Polyphenolic plant extracts have demonstrated anti-inflammatory and anti-catabolic effects in vitro, however their meaningful translation into humans remains elusive. Urolithin A (UA), a gut-derived metabolite of ellagitannins, has shown promise for improving muscle function and metabolic health in rodent models. This study aimed to explore the impact of UA on insulin and anabolic sensitivity in human skeletal muscle cells. Primary human myogenic cultures were derived from skeletal muscle biopsies of eight healthy adults. After differentiation, myotubes were treated with 0.002, 1 and 50 µM UA or vehicle for 24 h. Cell viability was assessed using a resazurin assay. Basal and insulin-stimulated glucose uptake was measured using tritiated deoxy-D-glucose, whilst amino acid-stimulated protein synthesis was estimated using the surface sensing of translation (SuNSET) technique. Expression of myostatin and glucose transporters was quantified via real-time PCR. UA treatment at ≤ 50 µM did not compromise cell viability. Treatment with 50 µM UA enhanced both basal- and insulin-stimulated glucose uptake by 21

Non-alcoholic fatty liver disease (NAFLD) begins with lipid accumulation within hepatocytes, but the relative contributions of different macronutrients is still unclear. We investigated the impact of fatty acids, glucose and fructose on lipid accumulation in primary human hepatocytes (PHH) and three different cell lines: HepG2 (human hepatoblastoma–derived cell line), Huh7 (human hepatocellular carcinoma cell line) and McA-RH7777 (McA, rat hepatocellular carcinoma cell line). Cells were treated for 48 h with fatty acids (0 or 200 μM), glucose (5 mM or 11 mM) and fructose (0 mM, 2 mM or 8 mM). Lipid accumulation was measured via Nile Red staining. All cell types accumulated lipid in response to fatty acids (p < 0.001). PHH and McA, but not HepG2 or Huh7 cells, accumulated more lipid with 11 mM glucose plus fatty acids (p = 0.004, fatty acid × glucose interaction, for both), but only PHH increased lipid accumulation in response to fructose (p < 0.001). Considerable variation was observed between PHH cells from different individuals. Lipid accumulation in PHH was increased by insulin (p = 0.003) with inter-individual variability. Similarly, insulin increased lipid accumulation in both HepG2 and McA cells, with a bigger response in McA in the presence of fatty acids (p < 0.001 for fatty acid × insulin). McA were more insulin sensitive than either HepG2 or Huh7 cells in terms of AKT phosphorylation (p < 0.001 insulin × cell type interaction). Hence, glucose and fructose can contribute to the accumulation of lipid in PHH with considerable inter-individual variation, but hepatoma cell lines are not good models of PHH.

Myostatin negatively regulates skeletal muscle growth and appears upregulated in human obesity and associated with insulin resistance. However, observations are confounded by ageing, and the mechanisms responsible are unknown. The aim of this study was to delineate between the effects of excess adiposity, insulin resistance and ageing on myostatin mRNA expression in human skeletal muscle and to investigate causative factors using in vitro models. An in vivo cross-sectional analysis of human skeletal muscle was undertaken to isolate effects of excess adiposity and ageing per se on myostatin expression. In vitro studies employed human primary myotubes to investigate the potential involvement of cross-talk between subcutaneous adipose tissue (SAT) and skeletal muscle, and lipid-induced insulin resistance. Skeletal muscle myostatin mRNA expression was greater in aged adults with excess adiposity than age-matched adults with normal adiposity (2.0-fold higher; P < 0.05) and occurred concurrently with altered expression of genes involved in the maintenance of muscle mass but did not differ between younger and aged adults with normal adiposity. Neither chronic exposure to obese SAT secretome nor acute elevation of fatty acid availability (which induced insulin resistance) replicated the obesity-mediated upregulation of myostatin mRNA expression in vitro. In conclusion, skeletal muscle myostatin mRNA expression is uniquely upregulated in aged adults with excess adiposity and insulin resistance but not by ageing alone. This does not appear to be mediated by the SAT secretome or by lipid-induced insulin resistance. Thus, factors intrinsic to skeletal muscle may be responsible for the obesity-mediated upregulation of myostatin, and future work to establish causality is required.

