A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping.

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.

Hypertension alters the function and expression profile of the peptide cotransporters PEPT1 and PEPT2 in the rodent renal proximal tubule.

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, ß-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.

Neutrophil Extracellular Trap Density Increases With Increasing Histopathological Severity of Crohn's Disease.

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.

Our data show for the first time that the density of neutrophil extracellular trap formed in the bowel of Crohn's disease patients increases with increasing disease severity, suggesting that myeloperoxidase-mediated host-tissue damage may play a role in disease pathogenesis.

Cell-Projection Pumping of Fibroblast Contents into Osteosarcoma SAOS-2 Cells Correlates with Increased SAOS-2 Proliferation and Migration, as well as Altered Morphology.

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.

Efficacy of the Piperidine Nitroxide 4-MethoxyTEMPO in Ameliorating Serum Amyloid A-Mediated Vascular Inflammation.

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.

The Synthetic Myeloperoxidase Inhibitor AZD3241 Ameliorates Dextran Sodium Sulfate Stimulated Experimental Colitis.

Chronic inflammatory bowel disease (IBD) is a condition with multifactorial pathophysiology. To date, there is no permanent cure and the disease is primarily managed by immunosuppressive drugs; long-term use promotes serious side effects including increased risk malignancies. The current study aimed to target neutrophil-myeloperoxidase, a key contributor to the pathogenesis of IBD, through the use of AZD3241that inhibits extracellular myeloperoxidase. Experimental colitis was induced in C57BL/6 male mice by 2% dextran sodium sulfate in drinking water ad libitum over 9 days. Mice received either normal drinking water and peanut butter (control), 2% DSS in drinking water and peanut butter or 2% DSS in drinking water and AZD3241 (30 mg/kg) dispersed in peanut butter daily for 9 days. Administered AZD3241 attenuated body weight loss (10% p<0.05) and improved clinical score (9 fold p<0.05; a score comprising the time-dependent assessment of stool consistency and extent of rectal bleeding), loss of colonic crypts (p<0.001), preserved surface epithelium (p<0.001) and enhanced expression of the transcription factor Nrf-2 (regulator of antioxidants) and enhanced expression of the downstream antioxidant response element haeoxygenase-1 (HO-1) in the colon tissue. Also, the concentration of fecal hemoglobin and the myeloperoxidase specific oxidative damage biomarker 3-chlorotyrosine in the colon were significantly decreased in the presence of AZD3241. This latter result was consistent with AZD3241 inhibiting MPO activity in vitro. Overall, AZD3241 ameliorated the course and severity of experimental colitis through ameliorating MPO derived tissue damage and could be considered a potential therapeutic option, subject to further validation in chronic IBD models.

Nitroxides Mitigate Neutrophil-Mediated Damage to the Myocardium after Experimental Myocardial Infarction in Rats.

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.

The Role of Thiocyanate in Modulating Myeloperoxidase Activity during Disease.

Thiocyanate (SCN-) is a pseudohalide anion omnipresent across mammals and is particularly concentrated in secretions within the oral cavity, digestive tract and airway. Thiocyanate can outcompete chlorine anions and other halides (F-, Br-, I-) as substrates for myeloperoxidase by undergoing two-electron oxidation with hydrogen peroxide. This forms their respective hypohalous acids (HOX where X- = halides) and in the case of thiocyanate, hypothiocyanous acid (HOSCN), which is also a bactericidal oxidative species involved in the regulation of commensal and pathogenic microflora. Disease may dysregulate redox processes and cause imbalances in the oxidative profile, where typically favoured oxidative species, such as hypochlorous acid (HOCl), result in an overabundance of chlorinated protein residues. As such, the pharmacological capacity of thiocyanate has been recently investigated for its ability to modulate myeloperoxidase activity for HOSCN, a less potent species relative to HOCl, although outcomes vary significantly across different disease models. To date, most studies have focused on therapeutic effects in respiratory and cardiovascular animal models. However, we note other conditions such as rheumatic arthritis where SCN- administration may worsen patient outcomes. Here, we discuss the pathophysiological role of SCN- in diseases where MPO is implicated.

