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Type 2 diabetes is an independent risk factor for non-alcoholic steatohepatitis (NASH) progression and its mediators have not been resolved. In this study, a pathogenic role of cellular communication network factor 2 (CCN2) protein in NASH pathology, was investigated in an established preclinical NASH model. Male wild type C57BL/6 mice received either Chow or high fat diet (HFD) for 26 weeks, with some mice in each group randomly selected to receive low dose streptozotocin (STZ: 3 i.p. injections, 65 mg/kg) at 15 weeks to induce type 2 diabetes. In the final 10 of the 26 weeks mice from each group were administered i.p. either rabbit anti-CCN2 neutralizing antibody (CCN2Ab) or as control normal rabbit IgG, at a dose of 150 µg per mouse twice/week. NASH developed in the HFD plus diabetes (HFD+DM) group. Administration of CCN2Ab significantly downregulated collagen I and collagen III mRNA induction and prevented pro-inflammatory MCP-1 mRNA induction in HFD+DM mice. At the protein level, CCN2Ab significantly attenuated collagen accumulation by PSR stain and collagen I protein induction in HFD+DM. Phosphorylation of the pro-fibrotic ERK signalling pathway in liver in HFD+DM was attenuated by CCN2Ab treatment. Intrahepatic CCN1 mRNA was induced, whereas CCN3 was downregulated at both the mRNA and protein levels in HFD+DM. CCN3 down-regulation was prevented by CCN2Ab treatment. This in vivo study indicates that CCN2 is a molecular target in NASH with high fat diet and diabetes, and that regulation of ERK signalling is implicated in this process.

Research has described that adiponectin plays a key role in cardiomyocytes metabolism, however, the effects of exercise during obesity on cardiac adiponectin levels is unclear.To investigate the effects of constant-moderate endurance (END) and high-intensity interval training (HIIT), on heart adiponectin levels in mice.Two experiments were conducted: (1) preventive (EX1): 10 week-old male mice were fed standard (CHOW) or high-fat diet (HFD;45% fat) and simultaneously trained with END and HIIT for 10 weeks; (2) Treatment (EX2): after 10 weeks of dietary intervention, another cohort of 10 week-old mice were trained by both programmes for 10 weeks.In EX1, END and HIIT decreased low-molecular weight adiponectin (∼0.5-fold

(1) Background: studies on the long-term dynamic changes in fat depot metabolism in response to a high-fat diet (HFD) on hepatic lipid deposition and insulin resistance are sparse. This study investigated the dynamic changes produced by HFD and the production of dysfunctional fat depots on insulin resistance and liver lipid metabolism. (2) Methods: mice fed a chow or HFD (45% kcal fat) diet had three fat depots, liver, and blood collected at 6, 10, 20, and 30 weeks. Anthropometric changes and gene markers for adipogenesis, thermogenesis, ECM remodeling, inflammation, and tissue insulin resistance were measured. (3) Results: early responses to the HFD were increased body weight, minor deposition of lipid in liver, increased adipocyte size, and adipogenesis. Later changes were dysfunctional adipose depots, increased liver fat, insulin resistance (shown by changes in ITT) accompanied by increased inflammatory markers, increased fibrosis (fibrosis > 2-fold, p < 0.05 from week 6), and the presence of crown cells in white fat depots. Later, changes did not increase thermogenic markers in response to the increased calories and decreased UCP1 and PRDM16 proteins in WAT. (4) Conclusions: HFD feeding initially increased adipocyte diameter and number, but later changes caused adipose depots to become dysfunctional, restricting adipose tissue expansion, changing the brown/beige ratios in adipose depots, and causing ectopic lipid deposition and insulin resistance.

Despite advances in antiviral therapy, molecular drivers of Hepatitis C Virus (HCV)-related liver disease remain poorly characterised. Chronic infection with HCV genotypes (1 and 3) differ in presentation of liver steatosis and virological response to therapies, both to interferon and direct acting antivirals. To understand what drives these clinically important differences, liver expression profiles of patients with HCV Genotype 1 or 3 infection (n = 26 and 33), alcoholic liver disease (n = 8), and no liver disease (n = 10) were analysed using transcriptome-wide microarrays. In progressive liver disease, HCV genotype was the major contributor to altered liver gene expression with 2151 genes differentially expressed >1.5-fold between HCV Genotype 1 and 3. In contrast, only 6 genes were altered between the HCV genotypes in advanced liver disease. Induction of lipogenic, lipolytic, and interferon stimulated gene pathways were enriched in Genotype 1 injury whilst a broad range of immune-associated pathways were associated with Genotype 3 injury. The results are consistent with greater lipid turnover in HCV Genotype 1 patients. Moreover, the lower activity in inflammatory pathways associated with HCV genotype 1 is consistent with relative resistance to interferon-based therapy. This data provides a molecular framework to explain the clinical manifestations of HCV-associated liver disease.

Objective To determine whether rapid polymerase chain reaction (PCR) testing for influenza and respiratory syncytial viruses (RSV) in emergency departments (EDs) is associated with better patient and laboratory outcomes than standard multiplex PCR testing. Design, setting A before‐and‐after study in four metropolitan EDs in New South Wales. Participants 1491 consecutive patients tested by standard multiplex PCR during July–December 2016, and 2250 tested by rapid PCR during July–December 2017. Main outcome measures Hospital admissions; ED length of stay (LOS); test turnaround time; patient receiving test result before leaving the ED; ordering of other laboratory tests. Results Compared with those tested by standard PCR, fewer patients tested by rapid PCR were admitted to hospital (73.3% v 77.7%; P

Aims Delayed healing of diabetes-related foot ulcers (DRFUs) is associated with increased macrophage and matrix metalloproteinases (MMPs) at the wound site. Whether circulating monocyte phenotype and/or MMPs are altered in association with wound healing outcome is unknown, and was investigated in this study. Methods Blood was obtained from 21 participants with DRFU, at initial visit (V1), week-4 (V2), and week-8 (V3) for measurement of monocyte number (CD14+), phenotype (CD16, CD163) and chemokine receptors (CCRs) by flow cytometry, and circulating MMPs and TIMP-1 by ELISA. Results Six wounds healed during the study. At V1, non-classical CD16++ monocytes and MMP-3 were higher in healed vs unhealed (both p Conclusions These results indicate that circulating monocyte phenotype and MMPs alter as DRFUs heal. The relationship of %CD16++ monocytes with WCR and ROC curve suggest a predictive role of %CD16++ monocytes for ulcer healing.

