and matrix-remodelling aspects of fibroblasts. However, less is known on how fibroblasts influence osteosarcoma cell behaviour, particular under stress. In a proof-of-concept study, human dermal fibroblasts and osteosarcoma cells were studied using direct coculture and size-restricted transwell systems. Fluorescent labelling, confocal microscopy, live-cell imaging, and single-cell tracking were used to assess intercellular transfer, migratory behaviour, proliferation, and survival under chemotherapy and hypoxic challenge. Osteosarcoma cells acquired fibroblast-derived, mitochondria cargo-enriched material in both direct and indirect coculture systems, with transfer of material enhanced under carboplatin and hypoxic stress. Notably, fibroblast coculture increased osteosarcoma cell migration and promoted survival under stress but did not substantially increase basal proliferation. These findings suggest that fibroblast-to-cancer cell transfer of mitochondria-enriched cargo is accelerated during stress conditions with the concomitant observation of proportional osteosarcoma protection to degree of fibroblast cargo transfer. Further research should examine stress-amplified transfer during tumour adaptation, chemoresistance, and metastatic behaviour, potentially highlighting stromal intercellular communication as an important aspect of osteosarcoma biology.

Inflammatory Bowel Disease (IBD), including ulcerative colitis (UC), requires non-invasive, convenient and cost-effective biomarkers for disease monitoring. Myeloperoxidase (MPO), a neutrophil-derived enzyme, correlates with intestinal inflammation. This study aims to assess the Myeloperoxidase Luminol Reaction (MPOLR) assay - a novel chemiluminescent-based method for detecting faecal MPO (fMPO) activity, to improve the prediction of endoscopic findings in UC. A cross-sectional clinical study recruited 39 participants, categorized into UC (n = 20), colonoscopy control (CC; n = 9), and healthy control (HC; n = 10). Faecal samples are analysed for fMPO, calprotectin (fCalpro), and lactoferrin (fLacto) using ELISA and the MPOLR assay. The MPOLR assay is validated against endoscopic disease severity (UCEIS), symptomology, and clinical indices. MPOLR strongly correlated with UCEIS (ρ = 0.78), disease severity (ρ = 0.75), and symptomology (ρ = 0.78), outperforming fCalpro (ρ = 0.58, ρ = 0.57, and ρ = 0.41, respectively). AUROC analysis reveals MPOLR (0.78) and fMPO (0.75) have superior predictive potential for UC diagnosis compared to fCalpro (0.66). MPOLR is a rapid, cost-effective assay that enhances UC monitoring by accurately reflecting endoscopic findings and outcompetes fCalpro and other faecal biomarkers' predictive potential for UC diagnosis.

Areca nut chewing is a culturally significant practice shown to cause Oral Submucous Fibrosis (OSMF), which has a high potential for morbidity and malignancy. We postulate that arecoline upregulates production of pro-fibrotic cytokines, IL-33 and IL-13 and aimed to assess both IL-33/IL-13 expression and extracellularisation during arecoline treatment in human gingival fibroblasts (HGF) and mast cells, respectively. Primary HGF cells were exposed to increasing concentrations of arecoline (0 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL) under serum starvation conditions for 16 h. Co-culture transwell systems were used to examine arecoline-induced synergic activation of mast cells. We observed that 100 μg/mL and 200 μg/mL of arecoline is sufficient to induce significant levels of cell toxicity and death, with concurrent release of both pro-fibrotic factor, CTGF and IL-33 from HGF cells and IL-13 from mast cells. We are the first to report that arecoline induces release of IL-13, mast cell degranulation, and increased soluble CTGF. In conclusion, arecoline induces IL-33, IL-13 and CTGF release in HGF and mast cells, highlighting the importance of the IL-33/IL-13 axis in the progressive fibrosis of OSMF. These findings could support the approach of novel therapies targeting the IL-33/IL-13 axis for patients with OSMF.