High expression of polymeric immunoglobulin receptor (PIGR) in breast cancer is associated with increased 5-year survival rate. However, the factors influencing PIGR expression in breast cancer have not been elucidated. The aim of this study was to determine the role of macrophages and cytokines affecting expression of PIGR in two breast cancer cell lines. M1, M2 macrophage conditioned media (CM) and recombinant human cytokines were used to determine factors which increased PIGR expression in MCF7 (HTB-22) and MDA-MB468 (HTB-132) breast cancer cell lines. The level of PIGR expression in the cells and PIGR secretory component were evaluated by real-time quantitative polymerase chain reaction and Western blotting. M1 macrophage CM induced a dose-dependent increase in PIGR mRNA expression in MDA-MB468 cells, up to 20-fold. The level of PIGR expression in MCF7 cells was very low and not affected by M1 and M2 CM. Interferon gamma (IFN-γ) and interleukin (IL)-1β also increased PIGR expression in MDA-MB468 and MCF7 cells. However, IL-1β was demonstrated to increase in M1 macrophages, while IFN-γ was not. The role of IL-1β secreted from M1 macrophages in increasing expression of PIGR was confirmed by IL-1 receptor blockade, indicating that IL-1β was the major M1 macrophage-derived cytokine that enhanced PIGR expression. Elevated PIGR expression in breast cancer in vivo may reflect the polarization state of tumor-associated immune cells.

Systemic responses to immune challenge are immature at birth. However exposure to experimental inflammogens are able to produce an immunologic response which is characterised by swelling and oedema but, unlike in adults, does not result in sensory hypersensitivity. We sought to investigate whether the lack of nociceptive hypersensitivity was as a result of altered hemapoietic immune cell recruitment to the site of inflammation and/or differences in the cytokine and chemokine profile released by tissue invading cells. Postnatal (day of birth) and young adult (40-days old) Sprague-Dawley rats were used. Inflammation was induced by s.c. injection of Complete Freunds Adjuvant (CFA) unilaterally into the one hind paw. Mechanical withdrawal thresholds were measured before and after injection (2-168hrs). In adults a significant hyperalgesia was evoked which was absent in neonates. Immunohistochemical analysis of invading immune cells present in the perfusion fixed skin showed that although total cell numbers in the paw were the same in both age groups, neonates recruited more cells positive for both cell surface markers CD68 and Mannose-receptor (MR) whereas adults recruited significantly more cells positive for MR alone. There were no differences in neutrophil recruitment (as measured with H&E staining). TaqMan qPCR demonstrated that the temporal profile of cytokine production in the skin differed between ages with neonates responding faster than adults and that neonates produced significantly more IL-1b and IL-27 then adults who expressed significantly more IL-6 and IL-10. This study illustrates that in neonates the cell recruitment and cytokine profiles are markedly different to those seen in adults; this may in part explain why behavioural responses to inflammation are suppressed relative to adults.