Zinc is required for activation of Matrix Metalloproteinases (MMPs). Circulating zinc levels are decreased in diabetes1 and decreased activity of some MMPs is associated with the development of diabetic nephropathy (DN). The association between MMP activity and zinc in the development of DN has not been investigated and was examined in a preclinical model in this study. In this study, diabetic (DM: STZ: 3x 65mg/kg) male BALB/c mice were euthanized after 5, 10 and 30wks of diabetes. Age-matched mice acted as controls. At termination, kidneys, plasma and urine were collected for histopathology, measurement of biochemical parameters, total MMP activity using a broad spectrum MMP fluorogenic substrate and zinc level by ICPMS. In DM mice, blood glucose levels were increased at 5, 10 and 30wks (P Disclosure A. Tao: None. R. McQuilty: None. S.S. Sutanto: None. L.A. Ban: None. S.M. Twigg: Advisory Panel; Self; Abbott, AstraZeneca, Sanofi. Speaker’s Bureau; Self; Abbott, AstraZeneca, Novo Nordisk Inc., Roche Diabetes Care, Sanofi. S. McLennan: None.

Transforming growth factor β (TGFβ) a multifunctional cytokine is known to regulate cell proliferation, differentiation, migration and survival. Although there is variable expression of modulators of TGFβ action during differentiation, a differential effect on fat cell metabolism at the different stages of adipocyte differentiation was unclear. In the present study, 3T3L1 cells were used as an in vitro model to study the effect of TGFβ on adipogenic and thermogenic markers at various stages of preadipocyte to mature adipocyte differentiation. As in our earlier studies on the effect of TGFβ on CEBP’s, we used a standard differentiation mix, and one with the addition of rosiglitazone. RhTGFβ1 was added to undifferentiated adipocytes (preadipocytes) and to adipocytes at day 0 (commitment stage) as well as day 10 (terminal differentiation). Cellular responses in terms of Pref1, PPARγ, TLE3, PGC1α, PRDM16, UCP1 and UCP2 mRNA levels and selected protein products, were determined. Increases in PPARγ, PRDM16, UCP1 and UCP2 mRNA and decreases in Pref1 are good indicators of successful differentiation. The early addition of rhTGFβ1 during commitment stage decreased PPARγ, PRDM16, TLE3, UCP1 and UCP2 mRNA and decreased PRDM16 protein consistent with our earlier report on the inhibition of CEBP’s by TGFβ and CCN2. The addition of rhTGFβ1 to mature adipocyte at day 10 increased UCP1 mRNA and increased PRDM16 and UCP1 proteins. In the present study, our results suggest that TGFβ1 added late enhances the thermogenic potential of mature cells and causes 3T3L1 cells to differentiate to resemble brown or beige rather than white adipose tissue.

The study of miRNAs in PTC has shown that these molecules can help in the diagnosis, prognosis and also as therapeutic candidates [1]. miRNAs are known to be differentially expressed in PTC compared to non-cancerous tissue and a few studies have shown association with some clinico-pathological features. However, little is known regarding their expression in relation to risk of disease progression. In this study, we examined the expression of miRNAs in patients diagnosed with PTC in association with risk of disease recurrence and/or persistence. Patients were classified by three endocrinologists to either high (H) or low (L) risk according to ATA Risk Stratification. PTC tissue was micro-dissected from Formalin Fixed Paraffin Embedded (FFPE) tissue for analysis. OPenArray analysis showed that 21 miRNAs were differentially expressed in H-L groups (n=4/grp). qRT-PCR was used to confirm these differences in a larger cohort of PTC (H=46; L=58). This comparison also included comparisons between cancer and normal tissue and investigation of miRNAs known to be differentially expressed (i.e miRNAs 222, 221 and 146b). All analysis was performed in Rstudio using ΔΔCt relative to expression of miR-16 which was not altered by PTC. By qRT-PCR, only 3 of the 21 miRNAs identified by OpenArray analysis, were differentially expressed in H vs L risk (each P In this study, we identified 3 miRNAs with potential utility for the stratification of patients into those with H or L risk disease recurrence/persistence. As the current ATA 3-tiered system used is still a reflection of the continuum of risk that patients have, whether these 3 miRNAs may have the utility to further stratify those in the intermediate group remains to be investigated. Reference: 1. Ramírez-Moya, J. and P. Santisteban, miRNA-Directed Regulation of the Main Signaling Pathways in Thyroid Cancer. Frontiers in Endocrinology, 2019. 10: p. 430.

Context: Telomeres protect chromosomes from damage, and shorter leucocyte telomere length (LTL) is a marker of advancing biological age. The association between testosterone (T) and its bioactive metabolites, dihydrotestosterone (DHT) and oestradiol (E2) with telomere length, particularly in older men, is uncertain. The study aimed to clarify associations of sex hormones with LTL in older men. Participants and methods: We used cross‐sectional data from 2913 men aged 76.7 ± 3.2 years with morning blood samples assayed for T, DHT, E2 (mass spectrometry), and sex hormone‐binding globulin (SHBG, immunoassay), to correlate sex hormones with LTL measured using PCR and expressed as T/S ratio in multivariable linear regression models adjusted for age, cardiometabolic risk factors and cardiovascular disease history. Results: Average difference per decade of age was T −0.46 nmol/L, DHT −0.11 nmol/L, E2 −7.5 pmol/L, SHBG +10.2 nmol/L and LTL (T/S ratio) −0.065. E2 correlated with T/S ratio (r = 0.038, P = 0.039) and SHBG was inversely correlated (r = −0.053, P = 0.004). After multivariable adjustment, E2 was associated with T/S ratio (per 1 SD increase E2: coefficient 0.011, P = 0.043), T and DHT were not associated. When E2 and SHBG were simultaneously included, E2 remained positively (coefficient 0.014, P = 0.014) and SHBG inversely (coefficient −0.013, P = 0.037) associated with T/S ratio. Conclusions: In older men, neither T nor DHT is associated with LTL while E2 is independently associated with LTL and SHBG is inversely associated, thus relating sex hormone exposure to lower biological age. Further research is needed to determine causality and clarify the role of sex hormones in male ageing.