Neutrophil-myeloperoxidase (MPO) is a heme-containing peroxidase which produces excess amounts of hypochlorous acid during inflammation. While pharmacological MPO inhibition mitigates all indices of experimental colitis, no studies have corroborated the role of MPO using knockout (KO) models. Therefore, we investigated MPO deficient mice in a murine model of colitis. Wild type (Wt) and MPO-deficient mice were treated with dextran sodium sulphate (DSS) in a chronic model of experimental colitis with three acute cycles of DSS-induced colitis over 63 days, emulating IBD relapse and remission cycles. Mice were immunologically profiled at the gut muscoa and the faecal microbiome was assessed via 16S rRNA amplicon sequencing. Contrary to previous pharmacological antagonist studies targeting MPO, MPO-deficient mice showed no protection from experimental colitis during cyclical DSS-challenge. We are the first to report drastic faecal microbiota shifts in MPO-deficient mice, showing a significantly different microbiome profile on Day 1 of treatment, with a similar shift and distinction on Day 29 (half-way point), via qualitative and quantitative descriptions of phylogenetic distances. Herein, we provide the first evidence of substantial microbiome shifts in MPO-deficiency, which may influence disease progression. Our findings have significant implications for the utility of MPO-KO mice in investigating disease models.

Journal Article Low-density Granulocytes as a Novel Biomarkers of Disease Activity in IBD Get access Nannan Zhu, MS, Nannan Zhu, MS Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University, Anhui Province, 230022, China https://orcid.org/0009-0009-5718-7877 Search for other works by this author on: Oxford Academic PubMed Google Scholar Jiejie Zhu, PhD, Jiejie Zhu, PhD Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University, Anhui Province, 230022, China Search for other works by this author on: Oxford Academic PubMed Google Scholar Qiao Mei, PhD Qiao Mei, PhD Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University, Anhui Province, 230022, China Address Correspondence to: Qiao Mei MD, PhD, Department of Gastroenterology, the First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Hefei, Anhui Province, 230022, China (meiqiao@hotmail.com). https://orcid.org/0000-0002-0635-6564 Search for other works by this author on: Oxford Academic PubMed Google Scholar Inflammatory Bowel Diseases, Volume 29, Issue 8, August 2023, Page e31, https://doi.org/10.1093/ibd/izad136 Published: 01 August 2023 Article history Corrected and typeset: 01 August 2023 Published: 01 August 2023

Hypertension is a major risk factor for kidney and cardiovascular disease. The treatment of hypertensive individuals by selected ACE inhibitors and certain di-and tripeptides halts the progression of renal deterioration and extends life-span. Renal reabsorption of these low molecular weight substrates are mediated by the PEPT1 and PEPT2 cotransporters. This study aims to investigate whether hypertension and ageing affects renal PEPT cotransporters at gene, protein expression and distribution as well as function in the superficial cortex and the outer medulla of the kidney. Membrane vesicles from the brush border (BBMV) and outer medulla (OMMV) were isolated from the kidneys of young Wistar Kyoto (Y-WKY), young spontaneously hypertensive (Y-SHR), and middle aged SHR (M-SHR) rats. Transport activity was measured using the substrate, β-Ala-Lys (AMCA). Gene expression levels of PEPT genes were assessed with qRT-PCR while renal localisation of PEPT cotransporters was examined by immunohistochemistry with Western Blot validation. The Km and Vmax of renal PEPT1 were decreased significantly in SHR compared to WKY BBMV, whilst the Vmax of PEPT2 showed differences between SHR and WKY. By contrast to the reported cortical distribution of PEPT1, PEPT1-staining was detected in the outer medulla, whilst PEPT2 was expressed primarily in the cortex of all SHR; PEPT1 was significantly upregulated in the cortex of Y-SHR. These outcomes are indicative of a redistribution of PEPT1 and PEPT2 in the kidney proximal tubule under hypertensive conditions that has potential repercussions for nutrient handling and the therapeutic use of ACE inhibitors in hypertensive individuals.