Abstract Introduction The polymeric immunoglobulin receptor (PIGR) is a transmembrane protein, which transports polymeric immunoglobulin (pIg) across the epithelial cells. High expression of PIGR in breast cancer has been reported to associate with increased 5-year survival rate. In this study, the factors in tumour microenvironment which affected PIGR expression in breast cancer cell lines, were investigated. Method M1, M2 macrophage conditioned media (CM) and recombinant human cytokines were used to determine factors which increased PIGR expression in breast cancer cells. The level of PIGR expression in the cells and secreted PIGR free secretory component (SC) were evaluated by real time quantitative polymerase chain reaction and Western blotting. Results M1 macrophage CM induced a striking dose dependent increase in PIGR mRNA expression in MDA-MB468 cells, up to 20-fold in 100% CM. Interferon gamma (IFNγ) and interleukin (IL)-1β also increased PIGR expression in MDA-MB468 cells. However, IL-1β was demonstrated to increase in M1 macrophages, while IFNγ was not. The role of IL-1β secreted from M1 macrophages in increasing expression of PIGR was confirmed by IL-1 receptor blockade, indicating that IL-1β was the M1 macrophage cytokine that enhanced PIGR expression in breast cancer cells. Conclusions IL-1β was the M1 macrophage cytokine which enhanced PIGR expression in breast cancer cells. IFNγ was also shown to increase PIGR expression in the present study. These imply that elevated PIGR expression in breast cancer in vivo may reflect the polarization state of tumour associated immune cells. Take-home Message IL-1β secreted from M1 macrophage enhances PIGR expression in breast cancer cells. The elevated PIGR expression in breast cancer in vivo may reflect the polarization state of tumour associated immune cells.

Polymeric immunoglobulin receptor (PIGR) has a major role in mucosal immunity as a transporter of polymeric immunoglobulin across the epithelial cells. The aim of this study was to determine the effect of PIGR on cellular behaviours and chemo-sensitivity of MCF7 and MDA-MB468 breast cancer cell lines. Basal levels of PIGR mRNA and protein expression in MCF7 and MDA-MB468 cells were evaluated by real time quantitative polymerase chain reaction and Western blotting, respectively. MCF7/PIGR and MDA-MB468/PIGR stable cell lines, overexpressing the PIGR gene, were generated using a lentiviral vector with tetracycline dependent induction of expression. Cell viability, cell proliferation and chemo-sensitivity of PIGR transfected cells were evaluated and compared with un-transfected cells to determine the effect of PIGR overexpression on cell phenotype. The levels of PIGR mRNA and protein expression were significantly higher in MDA-MB468 cells than in MCF7 cells (380-fold, p < 0.0001). However, the differential expression of PIGR in these two cell lines did not lead to significant differences in chemosensitivity. Viral overexpression of PIGR was also not found to change any of the parameters measured in either cell line. PIGR per se did not affect cellular behaviours and chemosensitivity of these breast cancer cell lines.

BACKGROUND/AIMS:Diarrhea-predominant irritable bowel syndrome (IBS-D) has been previously associated with evidence of immune activation and altered microbiota. Our aim is to assess the effect of the anti-inflammatory agent, mesalazine, on inflammatory gene expression and microbiota composition in IBS-D. METHODS:We studied a subset of patients (n = 43) from a previously published 12-week radomized placebo-controlled trial of mesalazine. Mucosal biopsies were assessed by immunohistochemistry and reverse transcription-polymerase chain reaction for a range of markers of inflammation, altered permeability, and sensory receptors including Toll-like receptors (TLRs) at randomization after treatment. All biopsy data were compared to 21 healthy controls. Patient's stool microbiota composition was analysed through 16S ribosomal RNA sequencing. RESULTS:We found no evidence of increased immune activation compared to healthy controls. However, we did find increased expression of receptors in both sensory pathways and innate immune response including TLR4. Higher TLR4 expression was associated with greater urgency. TLR4 expression correlated strongly with the expression of the receptors bradykinin receptor B2, chemerin chemokine-like receptor 1, and transient receptor potential cation channel, subfamily A, member 1 as well as TLR4's downstream adaptor myeloid differentiation factor 88. Mesalazine had minimal effect on either gene expression or microbiota composition. CONCLUSIONS:Biopsies from a well-characterized IBS-D cohort showed no substantial inflammation. Mesalazine has little effect on gene expression and its previous reported effect on fecal microbiota associated with much greater inflammation found in inflammatory bowel diseases is likely secondary to reduced inflammation. Increased expression of TLR4 and correlated receptors in IBS may mediate a general increase in sensitivity to external stimuli, particularly those that signal via the TLR system.

