Safety and Efficacy of Novel Incretin Co-agonist Cotadutide in Biopsy-proven Noncirrhotic MASH With Fibrosis.

BACKGROUND & AIMS: Cotadutide, a peptide co-agonist at the glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptors, has demonstrated robust improvements in body weight, glycemia, and hepatic fat fraction (HFF) in patients living with obesity and type 2 diabetes mellitus. METHODS: In PROXYMO, a 19-week randomized double-blind placebo-controlled trial, the safety and efficacy of cotadutide (600 µg, 300 µg) or placebo were evaluated in 74 participants with biopsy-proven noncirrhotic metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis. Analyses were performed using intent-to-treat and modified intent-to-treat population data. RESULTS: Dose- and time-dependent improvements in HFF, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), markers of liver health, and metabolic parameters were observed with significant improvements after 19 weeks with 600 µg ([least squares] mean difference vs placebo, [95% confidence interval] for absolute HFF: -5.0% [-8.5 to -1.5]; ALT: -23.5 U/L [-47.1 to -1.8]; AST: -16.8 U/L [-33.0 to -0.8]). Incidences of any grade treatment-emergent adverse events (TEAEs) were 91.7%, 76.9%, and 37.5% with cotadutide 600 µg, 300 µg, and placebo, respectively. The majority were gastrointestinal, mild to moderate in severity, and generally consistent with other incretins at this stage of development. TEAEs leading to treatment discontinuation were 16.7%, 7.7%, and 4.2% with cotadutide 600 µg, 300 µg, and placebo, respectively. CONCLUSIONS: PROXYMO provides preliminary evidence for the safety and efficacy of GLP-1/GCG receptor co-agonism in biopsy-proven noncirrhotic MASH with fibrosis, supporting further evaluation of this mechanism in MASH. CLINICAL TRIAL REGISTRATION NUMBER: NCT04019561.

Non-invasive assessment of hepatic steatosis by ultrasound-derived fat fraction in individuals at high-risk for metabolic dysfunction-associated steatotic liver disease.

AIMS: Given the increasing number of individuals developing metabolic dysfunction-associated steatotic liver disease (MASLD) and the low rate of those with progressive liver disease, there is a pressing need to conceive affordable biomarkers to assess MASLD in general population settings. Herein, we aimed to investigate the performance of the ultrasound-derived fat fraction (UDFF) for hepatic steatosis in high-risk individuals. METHODS: A total of 302 Europeans with obesity, type 2 diabetes, or a clinical history of hepatic steatosis were included in the analyses. Clinical, laboratory, and imaging data were collected using standardized procedures during a single screening visit in Rome, Italy. Hepatic steatosis was defined by controlled attenuation parameter (CAP) or ultrasound-based Hamaguchi's score. UDFF performance for hepatic steatosis was estimated by the area under the receiver operating characteristic curve (AUC). RESULTS: Overall, median (IQR) UDFF was 12% (7-20). UDFF was positively correlated with CAP (ρ = 0.73, p < 0.0001) and Hamaguchi's score (ρ = 0.79, p < 0.0001). Independent predictors of UDFF were circulating triglycerides, alanine aminotransferase (ALT), and ultrasound-measured visceral adipose tissue (VAT). UDFF AUC was 0.89 (0.85-0.93) and 0.92 (0.88-0.95) for CAP- and ultrasound-diagnosed hepatic steatosis, respectively. UDFF AUC for hepatic steatosis was higher than those of fatty liver index (FLI), hepatic steatosis index (HSI), CAP-score (CAPS), and ALT (p < 0.0001). Lower age, ALT, and VAT were associated with discordance between UDFF and ultrasound. CONCLUSIONS: UDFF may be a simple and accurate imaging biomarker to assess hepatic steatosis and monitor changes in hepatic fat content over time or in response to therapeutic interventions beyond clinical trials.

Cell Type-Specific Decrease of the Intrinsic Excitability of Motor Cortical Pyramidal Neurons in Parkinsonism.