Abstract Background Intestinal neutrophil recruitment is a characteristic feature of the earliest stages of inflammatory bowel disease (IBD). Neutrophil elastase (NE) and myeloperoxidase (MPO) mediate the formation of neutrophil extracellular traps (NETs); NETs produce the bactericidal oxidant hypochlorous acid (HOCl), causing host tissue damage when unregulated. The project aim was to investigate the relationship between NET formation and clinical IBD in humans. Methods Human intestinal biopsies were collected from Crohn’s disease (CD) patients, endoscopically categorized as unaffected, transitional, or diseased, and assigned a histopathological score. Results A significant linear correlation was identified between pathological score and cell viability (TUNEL+). Immunohistochemical analysis revealed the presence of NET markers NE, MPO, and citrullinated histone (CitH3) that increased significantly with increasing histopathological score. Diseased specimens showed greater MPO+-immunostaining than control (P < .0001) and unaffected CD (P < .0001), with transitional CD specimens also showing greater staining than controls (P < .05) and unaffected CD (P < .05). Similarly, NE+-immunostaining was elevated significantly in diseased CD than controls (P < .0001) and unaffected CD (P < .0001) and was significantly higher in transitional CD than in controls (P < .0001) and unaffected CD (P < .0001). The CitH3+-immunostaining of diseased CD was significantly higher than controls (P < .05), unaffected CD (P < .0001) and transitional CD (P < .05), with transitional CD specimens showing greater staining than unaffected CD (P < .01). Multiplex immunohistochemistry with z-stacking revealed colocalization of NE, MPO, CitH3, and DAPI (cell nuclei), confirming the NET assignment. Conclusion These data indicate an association between increased NET formation and CD severity, potentially due to excessive MPO-mediated HOCl production in the extracellular domain, causing host tissue damage that exacerbates CD.

We recently described cell-projection pumping as a mechanism transferring cytoplasm between cells. The uptake of fibroblast cytoplasm by co-cultured SAOS-2 osteosarcoma cells changes SAOS-2 morphology and increases cell migration and proliferation, as seen by single-cell tracking and in FACS separated SAOS-2 from co-cultures. Morphological changes in SAOS-2 seen by single cell tracking are consistent with previous observations in fixed monolayers of SAOS-2 co-cultures. Notably, earlier studies with fixed co-cultures were limited by the absence of a quantitative method for identifying sub-populations of co-cultured cells, or for quantitating transfer relative to control populations of SAOS-2 or fibroblasts cultured alone. We now overcome that limitation by a novel Cartesian plot analysis that identifies individual co-cultured cells as belonging to one of five distinct cell populations, and also gives numerical measure of similarity to control cell populations. We verified the utility of the method by first confirming the previously established relationship between SAOS-2 morphology and uptake of fibroblast contents, and also demonstrated similar effects in other cancer cell lines including from melanomas, and cancers of the ovary and colon. The method was extended to examine global DNA methylation, and while there was no clear effect on SAOS-2 DNA methylation, co-cultured fibroblasts had greatly reduced DNA methylation, similar to cancer associated fibroblasts.

The pathophysiology of inflammatory bowel disease (IBD) remains poorly understood and treatment remain suboptimal for many patients. Furthermore, many of the available treatment options for IBD exhibit severe immune suppressive side effects that impact lifestyle and predispose individuals to opportunistic infection. Thus, studies that identify novel and effective therapeutic options that preserve immune homeostasis are urgently required to address this shortfall in patient treatment options. There is some potential for the inflammatory milieu in bowel tissue to prolong the injury and slow beneficial processes linked to wound healing in the affected bowel. Therefore, primary or adjuvant therapies that restore the gut barrier and provide protection to colonic cells in underlying submucosal structures represent a useful approach to finding new treatments for IBD; this is where innovative stem cell therapies have significant potential to lead therapeutic development.

Intracellular redox imbalance in endothelial cells (EC) can lead to endothelial dysfunction, which underpins cardiovascular diseases (CVD). The acute phase serum amyloid A (SAA) elicits inflammation through stimulating production of reactive oxygen species (ROS). The cyclic nitroxide 4-MethoxyTEMPO (4-MetT) is a superoxide dismutase mimetic that suppresses oxidant formation and inflammation. The aim of this study was to investigate whether 4-MetT inhibits SAA-mediated activation of cultured primary human aortic EC (HAEC). Co-incubating cells with 4-MetT inhibited SAA-mediated increases in adhesion molecules (VCAM-1, ICAM-1, E-selectin, and JAM-C). Pre-treatment of cells with 4-MetT mitigated SAA-mediated increases in transcriptionally activated NF-κB-p65 and P120 Catenin (a stabilizer of Cadherin expression). Mitochondrial respiration and ROS generation (mtROS) were adversely affected by SAA with decreased respiratory reserve capacity, elevated maximal respiration and proton leakage all characteristic of SAA-treated HAEC. This altered respiration manifested as a loss of mitochondrial membrane potential (confirmed by a decrease in TMRM fluorescence), and increased mtROS production as assessed with MitoSox Red. These SAA-linked impacts on mitochondria were mitigated by 4-MetT resulting in restoration of HAEC nitric oxide bioavailability as confirmed by assessing cyclic guanosine monophosphate (cGMP) levels. Thus, 4-MetT ameliorates SAA-mediated endothelial dysfunction through normalising EC redox homeostasis. Subject to further validation in in vivo settings; these outcomes suggest its potential as a therapeutic in the setting of cardiovascular pathologies where elevated SAA and endothelial dysfunction is linked to enhanced CVD.