There is limited data on prevalence of PD-L1 expression in papillary thyroid cancer (PTC). Anti- PD-L1 therapy is now being trialled for head and neck cancers including thyroid cancer. Whether presence of PD-L1 staining in PTC might result in a better response to anti-PD-L1 therapy is uncertain. We evaluated the frequency of tumoral PD-L1 expression using standard antibody (SP263) and clinicopathologic factors including mutation status - BRAF, NRAS, KRAS using OncoFocus Mass Array System in an Australian PTC cohort with low or high risk of recurrence /treatment response as per 2015 ATA guidelines. The cohort had 128 patients; 66% were females; 40% had both multifocal and bilateral PTC; 47% had low recurrence risk, of whom 92% had excellent treatment response; 34% had high recurrence risk, of whom 37% had structural incomplete treatment response. There was nil (0%) PD-L1 staining in 59%, 1% staining in 20%, 5-10% staining in 11%, >10% staining in rest of the cohort; 30%, 40% and 70% staining was seen in 2% each. PD-L1 in immune cells was positive in 62%. There was no significant association between PD-L1 tumour status and age, gender, size of tumour, multifocality, bilaterality, both multifocality and bilaterality, lymphovascular invasion, perineural invasion, extrathyroidal extension (ETE), histological variants, risk stratification, treatment response, metastases or mutation status. Among PD-L1 negative vs positive patients, BRAFV600E was present in 64.9% vs 67.5%, NRAS in 8.8% vs 0% and nil mutation in 26 % vs 33 % p=0.15. The staining thresholds were chosen according to existing literature. When analysed according to the percentage of PD-L1 staining (0%, 1-10% and >=10% thresholds), there was no significant association between PD-L1 staining category and other features, except for ETE (35% vs 65% vs 27%; p=0.007), a relationship that remained significant in a multivariable-adjusted model (OR 9.9, p=0.04). PD-L1 staining thresholds of =5% were significantly associated with the size of tumour (24.7±15.4 vs 33.3±21.8; p=0.02) and histological variants (57 % vs 61% for classic variants and 27% vs 11% for follicular variants, p=0.03). This is the first study to our knowledge that analysed PD-L1 staining based on the risk stratification categories. The majority of PTC showed negative or low staining intensity for PD-L1 expression. There was no significant association between PD-L1 staining, clinicopathologic characteristics and mutation status of the PTC. Relatively less ETE was seen in the group that showed >=10% and also >=5% PD-L1 staining as compared to the group that showed 1%

Background Cell free fetal miRNAs have been identified as potential biomarkers for fetal abnormalities and/or placental function. Factors affecting the stability of cell free fetal miRNA samples (type of collection tube and time interval between sampling and analysis) have not previously been reported. Methods Blood from pregnant women (n = 12, 18 ± 4 weeks’ gestation) was collected into two types of tube (EDTA and RNA BCT) and was stored at different temperatures for up to 72 hours. Expression of seven apparently placental specific miRNAs was then measured to compare the effects of sampling and storage. These miRNAs were also assessed in non-pregnant women (n = 9). Results The quantity of miRNA extracted was not affected by time or tube. Three miRNAs (miR-518b, miR-525 and miR-526a*) were measureable only in pregnant women, but miR-518b was not always present. Detailed study of the two pregnancy specific miRNAs showed no effect of tube type at 4 hours. However, variability in miRNA level was observed with increased time and was significant for one miRNA in the BCT tube at >48 hours (p

NEW FINDINGS What is the central question of this study? Non-invasive muscle function tests have not been validated for use in the study of muscle performance in high-fat-fed mice. What is the main finding and its importance? This study shows that grip strength, hang wire and four-limb hanging tests are able to discriminate the muscle performance between chow-fed and high-fat-fed mice at different time points, with grip strength being reliable after 5, 10 and 20 weeks of dietary intervention. Non-invasive tests are commonly used for assessing muscle function in animal models. The value of these tests in obesity, a condition where muscle strength is reduced, is unclear. We investigated the utility of three non-invasive muscle function tests, namely grip strength (GS), hang wire (HW) and four-limb hanging (FLH), in C57BL/6 mice fed chow (chow group, n = 48) or a high-fat diet (HFD group, n = 48) for 20 weeks. Muscle function tests were performed at 5, 10 and 20 weeks. After 10 and 20 weeks, HFD mice had significantly reduced GS (in newtons; mean ± SD: 10 weeks chow, 1.89 ± 0.1 and HFD, 1.79 ± 0.1; 20 weeks chow, 1.99 ± 0.1 and HFD, 1.75 ± 0.1), FLH [in seconds per gram body weight; median (interquartile range): 10 weeks chow, 2552 (1337-4964) and HFD, 1230 (749-1994); 20 weeks chow, 2048 (765-3864) and HFD, 1036 (717-1855)] and HW reaches [n; median (interquartile range): 10 weeks chow, 4 (2-5) and HFD, 2 (1-3); 20 weeks chow, 3 (1-5) and HFD, 1 (0-2)] and higher falls [n; median (interquartile range): 10 weeks chow, 0 (0-2) and HFD, 3 (1-7); 20 weeks chow, 1 (0-4) and HFD, 8 (5-10)]. Grip strength was reliable in both dietary groups [intraclass correlation coefficient (ICC) = 0.5-0.8; P

Skeletal muscle extracellular matrix remodelling has been proposed as a new feature associated with obesity and metabolic dysfunction. Exercise training improves muscle function in obesity, which may be mediated by regulatory effects on the muscle extracellular matrix. This review examined available literature on skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise. A non-systematic literature review was performed on PubMed of publications from 1970 to 2015. A total of 37 studies from humans and animals were retained. Studies reported overall increases in gene and protein expression of different types of collagen, growth factors and enzymatic regulators of the skeletal muscle extracellular matrix in obesity. Only two studies investigated the effects of exercise on skeletal muscle extracellular matrix during obesity, with both suggesting a regulatory effect of exercise. The effects of exercise on muscle extracellular matrix seem to be influenced by the duration and type of exercise training with variable effects from a single session compared with a longer duration of exercise. More studies are needed to elucidate the mechanisms behind skeletal muscle extracellular matrix remodelling during obesity and the effects of exercise.