The hypokinetic motor symptoms of Parkinson's disease (PD) are closely linked with a decreased motor cortical output as a consequence of elevated basal ganglia inhibition. However, whether and how the loss of dopamine (DA) alters the cellular properties of motor cortical neurons in PD remains undefined. We induced parkinsonism in adult C57BL/6 mice of both sexes by injecting neurotoxin, 6-hydroxydopamine (6-OHDA), into the medial forebrain bundle. By using ex vivo patch-clamp recording and retrograde tracing approach, we found that the intrinsic excitability of pyramidal tract neurons (PTNs) in the primary motor cortical (M1) layer (L)5b was greatly decreased in parkinsonism; but the intratelencephalic neurons (ITNs) were not affected. The cell type-specific intrinsic adaptations were associated with a depolarized threshold and broadened width of action potentials (APs) in PTNs. Moreover, the loss of midbrain dopaminergic neurons impaired the capability of M1 PTNs to sustain high-frequency firing, which could underlie their abnormal pattern of activity in the parkinsonian state. We also showed that the decreased excitability in parkinsonism was caused by an impaired function of both persistent sodium channels and the large conductance, Ca2+-activated K+ channels. Acute activation of dopaminergic receptors failed to rescue the impaired intrinsic excitability of M1 PTNs in parkinsonian mice. Altogether, our data demonstrated a cell type-specific decrease of the excitability of M1 pyramidal neurons in parkinsonism. Thus, intrinsic adaptations in the motor cortex provide novel insight in our understanding of the pathophysiology of motor deficits in PD.SIGNIFICANCE STATEMENT The degeneration of midbrain dopaminergic neurons in Parkinson's disease (PD) remodels the connectivity and function of cortico-basal ganglia-thalamocortical network. However, whether and how dopaminergic degeneration and the associated basal ganglia dysfunction alter motor cortical circuitry remain undefined. We found that pyramidal neurons in the layer (L)5b of the primary motor cortex (M1) exhibit distinct adaptations in response to the loss of midbrain dopaminergic neurons, depending on their long-range projections. Besides the decreased thalamocortical synaptic excitation as proposed by the classical model of Parkinson's pathophysiology, these results, for the first time, show novel cellular and molecular mechanisms underlying the abnormal motor cortical output in parkinsonism.

Addition of trans fat and alcohol has divergent effects on atherogenic diet-induced liver injury in rodent models of steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are common causes of chronic liver disease. The overlap between ALD and NAFLD suggests the existence of metabolic steatohepatitis. Development of in vivo models that reflect various aspects of human steatohepatitis is essential for drug discovery. We aimed to characterize several models of steatohepatitis (SH) and to investigate whether the pathology could be modulated. Sprague-Dawley rats were fed a high-fat diet (HFD) for 9 wk, followed by either a high-fat, high-cholesterol and cholate diet (HFC) or a HFC diet containing 13% trans fat (HFC-TF). A subset received 15% ethanol-water twice a week for 12 wk. Serum triglycerides, cholesterol, LDL, HDL, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and rodent NH2-terminal propeptide of type III collagen (rPRO-C3) were assessed. The liver was weighed and evaluated using modified Nonalcoholic Steatohepatitis Clinical Research Network histological score system criteria. All diets induced hepatomegaly, but only HFC-TF increased the size of visceral adipose tissue. Trans fat augmented HFC-induced dyslipidemia, and cholesterol was higher and HDL was lower in the HFC-TF groups. Alcohol lowered triglycerides in both dietary groups. HFC elevated ALT and AST, which were lowered by trans fat. All diets induced histological SH, addition of trans fat induced more steatosis but less inflammation. Inclusion of alcohol augmented the HFC-induced inflammation. All diets induced mild fibrosis. Inclusion of trans fat and alcohol significantly increased rPRO-C3. The addition of trans fat reduced the HFC-induced inflammation but augmented steatosis and dyslipidemia. Inclusion of alcohol induced a more inflammatory and fibrogenic phenotype.NEW & NOTEWORTHY Alcoholic liver disease and nonalcoholic liver disease share significant overlap, which suggests the existence of metabolic steatohepatitis. Trans fat has been implicated in steatohepatitis development. Here, we show that the addition of trans fat to an atherogenic diet results in a more steatotic but less inflammatory phenotype, whereas the addition of alcohol to an atherogenic diet augments the inflammatory and fibrogenic properties of the diet.