The role of sodium thiocyanate supplementation during dextran sodium sulphate-stimulated experimental colitis.

Ulcerative colitis is a condition characterised by the infiltration of leukocytes into the gastrointestinal wall. Leukocyte-MPO catalyses hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) formation from chloride (Cl-) and thiocyanous (SCN-) anions, respectively. While HOCl indiscriminately oxidises biomolecules, HOSCN primarily targets low-molecular weight protein thiols. Oxidative damage mediated by HOSCN may be reversible, potentially decreasing MPO-associated host tissue destruction. This study investigated the effect of SCN- supplementation in a model of acute colitis. Female mice were supplemented dextran sodium sulphate (DSS, 3% w/v) in the presence of 10 mM Cl- or SCN- in drinking water ad libitum, or with salts (NaCl and NaSCN only) or water only (controls). Behavioural studies showed mice tolerated NaSCN and NaCl-treated water with water-seeking frequency. Ion-exchange chromatography showed increased fecal and plasma SCN- levels in thiocyanate supplemented mice; plasma SCN- reached similar fold-increase for smokers. Overall there was no difference in weight loss and clinical score, mucin levels, crypt integrity and extent of cellular infiltration between DSS/SCN- and DSS/Cl- groups. Neutrophil recruitment remained unchanged in DSS-treated mice, as assessed by fecal calprotectin levels. Total thiol and tyrosine phosphatase activity remained unchanged between DSS/Cl- and DSS/SCN- groups, however, colonic tissue showed a trend in decreased 3-chlorotyrosine (1.5-fold reduction, p < 0.051) and marked increase in colonic GCLC, the rate-limiting enzyme in glutathione synthesis. These data suggest that SCN- administration can modulate MPO activity towards a HOSCN-specific pathway, however, this does not alter the development of colitis within a DSS murine model.

Neutrophil-Mediated Cardiac Damage After Acute Myocardial Infarction: Significance of Defining a New Target Cell Type for Developing Cardioprotective Drugs.

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.

Potential Hydrodynamic Cytoplasmic Transfer between Mammalian Cells: Cell-Projection Pumping.

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fibs) and malignant cells (MCs). Others report similar transfer via either tunneling nanotubes (TNTs) or shed membrane vesicles, and this changes the phenotype of recipient cells. Our time-lapse microscopy showed most exchange was from Fibs into MCs, with less in the reverse direction. Although TNTs were seen, we were surprised transfer was not via TNTs but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless by their organellar cargo and the grooves they formed indenting MCs, which was consistent with holotomography. Discrete, rapid, and highly localized transfer events evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell projections normally returns cytoplasm to the cell body. We hypothesize "cell-projection pumping" (CPP), in which cytoplasm in retracting cell projections partially equilibrates into adjacent recipient cells via microfusions that form temporary intercellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modeling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure toward least resistance. Predictions from the model were satisfied when Fibs were cocultured with MCs and fluorescence exchange was related to cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MCs or Fibs was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian intercellular cytoplasmic transfer and communication.

The nitroxide 4-methoxy-tempo inhibits the pathogenesis of dextran sodium sulfate-stimulated experimental colitis.