Objectives: The performance of cadexomer iodine was determined against microbial populations from chronic non-healing diabetic foot ulcers (DFUs) complicated by biofilm in vivo, using molecular, microscopy and zymography methods. Methods: Chronic non-healing DFUs due to suspected biofilm involvement were eligible for enrolment. DNA sequencing and real-time quantitative PCR was used to determine the microbial load and diversity of tissue punch biopsies obtained pre- and post-treatment. Scanning electron microscopy and/or fluorescence in situ hybridization confirmed the presence or absence of biofilm. Zymography was used to determine levels of wound proteases. Results: Seventeen participants were recruited over a 6 month period. Scanning electron microscopy and or fluorescence in situ hybridization confirmed the presence of biofilm in all samples. Eleven participants exhibited log10 reductions in microbial load after treatment (range 1–2 log10) in comparison with six patients who experienced

Objective To investigate the associations of Graves' disease (GD) severity, autoimmunity and longitudinal liver enzyme changes with time in a cohort with well-characterized GD. Design Retrospective cohort study. Patients Patients diagnosed with Graves' disease, treated at Royal Prince Alfred Hospital Sydney, Adult Thyroid Clinic from 2000 to 2012 inclusive. Measurements Inclusion criteria were patients with a complete set of TSH, FT4, FT3, liver enzymes and TSH receptor antibody (TRAb) results prior to commencement of thionamide therapy. Results Of the 146 patients who had complete results, 69 (47%) had at least one abnormal liver enzyme. Gamma glutamyltransferase (GGT) was most frequently abnormal (74%), followed by alanine aminotransferase (ALT) (57%), alkaline phosphatase (ALP) (39%) and then aspartate aminotransferase (AST) (29%). Subsequent to thyroid function normalization, 78% of the liver enzymes were normalized, 10% were persistently abnormal and 12% were lost to follow-up. Circulating TRAb, FT3 and FT4 results were categorized into mild, moderate and severe elevations. At univariate regression analyses, TRAb, FT3 and FT4 levels were each significantly associated with abnormal liver enzyme profile. Multivariate regression including TRAB, FT3 and FT4 as independent variables demonstrated FT3 and FT4 were more strongly associated with abnormal liver profile than TRAb. However, the initial FT3 and FT4 levels were not associated with abnormal liver profile in the subgroup with persistently abnormal liver profile. Conclusion Graves' disease is commonly associated with abnormal liver enzymes, and most commonly with abnormal levels of GGT, and that an abnormal liver enzyme profile is more directly linked to the degree of thyrotoxicosis than levels of TRAB.

BackgroundChronic inflammation is the driver of liver injury resulting in progressive fibrosis and eventual cirrhosis. The consequences include both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury.MethodsNon-diseased, progressive injury and cirrhotic liver from humans and mice were examined using mAb targeting CD147. Inflammatory cell subsets were assessed by multicolor flow cytometry.ResultsIn liver injury, we observe abundant intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+cells which we have labelled “leukocyte aggregates”. We have shown that these leukocyte aggregates are significant in determining the extent of liver injury. If CD147 is blockedin vivo,these leukocyte aggregates diminish in size and number together with a marked significant reduction in liver injury including fibrosis. This accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, “off-target” or unpredicted effects in targeting CD147.ConclusionCD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades but till now been shown to determine the extent of injury.

A standard multiplex PCR offers comprehensive testing for respiratory viruses. However, it has traditionally been performed in a referral laboratory with a lengthy turnaround time, which can reduce patient flow through the hospital. We aimed to determine whether the introduction of a rapid PCR, but with limited targets (Cepheid Xpert Flu/RSV XC), was associated with improved outcomes for adults hospitalized with respiratory illness. A controlled quasi-experimental study was conducted across three hospitals in New South Wales, Australia. Intervention groups received standard multiplex PCR during the preimplementation, July to December 2016 (n = 953), and rapid PCR during the postimplementation, July to December 2017 (n = 1,209). Control groups (preimplementation, n = 937, and postimplementation, n = 1,102) were randomly selected from adults hospitalized with respiratory illness during the same periods. The outcomes were hospital length of stay (LOS) and microbiology test utilization (blood culture, urine culture, sputum culture, and respiratory bacterial and virus serologies). The introduction of rapid PCR was associated with a nonsignificant 8.9-h reduction in median LOS (95% confidence interval [CI], -21.5 h to 3.7 h; P = 0.17) for all patients and a significant 21.5-h reduction in median LOS (95% CI, -36.8 h to -6.2 h; P < 0.01) among patients with positive test results in an adjusted difference-in-differences analysis. For patients receiving test results before disposition, rapid PCR use was associated with a significant reduction in LOS, irrespective of test results. Compared with standard PCR testing, rapid PCR use was significantly associated with fewer blood culture (adjusted odds ratio [aOR], 0.67; 95% CI, 0.5 to 0.82; P < 0.001), sputum culture (aOR, 0.56; 95% CI, 0.47 to 0.68, P < 0.001), bacterial serology (aOR, 0.44; 95% CI, 0.35 to 0.55, P < 0.001) and viral serology (aOR, 0.42; 95% CI, 0.33 to 0.53, P < 0.001) tests, but not with fewer urine culture tests (aOR, 0.94; 95% CI, 0.78 to 1.12, P = 0.48). Rapid PCR testing of adults hospitalized with respiratory illnesses can deliver benefits to patients and reduce resource utilization. Future research should consider a formal economic analysis and assess its potential impacts on clinical decision making.