We earlier reported that cell-projection pumping transfers fibroblast contents to cancer cells and this alters the cancer cell phenotype. Here, we report on single-cell tracking of time lapse recordings from co-cultured fluorescent fibroblasts and SAOS-2 osteosarcoma cells, tracking 5201 cells across 7 experiments. The fluorescent lipophilic marker DiD was used to label fibroblast organelles and to trace the transfer of fibroblast cytoplasm into SAOS-2 cells. We related SAOS-2 phenotypic change to levels of fluorescence transfer from fibroblasts to SAOS-2 cells, as well as what we term ‘compensated fluorescence’, that numerically projects mother cell fluorescence post-mitosis into daughter cells. The comparison of absolute with compensated fluorescence allowed us to deduct if the phenotypic effects in mother SAOS-2 cells were inherited by their daughters. SAOS-2 receipt of fibroblast fluorescence correlated by Kendall’s tau with cell-profile area and without evidence of persistence in daughter cells (median tau = 0.51, p < 0.016); negatively and weakly with cell circularity and with evidence of persistence (median tau = −0.19, p < 0.05); and very weakly with cell migration velocity and without evidence of persistence (median tau = 0.01, p < 0.016). In addition, mitotic SAOS-2 cells had higher rates of prior fluorescence uptake (median = 64.9 units/day) than non-dividing cells (median = 35.6 units/day, p < 0.016) and there was no evidence of persistence post-mitosis. We conclude that there was an appreciable impact of cell-projection pumping on cancer cell phenotype relevant to cancer histopathological diagnosis, clinical spread and growth, with most effects being ‘reset’ by cancer cell mitosis.

Nitroxides are a diverse range of stable free radicals with applications in industrial polarisation processes and imaging contrasting, and over the last 30 years mounting evidence has demonstrated their therapeutic potential in mitigating diseases where oxidative damage is implicated. This is due to their radical oxidative/antioxidant duality and ability to readily cross cell membranes. In biological tissues, nitroxides are balanced between radical and antioxidant states depending on reducing or hypoxic conditions. For example, in cancer biology where conditions are typically hypoxic, the nitroxide radical induces further oxidative stress in metabolically stressed cancer cells and induces cell death, whereas nitroxides are reduced to a non-radical hydroxylamine antioxidant form in healthy cells, thereby conferring cytoprotection from radiotherapy. Nitroxides-derived antioxidant activities have also been described in terms of superoxide dismutase mimetics and inhibitors of hypochlorous acid production from neutrophil-derived myeloperoxidase during inflammatory insults. As such, nitroxides have potential applications in chronic inflammatory disorders by abrogating the production of more powerful oxidants which mediate tissue injury and cell death. Although nitroxides typically have a short half-life, recent developments to sterically stabilised nitroxides have seen biological improvements in their half-life and subsequent efficacies. Further structural variations with altered side-chain properties offer subcellular delivery to improve the bio-activity of nitroxides, thereby offering major new opportunities to exploit both the radical and antioxidant redox activity of nitroxides for therapeutic advantage in a wide range of human disorders.