Inflammatory bowel disease (IBD) is a chronic condition characterised by leukocyte recruitment to the gut mucosa. Leukocyte myeloperoxidase (MPO) produces the two-electron oxidant hypochlorous acid (HOCl), damaging tissue and playing a role in cellular recruitment, thereby exacerbating gut injury. We tested whether the MPO-inhibitor, 4-Methoxy-TEMPO (MetT), ameliorates experimental IBD. Colitis was induced in C57BL/6 mice by 3% w/v dextran-sodium-sulfate (DSS) in drinking water ad libitum over 9-days with MetT (15 mg/kg; via i. p. injection) or vehicle control (10% v/v DMSO+90% v/v phosphate buffered saline) administered twice daily during DSS challenge. MetT attenuated body-weight loss (50%, p < 0.05, n = 6), improved clinical score (53%, p < 0.05, n = 6) and inhibited serum lipid peroxidation. Histopathological damage decreased markedly in MetT-treated mice, as judged by maintenance of crypt integrity, goblet cell density and decreased cellular infiltrate. Colonic Ly6C+, MPO-labelled cells and 3-chlorotyrosine (3-Cl-Tyr) decreased in MetT-treated mice, although biomarkers for nitrosative stress (3-nitro-tyrosine-tyrosine; 3-NO2-Tyr) and low-molecular weight thiol damage (assessed as glutathione sulfonamide; GSA) were unchanged. Interestingly, MetT did not significantly impact colonic IL-10 and IL-6 levels, suggesting a non-immunomodulatory pathway. Overall, MetT ameliorated the severity of experimental IBD, likely via a mechanism involving the modulation of MPO-mediated damage.

Increased cell size, structural complexity and migration of cancer cells acquiring fibroblast organelles by cell-projection pumping.

We recently described a hydrodynamic mechanism for cytoplasmic transfer between cells, termed cell-projection pumping (CPP). Earlier image analysis related altered SAOS-2 osteosarcoma cell morphology, to what we now recognize as CPP uptake of fibroblast cytoplasm. We here examine SAOS-2 phenotype following co-culture with human dermal fibroblasts (HDF) in which organelles were pre-labelled with a fluorescent lipophilic marker. Fluorescence activated cell sorting (FACS) analysis was performed of HDF and SAOS-2, cultured either alone or together. FACS forward scatter is proportionate to cell size, and increased for SAOS-2 with high levels of HDF fluorescence uptake (p < 0.004). FACS side scatter is proportionate to internal cell complexity, and increased in SAOS-2 with increasing uptake of HDF fluorescence (p < 0.004), consistent with uptake of HDF organelles. Scratch migration assays revealed that HDF migrated more quickly than SAOS-2 in both isolated cell culture, and following co-culture (p < 0.004). Notably, SAOS-2 with high levels of HDF labelling migrated faster compared with SAOS-2 with low HDF labelling (p < 0.008). A slight and unconvincing reduction in SAOS-2 proliferation was seen (p < 0.02). Similar results were obtained in single additional experiments with A673 and H312 cancer cells. Forward and side scatter results suggest organellar transfer by CPP increases cancer cell morphological diversity. This may contribute to histological pleomorphism relevant to cancer diagnosis and prognosis. Also, increased migration of sub-populations of cancer cells with high CPP organellar uptake, may contribute to invasion and metastasis in-vivo. We thus suggest relevance of CPP to cancer diagnosis and progression.

Catalase-Like Antioxidant Activity is Unaltered in Hypochlorous Acid Oxidized Horse Heart Myoglobin.

Activated neutrophils release myeloperoxidase that produces the potent oxidant hypochlorous acid (HOCl). Exposure of the oxygen transport protein horse heart myoglobin (hhMb) to HOCl inhibits Iron III (Fe(III))-heme reduction by cytochrome b5 to oxygen-binding Iron II (Fe(II))Mb. Pathological concentrations of HOCl yielded myoglobin oxidation products of increased electrophoretic mobility and markedly different UV/Vis absorbance. Mass analysis indicated HOCl caused successive mass increases of 16 a.m.u., consistent serial addition of molecular oxygen to the protein. By contrast, parallel analysis of protein chlorination by quantitative mass spectrometry revealed a comparatively minor increase in the 3-chlorotyrosine/tyrosine ratio. Pre-treatment of hhMb with HOCl affected the peroxidase reaction between the hemoprotein and H2O2 as judged by a HOCl dose-dependent decrease in spin-trapped tyrosyl radical detected by electron paramagnetic resonance (EPR) spectroscopy and the rate constant of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) oxidation. By contrast, Mb catalase-like antioxidant activity remained unchanged under the same conditions. Notably, HOCl-modification of Mb decreased the rate of ferric-to-ferrous Mb reduction by a cytochrome b5 reductase system. Taken together, these data indicate oxidizing HOCl promotes Mb oxidation but not chlorination and that oxidized Mb shows altered Mb peroxidase-like activity and diminished rates of one-electron reduction by cytochrome b5 reductase, possibly affecting oxygen storage and transport however, Mb-catalase-like antioxidant activity remains unchanged.