Telomeres are essential DNA-protein complexes comprising TTAGGG repeats binding specific proteins, which protect the physical ends of chromosomes from fusion and degradation. Attrition of telomeres results in cellular senescence. Leucocyte telomere length (LTL) reflects lengths of telomeres in various tissues, and shorter LTL is a marker of advancing biological age. Previous work has associated bioactive metabolites of T, dihydrotestosterone (DHT) and estradiol (E2) with LTL in a population of predominantly middle-aged men [1]. However, the relationship of these hormones to biological age in older men was unclear. We aimed to clarify associations of sex hormones with LTL in a cohort of 2,913 community-dwelling men aged 70-89 years. Early morning blood samples were assayed for T, DHT and E2 using mass spectrometry, and for sex hormone-binding globulin (SHBG) using immunoassay. LTL was measured using a multiplex quantitative PCR method and expressed as the amount of telomeric DNA relative to beta-globin, a single copy control gene (T/S ratio). Cross-sectional analyses utilised multivariable linear regression. Mean (±SD) age was 76.7±3.2 years. The average difference per decade of age was T -0.46 nmol/L, DHT -0.11 nmol/L, E2 -7.5 pmol/L, SHBG +10.2 nmol/L, and LTL (T/S ratio) -0.065. E2 correlated with T/S ratio (r=0.038, p=0.039). After excluding highest and lowest 1% of values, the correlation between E2 and T/S ratio was largely unchanged (r=0.039, p=0.037). SHBG was inversely correlated with T/S ratio (r=-0.053, p=0.004), also unchanged in the trimmed analysis (r=-0.055, p=0.004.) After adjusting for age, BMI, cardiovascular disease, diabetes, alcohol, smoking, physical activity, lipids and hypertension, E2 remained associated with T/S ratio (per 1 SD increase E2: coefficient 0.011, p=0.043). When E2 and SHBG were simultaneously included in the multivariate model, E2 remained positively associated with T/S ratio (coefficient 0.014, p=0.014) and SHBG inversely associated (coefficient -0.013, p=0.037). The magnitude of increase in T/S ratio associated with a 1 SD higher plasma E2 concentration was comparable with having a BMI 3.6 kg/m2 lower, and two thirds that associated with being 3.6 years younger. T, DHT and LH were not associated with LTL in multivariate analyses. To conclude, in older men, neither T nor DHT are associated with LTL while E2 is independently associated with LTL and SHBG is inversely associated. These findings associate activity of the gonadal axis with lower biological age in older men. However, causality cannot be inferred from an observational, cross-sectional study, thus additional research is necessary to determine whether sex hormone exposure modulates male biological ageing. Reference: [1] Yeap BB, et al. J Clin Endocrinol Metab 2016; 101: 1299-1306.

People with diabetes mellitus have shorter telomeres compared with non-diabetic subjects. The aim of this study was to investigate an in-vitro model of telomere shortening under diabetes metabolic conditions. The mechanisms of the accelerated telomere length attrition and the potential telomere protective action of fenofibrate with related cellular mechanisms were also examined. Human dermal fibroblasts were passaged and cultured in normal (5.5 mM) or high (25 mM) D-glucose, across 7 days with hydrogen peroxide (H(2)O(2)), glucosamine (GA), or glycated albumin (AGEs-BSA). Relative telomere length (RTL) was determined by qPCR. The expression of shelterin complex members which regulate telomere stability were measured by qRT-PCR and Western immunoblot. Culture in high glucose decreased RTL compared with normal glucose: H(2)O(2) and GA lowered the RTL after 7 days (each P

PTC and melanoma are known to harbour common mutations, but this has not been extensively investigated. Targeted therapies for BRAF and PD-L1 have been used for melanoma and there are ongoing clinical trials for use of PD-L1 inhibitors in PTC but its utility is uncertain. Additionally, many of these patients have multiple cancers, so, whether they have a tumour predisposition syndrome is also unclear. Both germline and somatic mutations in BRCA1-associated protein 1 (BAP1) are associated with a wide spectrum of tumours. We hypothesized that a common genetic link may be present in our cohort of patients who have both PTC and melanoma. The aim of this study was to elucidate molecular genetics, specifically BRAF, NRAS, KRAS, KIT using OncoFocus Mass Array System as well as expression of PD-L1 and BAP1, using a standard antibody (SP263) and C-4 respectively, in an Australian cohort with concurrent PTC and melanoma. In our cohort of 21 patients (43% females, all Caucasian), melanoma was diagnosed about 8 years prior to PTC (50.3 ± 18.3 vs. 58.6 ± 12.8 years). The most common mutation was BRAFV600E seen in 88% of PTC, followed by NRAS mutation in 12% of PTC. Majority of the PTC (68%) stained negative for PD-L1. There was no significant association between PD-L1 tumour status and clinicopathologic outcomes. Interestingly, majority of multifocal, bilateral and both bilateral and multifocal PTC were PD-L1 negative (85%,69% and 69% respectively, P25%, a much higher degree of staining compared to PTC group. Among 7 patients where data were available for both tissues, concordant mutations were found in only 2 patients (both BRAFV600E). In addition, 11 of the 21 patients had at least one other cancer apart from PTC and melanoma. Nine of the 11 patients who had more than one cancer were BRAF positive. BAP1 staining was retained in the majority of PTCs and melanoma tissues, indicating no loss of BAP1 protein. PTC and melanoma both share molecular markers including BRAF, NRAS, PD-L1 as shown in our cohort. This is the largest study describing the mutation status of both PTC and melanoma. It is also the only study describing the PD-L1 and BAP1 expression in PTC and melanoma. BRAFV600E was the most common mutation. Majority of the PTC and melanoma stained negative for PD-L1. BAP1 expression was retained in both either PTC and melanoma tissues thus making presence of BAP1 tumour predisposition syndrome unlikely.

BackgroundChronic inflammation is the driver of liver injury and results in progressive fibrosis and eventual cirrhosis with consequences including both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury.MethodsNon-diseased, progressive injury, and cirrhotic liver from humans and mice were examined using a mAb targeting CD147. Inflammatory cell subsets were assessed by multiparameter flow cytometry.ResultsIn liver injury, we observe abundant, intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have termed "leukocyte aggregates". We have shown that these leukocyte aggregates have a significant effect in determining the extent of liver injury. If CD147 is blocked in vivo, these leukocyte aggregates diminish in size and number, together with a marked significant reduction in liver injury including fibrosis. This is accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking of aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, "off-target" or unpredicted effects in targeting CD147.ConclusionCD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades. Here we demonstrate that leukocyte aggregates determine the extent of liver injury.