ADAPT: An Algorithm Incorporating PRO-C3 Accurately Identifies Patients With NAFLD and Advanced Fibrosis.

Given the high global prevalence of nonalcoholic fatty liver disease (NAFLD), the need for relevant noninvasive biomarkers and algorithms to accurately stage disease severity is a critical unmet medical need. Identifying those with advanced fibrosis (≥ F3) is the most crucial, as these individuals have the greatest risk of adverse, long-term, liver-related outcomes. We aimed to investigate the role of PRO-C3 (a marker of type III collagen formation) as a biomarker for advanced fibrosis in NAFLD. We measured PRO-C3 by enzyme-linked immunosorbent assay in two large independent cohorts with extensive clinical phenotyping and liver biopsy: 150 in the derivation and 281 in the validation cohort. A PRO-C3-based fibrosis algorithm that included age, presence of diabetes, PRO-C3, and platelet count (ADAPT) was developed. PRO-C3 increased with fibrosis stage (Rho 0.50; P < 0.0001) and was independently associated with advanced fibrosis (odds ratio = 1.05; 95% confidence interval [CI] 1.02-1.08; P = 0.003). ADAPT showed areas under the receiver operating characteristics curve of 0.86 (95% CI 0.79-0.91) in the derivation and 0.87 in the validation cohort (95% CI 0.83-0.91) for advanced fibrosis. This was superior to the existing fibrosis scores, aspartate aminotransferase to platelet ratio index (APRI), FIB-4, and NAFLD fibrosis score (NFS) in most comparisons. Conclusion: PRO-C3 is an independent predictor of fibrosis stage in NAFLD. A PRO-C3-based score (ADAPT) accurately identifies patients with NAFLD and advanced fibrosis and is superior to APRI, FIB-4, and NFS.

Collagen biology and non-invasive biomarkers of liver fibrosis.

There is an unmet need for high-quality liquid biomarkers that can safely and reproducibly predict the stage of fibrosis and the outcomes of chronic liver disease (CLD). The requirement for such markers has intensified because of the high global prevalence of diseases such as non-alcoholic fatty liver disease (NAFLD). In particular, there is a need for diagnostic and prognostic tools, as well as predictive biomarkers that reflect the efficacy of interventions, as described by the BEST criteria (Biomarkers, EndpointS, and other Tools Resource). This review covers the various liver collagens, their functional role in tissue homeostasis and delineates the common nomenclature for biomarkers based on BEST criteria. It addresses the common confounders affecting serological biomarkers, and describes defined collagen epitope biomarkers that originate from the dynamic processes of extracellular matrix (ECM) remodelling during liver injury.

Success rates and outcomes of laparoscopic mesh sacrohysteropexy.

BACKGROUND: Uterovaginal prolapse is a prevalent gynaecological issue, which can have a negative impact on the quality of life of women. Hysterectomy and vaginal repair are conventional treatments to address apical prolapse; however, women are increasingly requesting uterine-preserving alternatives. AIMS: This study aimed to evaluate the impact of laparoscopic mesh sacrohysteropexy on symptomatic prolapse from an Australian experience. MATERIALS AND METHODS: This retrospective cohort study presents outcomes of 157 patients who underwent laparoscopic mesh sacrohysteropexy at a private practice in South Australia during 2007-2017. Primary outcome is the success rate according to the pelvic organ prolapse quantification (POP-Q) system. Secondary measures included complication rates and patients identified as having Stages III-IV prolapse and their outcomes. RESULTS: The median age was 58 years (27-86 years), median parity was 2 (0-6), and median body mass index was 26.8 (23-29.9). One hundred and thirty-four women had a laparoscopic hysteropexy and concurrent vaginal prolapse repair and four women had an isolated laparoscopic hysteropexy. The mean pre-operative point C was 0.60. The mean change from pre-operative point C to post-operative point C was 7.6 cm (P < 0.01). Of the 136 patients (98.6%) seen at post-operative 4-6 weeks, all had Stage 0 POP-Q scores. Prolapse recurrence was observed in 22 patients, while 116 patients remained cured at their last follow-up. Prolapse recurrence was associated with anterior vaginal mesh, previous prolapse surgery, pre-operative Stage III-IV disease and number of vaginal deliveries. CONCLUSIONS: Laparoscopic mesh sacrohysteropexy is an effective and safe procedure with a high success rate comparable to available international data.