Abstract Introduction Neoadjuvant chemotherapy (NAC) is used with increasing frequency as the primary treatment for breast cancer. Pathological complete response (pCR) which contributes to survival benefit is achieved in only 25–30% of patients with little benefit and significant morbidity in the remainder. Therefore, predicting the patients who are likely to achieve a pCR before commencing NAC is desirable. A pilot study using next generation RNA sequencing of 10 large and locally advanced breast cancer biopsy specimens demonstrated that the expression of polymeric immunoglobulin receptor (PIGR) was significantly increased in breast cancers with pCR. The predictive and prognostic roles of PIGR in breast cancer were expanded in the present study. Method PIGR mRNA expression was determined by RNA in situ hybridization assay in formalin-fixed paraffin-embedded (FFPE) specimens of pre-treatment breast cancer core biopsy from 46 women with breast cancer who received NAC. The expression of PIGR and its associations with pCR and overall survival was examined. Results PIGR mRNA expression was lower in breast cancer compared with normal breast tissue (median H score: 0 vs. 120, P < 0.0001). There was no statistically significant difference in the proportion of pCR in PIGR positive compared with PIGR negative tumours (22.2% vs. 32.1%, P = 0.466). Five-year overall survival of the patients with PIGR positive and negative tumours was 88.9% and 78.3%, respectively. However, overall survival was not significantly different (P = 0.406). Conclusions The expression of PIGR did not appear to be associated with pCR after NAC and did not correlate with overall survival in breast cancer patients. Take-home Message The expression of PIGR did not appear to be associated with pCR after NAC and did not correlate with overall survival in breast cancer patients.

Background Neuroinflammation is a critical feature of sensitisation of spinal nociceptive processing in chronic pain states. We hypothesised that the resolvin pathways, a unique endogenous control system, may ameliorate aberrant spinal processing of somatosensory inputs associated with chemotherapy-induced neuropathic pain (CINP). Method The paclitaxel (PCX) model of CINP was established in male Sprague-Dawley rats and compared to control rats ( n = 23 and 22, respectively). Behavioural pain responses were measured, and either single unit electrophysiological recordings of dorsal horn wide dynamic range (WDR) neurones were performed, or mRNA microarray analysis of the dorsal horn of the spinal cord was undertaken. Results PCX rats exhibited significant changes in behavioural responses to mechanical and cold stimuli. A higher proportion of WDR neurones in PCX rats were polymodal (generating post-discharge following a non-noxious mechanical stimulus, responding to non-noxious cold and exhibiting spontaneous activity) compared to control ( p < 0.05). Microarray analysis revealed changes in proinflammatory pathways ( Tlr , Tnfrsf1a , Nlrp1a , Cxcr1 , Cxcr5 , Ccr1 , Cx3cr1 ) and anti-inflammatory lipid resolvin pathways ( Alox5ap , Cyp2j4 and Ptgr1 ) compared to control ( p < 0.05). Ingenuity pathway analysis predicted changes in glutamatergic and astrocyte signaling in the PCX group. Activation of the resolvin system via the spinal administration of aspirin-triggered resolvin D1 (AT-RvD1) markedly inhibited (73 ± 7% inhibition) normally non-noxious mechanically (8 g) evoked responses of WDR neurones only in PCX rats, whilst leaving responses to noxious mechanically induced stimuli intact. Inhibitory effects of AT-RvD1were comparable in magnitude to spinal morphine (84 ± 4% inhibition). Conclusion The PCX model of CINP was associated with mechanical allodynia, altered neuronal responses and dysregulation of pro- and anti-inflammatory signalling in the spinal dorsal horn. The resolvin AT-RvD1 selectively inhibited low weight mechanical-evoked responses of WDR neurones in PCX rats, but not in controls. Our data support the targeting of spinal neuroinflammation via the activation of the resolvin system as a new therapeutic approach for CINP.