Background & AimsFibroblast activation protein-a (FAP) is a post-proline peptidase closely related to dipeptidyl peptidase-4. FAP degrades bioactive peptides including fibroblast growth factor-21 (FGF-21) and neuropeptide Y. We examined metabolic outcomes of specific genetic ablation of FAP and its enzyme activity in a mouse model of diet-induced obesity (DIO) causing fatty liver.MethodsWildtype (WT) and genetically modified FAP deficient mice that specifically lacked either the FAP protein or FAP enzyme activity received chow, or an atherogenic diet for 8 to 20 weeks of DIO.ResultsFAP deficient male and female mice in the DIO model were more metabolically healthy than controls. The FAP deficient mice had less glucose intolerance, liver lipid, adiposity, insulin resistance, pancreatic and plasma insulin, pancreatic β-cell hyperplasia, serum alanine transaminase and circulating cholesterol compared to wild type controls. Furthermore, FAP deficiency lowered respiratory exchange ratio and greatly increased intrahepatic non-esterified free fatty acids, indicative of increased lipolysis and β-oxidation. Concordantly, lipogenic genes (Pparg, Gck, Acc, Fasn) and hepatic triglyceride and fatty acid uptake genes (Cd36, Apoc3, Ldlr) and plasma low-density lipoprotein cholesterol were downregulated. Glucagon like peptide-1 levels were unaltered. FAP was localized to human pancreatic β-cells and pancreas from diabetes mellitus patients contained elevated FAP activity. Comparable data from a FAP gene knockout mouse and a novel mouse lacking FAP enzyme activity indicated that these metabolic changes depended upon the enzymatic activity of FAP. These changes may be driven by FGF-21, which was upregulated in livers of FAP deficient DIO mice.ConclusionThis is the first study to show that specific genetic ablation of FAP activity or protein protects against DIO-driven glucose intolerance, hyperinsulinaemia, insulin resistance, hypercholesterolaemia and liver steatosis in mice and provide mechanistic insights.

Obesity associates highly with metabolic disorders including insulin resistance. To determine if two exercise prescriptions exert differential metabolic benefits in insulin sensitive tissues, we compared isocaloric programs of constant-moderate endurance (END) and high intensity interval training (HIIT), in a mouse model of diet-induced obesity. Male 10 week old C57BL/6 mice were fed a high fat diet (HFD; 45% kcal fat) ab libitum for 10 weeks, achieving 25% excess weight, and for a further 10 weeks they then underwent isocaloric END or HIIT (3x40min sessions/week). Untrained HFD and chow-fed mice acted as controls. At 30 weeks of age, mice were sacrificed and quadriceps muscle, subcutaneous adipose tissue (SAT) and liver were excised. Neither END or HIIT altered the body weight or the fat/lean mass proportion in HFD mice. An insulin tolerance test (0.65 IU/kg*BW) showed HIIT but not END improved insulin sensitivity in HFD mice (p Disclosure S.F. Martinez-Huenchullan: None. L.A. Ban: None. L.F. Olaya: None. B.R. Maharjan: None. C. Tam: None. S. McLennan: None. S.M. Twigg: Advisory Panel; Self; Abbott. Consultant; Self; Abbott. Advisory Panel; Self; Novo Nordisk Inc., Sanofi-Aventis, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. Speaker's Bureau; Self; AstraZeneca, Merck Sharp & Dohme Corp.. Other Relationship; Self; Abbott.

Poor wound healing in diabetes is associated with increased chronic inflammation and alterations in macrophage accumulation. Chronic inflammation can alter the phenotype of macrophage precursors, monocytes but whether their phenotype is changed in association with wound healing is not known. Blood was obtained from 21 patients (14 male: 7 female) attending the High Risk Foot Clinic at RPA Hospital at their initial visit (V1), week 4 (V2) and week 8 (V3). Wound area was measured and wound type was assessed by a podiatrist. Monocyte number (CD14+), phenotype as classical (CD16−), non-classical (CD16++), and intermediate (CD16+), anti-inflammatory (CD163+) subset and expression of receptors CCR2, CCR5 and TLR2, TLR4 were determined by flow cytometry. The MMPs and TIMP-1 were measured in plasma by zymography and ELISA. Over the 8 weeks, 6 ulcers healed (H) and 15 failed to heal (NH). The age, duration of diabetes, HbA1c, wound size and duration at V1 were not different between the groups but the % CD16++ monocytes was higher in H vs. NH at V1 (23.5 vs. 8.3) and V3 (17.4 vs. 7.2) and monocyte CCR2+ was lower in H at V3 (P Disclosure D. Min: None. V.L. Nube: None. A. Tao: None. X. Yuan: None. B. Brooks: None. J. Wong: Speaker's Bureau; Self; Novo Nordisk A/S, AstraZeneca, Eli Lilly and Company. S.M. Twigg: Advisory Panel; Self; Abbott. Consultant; Self; Abbott. Advisory Panel; Self; Novo Nordisk Inc., Sanofi-Aventis, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. Speaker's Bureau; Self; AstraZeneca, Merck Sharp & Dohme Corp.. Other Relationship; Self; Abbott. S. McLennan: None.

Whether obesity and diabetes (DM) similarly affect composition and function of skeletal muscle extracellular matrix (ECM) has not been resolved. This study examines the effects of long term high fat diet (HFD) and DM on rodent skeletal muscle ECM were addressed. Five week old C57BL/6 male mice were randomly allocated to standard chow (control; n=26) or HFD (45% kcal fat; n=25). After 15 weeks chow and HFD mice were rendered diabetic (by low dose STZ, 65mg/kgx3d, I.P.) (DM, n=14; HFD+DM, n=14); nondiabetic mice acted as controls. Mice were euthanased after 30 weeks and plasma and quadriceps muscle were collected for analysis. Body weight and plasma insulin expectedly decreased with DM induction and increased with HFD (all p Disclosure S.F. Martinez-Huenchullan: None. L.A. Ban: None. A. Tao: None. S.S. Sutanto: None. C. Tam: None. S. McLennan: None. S.M. Twigg: Advisory Panel; Self; Abbott. Consultant; Self; Abbott. Advisory Panel; Self; Novo Nordisk Inc., Sanofi-Aventis, AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company. Speaker's Bureau; Self; AstraZeneca, Merck Sharp & Dohme Corp.. Other Relationship; Self; Abbott.

Background and Aim: The glycoprotein CD147 has a role in tumor progression, is readily detectable in the circulation, and is abundantly expressed in hepatocellular carcinoma (HCC). Advanced HCC patients are a heterogeneous group with some individuals having dismal survival. The aim of this study was to examine circulating soluble CD147 levels as a prognostic marker in HCC patients. Methods: CD147 was measured in 277 patients (110 HCC, 115 chronic liver disease, and 52 non‐liver disease). Clinical data included etiology, tumor progression, Barcelona Clinic Liver Cancer (BCLC) stage, and treatment response. Patients with HCC were stratified into two groups based upon the 75th percentile of CD147 levels (24 ng/mL). Results: CD147 in HCC correlated inversely with poor survival (P = 0.031). Increased CD147 predicted poor survival in BCLC stages C and D (P = 0.045), and CD147 levels >24 ng/mL predicted a significantly diminished 90‐day and 180‐day survival time (hazard ratio [HR] = 6.1; 95% confidence interval [CI]: 2.1–63.2; P = 0.0045 and HR = 2.8; 95% CI: 1.2–12.6; P = 0.028, respectively). In BCLC stage C, CD147 predicted prognosis; levels >24 ng/mL were associated with a median survival of 1.5 months compared with 6.5 months with CD147 levels ≤24 ng/mL (P = 0.03). CD147 also identified patients with a poor prognosis independent from treatment frequency, modality, and tumor size. Conclusions: Circulating CD147 is an independent marker of survival in advanced HCC. CD147 requires further evaluation as a potential new prognostic measure in HCC to identify patients with advanced disease who have a poor prognosis.