Demographic characteristics and pregnancy outcomes in adolescents - Experience from an Australian perinatal centre.

BACKGROUND: Adolescent pregnancy is defined as pregnancy in girls aged 10-19 years and can be associated with increased risks. AIM: To investigate obstetric and perinatal outcomes in a cohort of adolescent girls from a major Australian tertiary centre. MATERIALS AND METHODS: This was a nine-year retrospective cohort study of women who birthed at the Mater Mother's Hospital (MMH) in Brisbane, Australia between 1 January 2007 and 31 December 2015. The adolescent cohort was aged <19 years and the control group was aged 20-24 years. RESULTS: Over the study period the total study cohort comprised 8904 women. Of these, the adolescent cohort consisted of 1625 girls (18.2%) and the control group consisted of 7279 women (81.8%). Adolescents were more likely to be nulliparous, single, of Indigenous ethnicity or to have refugee status. They had higher rates of smoking, asthma, diabetes mellitus and thyroid disease. They were more likely to have an uncomplicated spontaneous vaginal delivery but were less likely to have an intact perineum and had higher rates of pre-term delivery and low birth weight babies. There were no differences in rates of postpartum haemorrhage. CONCLUSIONS: Teenage pregnancy results in poorer obstetric and perinatal outcomes. A focus on optimising maternal health care and providing culturally appropriate antenatal and intrapartum care is imperative to improving outcomes.

Safety, Tolerability, and Pharmacodynamics of AZD3366 (Optimized Human CD39L3 Apyrase) Alone and in Combination With Ticagrelor and Acetylsalicylic Acid: A Phase 1, Randomized, Placebo-Controlled Study.

BACKGROUND: ADP and ATP are importantly involved in vascular and thrombotic homeostasis, via multiple receptor pathways. Blockade of ADP P2Y12 receptors inhibits platelet aggregation and represents an effective cardiovascular disease prevention strategy. AZD3366 (APT102), a long-acting recombinant form of an optimized CD39L3 human apyrase, has effectively reduced ATP, ADP, and platelet aggregation and provided tissue protection in preclinical models, features that could be very beneficial in treating patients with cardiovascular disease. METHODS AND RESULTS: We conducted this phase 1, first-in-human study of single ascending doses of intravenous AZD3366 or placebo, including doses added to dual antiplatelet therapy with ticagrelor and acetylsalicylic acid. The primary objective was safety and tolerability; secondary and exploratory objectives included pharmacokinetics, pharmacodynamics (measured as inhibition of platelet aggregation), adenosine diphosphatase (ADPase) activity, and ATP/ADP metabolism. In total, 104 participants were randomized. AZD3366 was generally well tolerated, with no major safety concerns observed. ADPase activity increased in a dose-dependent manner with a strong correlation to AZD3366 exposure. Inhibition of ADP-stimulated platelet aggregation was immediate, substantial, and durable. In addition, there was a prompt decrease in systemic ATP concentration and an increase in adenosine monophosphate concentrations, whereas ADP concentration appeared generally unaltered. At higher doses, there was a prolongation of capillary bleeding time without detectable changes in the ex vivo thromboelastometric parameters. CONCLUSIONS: AZD3366 was well tolerated in healthy participants and demonstrated substantial and durable inhibition of platelet aggregation after single dosing. Higher doses prolonged capillary bleeding time without detectable changes in ex vivo thromboelastometric parameters. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique Identifier: NCT04588727.