Brain-derived neurotrophic factor (BDNF) and the high-affinity receptor tropomyosin receptor kinase B (TrkB) have important roles in neuronal survival and in spinal sensitization mechanisms associated with chronic pain. Recent clinical evidence also supports a peripheral role of BDNF in osteoarthritis (OA), with synovial expression of TrkB associated with higher OA pain. The aim of this study was to use clinical samples and animal models to explore the potential contribution of knee joint BDNF/TrkB signalling to chronic OA pain. Brain-derived neurotrophic factor and TrkB mRNA and protein were present in knee synovia from OA patients (16 women, 14 men, median age 67 years [interquartile range: 61-73]). There was a significant positive correlation between mRNA expression of NTRK2 (TrkB) and the proinflammatory chemokine fractalkine in the OA synovia. Using the surgical medial meniscal transection (MNX) model and the chemical monosodium iodoacetate (MIA) model of OA pain in male rats, the effects of peripheral BDNF injection, vs sequestering endogenous BDNF with TrkB-Fc chimera, on established pain behaviour were determined. Intra-articular injection of BDNF augmented established OA pain behaviour in MIA rats, but had no effect in controls. Intra-articular injection of the TrkB-Fc chimera acutely reversed pain behaviour to a similar extent in both models of OA pain (weight-bearing asymmetry MIA: -11 +/- 4%, MNX: -12 +/- 4%), compared to vehicle treatment. Our data suggesting a contribution of peripheral knee joint BDNF/TrkB signalling in the maintenance of chronic OA joint pain support further investigation of the therapeutic potential of this target.

Background: Altering the temporal distribution of energy intake (EI) and introducing periods of intermittent fasting (IF) exert important metabolic effects. Restricting EI to earlier in the day [early time-restricted feeding (eTRF)] is a novel type of IF. Objectives: We assessed the chronic effects of eTRF compared with an energy-matched control on whole-body and skeletal muscle insulin and anabolic sensitivity. Methods: Sixteen healthy males (aged 23 +/- 1 y; BMI 24.0 +/- 0.6 kg.m(-2)) were assigned to 2 groups that underwent either 2 wk of eTRF (n = 8) or control/caloric restriction (CON:CR; n = 8) diet. The eTRF diet was consumed ad libitum and the intervention was conducted before the CON:CR, in which the dietwas provided to match the reduction in EI and body weight observed in eTRF. During eTRF, daily EI was restricted to between 08:00 and 16:00, which prolonged the overnight fast by similar to 5 h. The metabolic responses to a carbohydrate/protein drink were assessed pre- and post-interventions following a 12-h overnight fast. Results: When compared with CON:CR, eTRF improved wholebody insulin sensitivity [between-group difference (95% CI): 1.89 (0.18, 3.60); P = 0.03;.2 p = 0.29] and skeletal muscle uptake of glucose [between-group difference (95% CI): 4266 (261, 8270) mu mol.min(-1).kg(-1) .180 min; P = 0.04; eta(2)(p) = 0.31] and branched-chain amino acids (BCAAs) [between-group difference (95% CI): 266 (77, 455) nmol.min(-1).kg(-1).180 min; P = 0.01;eta(2)(p) = 0.44]. eTRF caused a reduction in EI (similar to 400 kcal.d(-1)) and weight loss (-1.04 +/- 0.25 kg; P = 0.01) that was matched in CON:CR (-1.24 +/- 0.35 kg; P = 0.01). Conclusions: Under free-living conditions, eTRF improves wholebody insulin sensitivity and increases skeletal muscle glucose and BCAA uptake. The metabolic benefits of eTRF are independent of its effects on weight loss and represent chronic adaptations rather than the effect of the last bout of overnight fast. This trial was registered at clinicaltrials.gov as NCT03969745. Am J Clin Nutr 2020;112:1015-1028.

The sediments in the Salford Quays, a heavily-modified urban water body, contain high levels of organic matter, iron, zinc and nutrients as a result of past contaminant inputs. Vivianite [Fe3(PO4)2.8H2O] has been observed to have precipitated within these sediments during early diagenesis as a result of the release of Fe and P to porewaters. These mineral grains are small (<100