Chronic liver disease causes significant morbidity and mortality through progressive fibrosis, cirrhosis, and liver cancer. The classical theory of fibrogenesis has hepatic stellate cells (HSCs) as the principal and only significant source of abnormal extracellular matrix (ECM). Further, HSCs have the major role in abnormal ECM turnover. It is the death of hepatocytes, as the initial target of injury, that initiates a sequence of events including the recruitment of inflammatory cells and activation of HSCs. Following this initial response, the ongoing insult to hepatocytes is regarded as perpetuating injury, but otherwise, hepatocytes are regarded as "victims" and "bystanders" in progressive fibrosis. Recent developments, however, challenge this view and suggest the concept of the hepatocyte being an active participant in liver injury. It is clear now that hepatocytes undergo phenotypic changes, adapt to injury, and react to the altered microenvironment. In this review, we describe studies showing that hepatocytes contribute to progressive fibrosis by direct manipulation of the surrounding ECM and through signaling to effector cells, particularly HSCs and intrahepatic immune cells. Together, these findings suggest an active "accomplice" role for the hepatocyte in progressive liver fibrosis and highlight novel pathways that could be targeted for development of future anti-fibrotic therapies.

Recent scientific efforts have focused on the detrimental effects that obesity has on the metabolic function of skeletal muscles and whether exercise can improve this dysfunction. In this regard, adiponectin, with important metabolic functions (e.g. insulin-sensitizer and anti-inflammatory), has been recently described as a myokine that acts in an autocrine/paracrine manner. Earlier studies reported that muscle adiponectin could be induced by pro-inflammatory mediators (e.g. lipopolysaccharide), cytokines, and high-fat diets, providing a protective mechanism of this tissue against metabolic insults. However, when metabolic insults such as high-fat diets are sustained this protective response becomes dysregulated, making the skeletal muscle susceptible to metabolic impairments. Recent studies have suggested that exercise could prevent or even reverse this process. Considering that most scientific knowledge on adiponectin dysregulation in obesity is from the study of adipose tissue, the present review summarizes and discusses the literature available to date regarding the effects of obesity on skeletal muscle adiponectin induction, along with the potential effects of different exercise prescriptions on this response in an obesity context.

Background Intrahepatic expression of dipeptidyl peptidase-4 (DPP4), and circulating DPP4 (cDPP4) levels and its enzymatic activity, are increased in non-alcoholic fatty liver disease (NAFLD) and in type 2 diabetes mellitus and/or obesity. DPP4 has been implicated as a causative factor in NAFLD progression but few studies have examined associations between cDPP4 activity and NAFLD severity in humans. This study aimed to examine the relationship of cDPP4 activity with measures of liver disease severity in NAFLD in subjects with diabetes and/or obesity. Methods cDPP4 was measured in 106 individuals with type 2 diabetes who had transient elastography (Cohort 1) and 145 individuals with morbid obesity who had liver biopsy (Cohort 2). Both cohorts had caspase-cleaved keratin-18 (ccK18) measured as a marker of apoptosis. Results Natural log increases in cDPP4 activity were associated with increasing quartiles of ccK18 (Cohorts 1 and 2) and with median liver stiffness ≥10.3 kPa (Cohort 1) and significant fibrosis (F ≥ 2) on liver biopsy (Cohort 2). Conclusions In diabetes and/or obesity, cDPP4 activity is associated with current apoptosis and liver fibrosis. Given the pathogenic mechanisms by which DPP4 may progress NAFLD, measurement of cDPP4 activity may have utility to predict disease progression and DPP4 inhibition may improve liver histology over time. 摘要 背景： 在非酒精性脂肪性肝病（non-alcoholic fatty liver disease，NAFLD）、2型糖尿病和/或肥胖症患者中，二肽基肽酶-4（dipeptidyl peptidase-4，DPP4）的肝内表达、循环中的DPP4（circulating DPP4，cDPP4）水平以及它的酶活性都增加了。目前认为在NAFLD的进展过程中DPP4是一种致病因素，但是很少有研究调查人类cDPP4活性与NAFLD严重程度之间的关系。这项研究的目的是在糖尿病和/或肥胖症的受试者中调查cDPP4活性与测量到的NAFLD肝病严重程度之间的关系。 方法： 在106名接受瞬时弹性成像检查的2型糖尿病患者（队列1）以及145名接受肝脏活检的病态肥胖症患者（队列2）中测量cDPP4。对两个队列都测量了半胱天冬酶切割的角蛋白-18（caspase-cleaved keratin-18，ccK18）作为细胞凋亡指标。 结果： cDPP4活性的自然对数的增加与ccK18四分位数的增加相关（队列1与2），并且与肝脏硬度中位数≥ 10.3 kPa（队列1）以及肝脏活检有显著纤维化（F ≥ 2）（队列2）相关。 结论： 在糖尿病和/或肥胖症患者中，cDPP4活性与当前的肝细胞凋亡以及肝纤维化之间都有相关性。鉴于DPP4可能导致NAFLD进展的发病机制，测量cDPP4活性以预测疾病进展是一种实用的方法，并且随着时间的推移，抑制DPP4可以改善肝脏的组织学。

AimsPropolis is a naturally occurring anti-inflammatory bee derived protectant resin. We have previously reported that topically applied propolis reduces inflammation and improves cutaneous ulcer healing in diabetic rodents. The aim of this study was to determine if propolis shows efficacy in a pilot study of human diabetic foot ulcer (DFU) healing and if it is well tolerated.MaterialsSerial consenting subjects (n=24) with DFU ≥4week's duration had topical propolis applied at each clinic review for 6weeks. Post-debridement wound fluid was analyzed for viable bacterial count and pro-inflammatory MMP-9 activity. Ulcer healing data were compared with a matched control cohort of n=84 with comparable DFU treated recently at the same center.ResultsUlcer area was reduced by a mean 41% in the propolis group compared with 16% in the control group at week 1 (P