Neutrophils are short-lived immune cells that represent the major cell type recruited to the inflamed bowel releasing their azurophilic granules containing enzymes myeloperoxidase (MPO). Fecal and serum MPO levels has previously been shown to correlate to disease severity in IBD patients. MPO, in the presence of H2O2 and free Cl- undergoes a halogenation cycle, yielding the two-electron oxidant, hypochlorous acid (HOCl) - a potent bactericidal agent. However, chronic intestinal exposure to MPO/HOCl due to perpetual inflammation may cause secondary host-tissue injury and cell death. Neutrophil Extracellular Trap (NET)osis is a specialised form of neutrophil death where MPO is entrapped in a DNA scaffold and continues to elicit HOCl activity and may further contribute to host-tissue injury. We investigated the presence of NETs in surgically excised ileum samples from CD and healthy patients using advanced confocal microscopic techniques and found MPO, Neutrophil Elastase (NE) and Citrullinated Histone h3 (CitH3) - critical components of NET formation, individually positively correlate to the severity of histopathological intestinal injury. Furthermore, multiplex Opal™ IHC performed using LMS880 Airyscan-moduled microscopy with z-stacking revealed colocalization of NE, MPO, CitH3 and DAPI indicating the extensive presence of NETs in severely affected CD tissue. Using two pharmacological inhibitors of MPO in a dextran sodium sulphate (DSS) model of murine colitis, we demonstrated the pathological role of MPO in experimental colitis. MPO inhibitors, TEMPOL and AZD3241 delivered via daily i.p significantly rescued the course of colitis by abrogating clinical indices including body weight loss, disease activity index, inhibiting serum peroxidation, and preserving colon length, while significantly mitigating histoarchitectural damage associated with DSS-induced colitis. We also showed that MPO inhibition decreased neutrophil migration to the gut, suggesting MPO may play a role in perpetuating the inflammatory cell by further recruiting cells to the inflamed gut. Collectively, we have shown for the first time that MPO is not only an important clinical marker of disease severity but may also play a critical role in perpetuating host-tissue damage and inflammation.

Reperfusion therapy increases survival post-acute myocardial infarction (AMI) while also stimulating secondary oxidant production and immune cell infiltration. Neutrophils accumulate within infarcted myocardium within 24 h post-AMI and release myeloperoxidase (MPO) that catalyses hypochlorous acid (HOCl) production while increasing oxidative stress and inflammation, thereby enhancing ventricular remodelling. Nitroxides inhibit MPO-mediated HOCl production, potentially ameliorating neutrophil-mediated damage. Aim: Assess the cardioprotective ability of nitroxide 4-methoxyTEMPO (4MetT) within the setting of AMI. Methods: Male Wistar rats were separated into 3 groups: SHAM, AMI/R, and AMI/R + 4MetT (15 mg/kg at surgery via oral gavage) and subjected to left descending coronary artery ligation for 30 min to generate an AMI, followed by reperfusion. One cohort of rats were sacrificed at 24 h post-reperfusion and another 28 days post-surgery (with 4MetT (15 mg/kg) administration twice daily). Results: 3-chlorotyrosine, a HOCl-specific damage marker, decreased within the heart of animals in the AMI/R + 4-MetT group 24 h post-AMI, indicating the drug inhibited MPO activity; however, there was no evident difference in either infarct size or myocardial scar size between the groups. Concurrently, MPO, NfκB, TNFα, and the oxidation marker malondialdehyde increased within the hearts, with 4-MetT only demonstrating a trend in decreasing MPO and TNF levels. Notably, 4MetT provided a significant improvement in cardiac function 28 days post-AMI, as assessed by echocardiography, indicating potential for 4-MetT as a treatment option, although the precise mechanism of action of the compound remains unclear.

Significance: Acute myocardial infarction (AMI) is a leading cause of death worldwide. Post-AMI survival rates have increased with the introduction of angioplasty as a primary coronary intervention. However, reperfusion after angioplasty represents a clinical paradox, restoring blood flow to the ischemic myocardium while simultaneously inducing ion and metabolic imbalances that stimulate immune cell recruitment and activation, mitochondrial dysfunction and damaging oxidant production. Recent Advances: Preclinical data indicate that these metabolic imbalances contribute to subsequent heart failure through sustaining local recruitment of inflammatory leukocytes and oxidative stress, cardiomyocyte death, and coronary microvascular disturbances, which enhance adverse cardiac remodeling. Both left ventricular dysfunction and heart failure are strongly linked to inflammation and immune cell recruitment to the damaged myocardium. Critical Issues: Overall, therapeutic anti-inflammatory and antioxidant agents identified in preclinical trials have failed in clinical trials. Future Directions: The versatile neutrophil-derived heme enzyme, myeloperoxidase (MPO), is gaining attention as an important oxidative mediator of reperfusion injury, vascular dysfunction, adverse ventricular remodeling, and atrial fibrillation. Accordingly, there is interest in therapeutically targeting neutrophils and MPO activity in the setting of heart failure. Herein, we discuss the role of post-AMI inflammation linked to myocardial damage and heart failure, describe previous trials targeting inflammation and oxidative stress post-AMI, highlight the potential adverse impact of neutrophil and MPO, and detail therapeutic options available to target MPO clinically in AMI patients.