Serum Amyloid A Stimulates Vascular and Renal Dysfunction in Apolipoprotein E-Deficient Mice Fed a Normal Chow Diet.

Elevated serum amyloid A (SAA) levels may promote endothelial dysfunction, which is linked to cardiovascular and renal pathologies. We investigated the effect of SAA on vascular and renal function in apolipoprotein E-deficient (ApoE-/-) mice. Male ApoE-/- mice received vehicle (control), low-level lipopolysaccharide (LPS), or recombinant human SAA by i.p. injection every third day for 2 weeks. Heart, aorta and kidney were harvested between 3 days and 18 weeks after treatment. SAA administration increased vascular cell adhesion molecule (VCAM)-1 expression and circulating monocyte chemotactic protein (MCP)-1 and decreased aortic cyclic guanosine monophosphate (cGMP), consistent with SAA inhibiting nitric oxide bioactivity. In addition, binding of labeled leukocytes to excised aorta increased as monitored using an ex vivo leukocyte adhesion assay. Renal injury was evident 4 weeks after commencement of SAA treatment, manifesting as increased plasma urea, urinary protein, oxidized lipids, urinary kidney injury molecule (KIM)-1 and multiple cytokines and chemokines in kidney tissue, relative to controls. Phosphorylation of nuclear-factor-kappa-beta (NFκB-p-P65), tissue factor (TF), and macrophage recruitment increased in kidneys from ApoE-/- mice 4 weeks after SAA treatment, confirming that SAA elicited a pro-inflammatory and pro-thrombotic phenotype. These data indicate that SAA impairs endothelial and renal function in ApoE-/- mice in the absence of a high-fat diet.

The cyclic nitroxide antioxidant 4-methoxy-TEMPO decreases mycobacterial burden in vivo through host and bacterial targets.

Tuberculosis is a chronic inflammatory disease caused by persistent infection with Mycobacterium tuberculosis. The rise of antibiotic resistant strains necessitates the design of novel treatments. Recent evidence shows that not only is M. tuberculosis highly resistant to oxidative killing, it also co-opts host oxidant production to induce phagocyte death facilitating bacterial dissemination. We have targeted this redox environment with the cyclic nitroxide derivative 4-methoxy-TEMPO (MetT) in the zebrafish-M. marinum infection model. MetT inhibited the production of mitochondrial ROS and decreased infection-induced cell death to aid containment of infection. We identify a second mechanism of action whereby stress conditions, including hypoxia, found in the infection microenvironment appear to sensitise M. marinum to killing by MetT both in vitro and in vivo. Together, our study demonstrates MetT inhibited the growth and dissemination of M. marinum through host and bacterial targets.

Molecular changes to the rat renal cotransporters PEPT1 and PEPT2 due to ageing.

Renal PEPT1 and PEPT2 cotransporters play an important role in the balance of circulating body oligopeptides and selected peptidomimetic drugs. We aim to comprehensively characterise age-related changes of the renal PEPT cotransporters at the gene, protein, and functional level. Brush border membrane vesicles (BBMV) and outer medulla membrane vesicles (OMMV) were isolated from the kidneys of young, middle-aged and old rats. The protein expression of PEPT1 was not only increased in BBMV from old rats, but PEPT1 also appeared in OMMV from middle-aged and old rats. SLC15A1 gene expression in the renal cortex increased in middle-aged group. PEPT2 protein expression was not only increased with ageing, but PEPT2 also was found in BBMV from middle-aged and old groups. SLC15A2 gene expression in the renal outer medulla increased in the old group. These changes in the expressions and localisations of PEPT1 and PEPT2 could explain the changes to transport activity in BBMV and OMMV. These findings provide novel insights that would be useful for maintaining protein nutrition and optimising the delivery of some peptidomimetic drugs in elderly individuals.