Fat cell differentiation (FCD) potentiates adipose cell characteristics including lipid storage and insulin sensitivity. In vitro, we have demonstrated that CCN2, also known as connective tissue growth factor (CTGF), inhibits FCD in NIH3T3-L1 cells and in adipocytes isolated from mouse epididymal fat pads. The aim of this study was to determine if the CCN2 effect on FCD is dependent on TGF-β and TGF-β downstream pathway signalling.NIH3T3-L1 cells were differentiated using standard methods with IBMX/Dex/Insulin. FCD at day 10 was confirmed by induced gene markers resistin and adiponectin and by lipid accumulation. Cells were treated at d0 with single dose active rhTGF-β1 (2 ng/mL), rhCCN2 (500 ng/mL) and/or TGF-β type 1 receptor blocker (SB431542, 5 μM). Early induction of FCD transcription factors: CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPAR-γ), were also determined.In an early time course from 2 h, single doses of rhTGF-β1 or rhCCN2 significantly inhibited by ~70 % the induction of C/EBP-β and -δ mRNA, and also nuclear protein levels otherwise seen during FCD, whereas only delayed effects on PPAR-γ, at 48 h, occurred. Furthermore, the CCN2 inhibition of FCD markers adiponectin and resistin and lipid accumulation by Oil red O stain were each prevented by TGF-β receptor blockade. Similar prevention was found using pan-specific anti-TGF-β neutralising antibody. CCN2 and TGF-β treatment each rapidly phosphorylated SMAD-3 signalling in early stages of FCD.This work shows novel findings that CCN2 effects on FCD are both TGF-β and TGF-β pathway dependent and are related to early effects on C/EBPs.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that binds to partners to mediate responses to environmental signals. To investigate its role in the innate immune system, floxed ARNT mice were bred with lysozyme M-Cre recombinase animals to generate lysozyme M-ARNT (LAR) mice with reduced ARNT expression. Myeloid cells of LAR mice had altered mRNA expression and delayed wound healing. Interestingly, when the animals were rendered diabetic, the difference in wound healing between the LAR mice and their littermate controls was no longer present, suggesting that decreased myeloid cell ARNT function may be an important factor in impaired wound healing in diabetes. Deferoxamine (DFO) improves wound healing by increasing hypoxia-inducible factors, which require ARNT for function. DFO was not effective in wounds of LAR mice, again suggesting that myeloid cells are important for normal wound healing and for the full benefit of DFO. These findings suggest that myeloid ARNT is important for immune function and wound healing. Increasing ARNT and, more specifically, myeloid ARNT may be a therapeutic strategy to improve wound healing.

Hepatocellular carcinoma (HCC) is a prevalent primary liver cancer that is derived from hepatocytes and is characterised by high mortality rate and poor prognosis. While HCC is driven by cumulative changes in the hepatocyte genome, it is increasingly recognised that the liver microenvironment plays a pivotal role in HCC propensity, progression and treatment response. The microenvironmental stimuli that have been recognised as being involved in HCC pathogenesis are diverse and include intrahepatic cell subpopulations, such as immune and stellate cells, pathogens, such as hepatitis viruses, and non-cellular factors, such as abnormal extracellular matrix (ECM) and tissue hypoxia. Recently, a number of novel environmental influences have been shown to have an equally dramatic, but previously unrecognized, role in HCC progression. Novel aspects, including diet, gastrointestinal tract (GIT) microflora and circulating microvesicles, are now being recognized as increasingly important in HCC pathogenesis. This review will outline aspects of the HCC microenvironment, including the potential role of GIT microflora and microvesicles, in providing new insights into tumourigenesis and identifying potential novel targets in the treatment of HCC.

Obesity-induced liver inflammation can drive insulin resistance. HDL has anti-inflammatory properties, so we hypothesized that low levels of HDL would perpetuate inflammatory responses in the liver and that HDL treatment would suppress liver inflammation and insulin resistance. The aim of this study was to investigate the effects of lipid-free apoAI on hepatic inflammation and insulin resistance in mice. We also investigated apoAI as a component of reconstituted HDLs (rHDLs) in hepatocytes to confirm results we observed in vivo. To test our hypothesis, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks and administered either saline or lipid-free apoAI. Injections of lipid-free apoAI twice a week for 2 or 4 weeks with lipid-free apoAI resulted in: i) improved insulin sensitivity associated with decreased systemic and hepatic inflammation; ii) suppression of hepatic mRNA expression for key transcriptional regulators of lipogenic gene expression; and iii) suppression of nuclear factor κB (NF-κB) activation. Human hepatoma HuH-7 cells exposed to rHDLs showed suppressed TNFα-induced NF-κB activation, correlating with decreased NF-κB target gene expression. We conclude that apoAI suppresses liver inflammation in HFD mice and improves insulin resistance via a mechanism that involves a downregulation of NF-κB activation.

The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis., Graphical abstract, Highlights • A novel synthetic fluorogenic substrate is proven to be FAP-specific. • Mice have higher levels of circulating FAP activity compared to baboons or humans. • No FAP activity was detected in urine or bile but bile contained high DPP4 activity. • FAP activity is greatest in pancreas, uterus, salivary gland, skin and lymph node. • FAP activity and protein is elevated in both serum and liver in human liver disease.

Neutrophils are important for wound repair, but their persistence can impair the healing process. Neutrophils express matrix metalloproteinases including MMP-9 and its regulator neutrophil gelatinase associated lipocalin (NGAL). Whether wounding affects neutrophil MMP-9 and NGAL in diabetic animals is not known. Skin wound tissue MMP-9 and NGAL was examined by qRT-PCR and immunohistochemistry in control, diabetic and insulin treated diabetic rats. The temporal expression of MMP-9 and NGAL mRNA, MMP-9 activity and the NGAL/MMP-9 complex was also investigated in an implant model and their circulating neutrophils. The cellular localisation of MMP-9 and NGAL was confirmed by immunofluorescence and the ability of glucose to regulate these factors was examined in isolated neutrophils. In skin wound tissue compared with control, diabetes increased neutrophil infiltration, NGAL mRNA and MMP-9 protein (P