Lipid availability influences ferroptosis sensitivity in cancer cells by regulating polyunsaturated fatty acid trafficking.

Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic acid and adrenic acid. These PUFAs then accumulate in phospholipids, particularly ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools dictates their sensitivity to ferroptosis.

Reduced thalamic excitation to motor cortical pyramidal tract neurons in parkinsonism.

Degeneration of midbrain dopaminergic (DA) neurons alters the connectivity and functionality of the basal ganglia-thalamocortical circuits in Parkinson's disease (PD). Particularly, the aberrant outputs of the primary motor cortex (M1) contribute to parkinsonian motor deficits. However, cortical adaptations at cellular and synaptic levels in parkinsonism remain poorly understood. Using multidisciplinary approaches, we found that DA degeneration induces cell subtype- and input-specific reduction of thalamic excitation to M1 pyramidal tract (PT) neurons. At molecular level, we identified that N-methyl-d-aspartate (NMDA) receptors play a key role in mediating the reduced thalamocortical excitation to PT neurons. At circuit level, we showed that the reduced thalamocortical transmission in parkinsonian mice can be rescued by chemogenetically suppressing basal ganglia outputs. Together, our data suggest that cell subtype- and synapse-specific adaptations in M1 contribute to altered cortical outputs in parkinsonism and are important aspects of PD pathophysiology.

Stage-dependent expression of fibrogenic markers in alcohol-related liver disease.

Alcohol-related liver disease (ALD) is the primary cause of liver-related mortality and morbidity. Liver fibrosis remains the best validated surrogate marker for patient prognosis and is usually assessed in liver biopsies using histochemical stains detecting collagen as the major extracellular matrix (ECM) component in ALD. Distinction of collagen subtypes is of clinical interest, as they, including their cleavage products, have different functions. Changes in production, distribution, and composition of specific collagen subtypes and other extracellular matrix (ECM) components as well as markers for their main cellular source (activated hepatic stellate cells (aHSCs) and myofibroblasts (MFBs)) have not been fully elucidated in ALD. Our aims were to investigate the stage-dependent expression of collagen subtypes and markers for aHSCs and MFBs in ALD. We included liver biopsies from 49 ALD patients with fibrosis stages F0-F4. Biopsies were classified according to the semi-quantitative non-alcoholic fatty liver disease (NAFLD) activity score (NAS-CRN). Slides were stained with H&E, Sirius Red, and immunohistochemically with antibodies against collagen types I, III, IV and VI, and aHSC/MFB markers α-SMA, osteonectin, and CD271. Expression was examined using automated digital imaging analysis (DIA), by calculating the positive area relative to the total liver biopsy area (proportionate area). Likewise, collagen-proportionate area (CPA) was assessed using DIA of Sirius Red stained slides. CPA correlated highly with fibrosis stage (rs = 0.88, P = 5.9E-17) and moderately with activity score (rs = 0.58, P = 0.00001). Collagen type I proportionate area increased from 0.3% (± 0.1) at F1 to 10.2% (± 1.6) at F4 (P < 0.001). Collagen type III proportionate area increased from 0.6% (± 0.2) at F0 to 11.9% (± 1.7) at F4 (P < 0.001). Collagen type IV proportionate area increased from 17.0% (± 2.5) at F0 to 27.0% (± 3.9) at F4 (P = 0.079). Collagen type VI proportionate area increased from 14.8% (± 1.4) at F0 to 31.4% (± 2.4) at F4 (P < 0.001). Proportionate areas for α-SMA and CD271 increased from 10.7% (± 1.6) and 6.5% (± 1.0) at F0 to 29.5% (± 3.4) and 22.1% (± 2.2) at F4 (P < 0.001). Proportionate area for osteonectin increased from 1.4-1.8% at F0-F2 to 3.1% (± 0.5) at F3 and 3.2% (± 0.6) at F4 (P < 0.05). In conclusion, our data indicate that collagen types I, III and VI and the aHSC/MFB markers α-SMA and CD271 show a stage-dependent increase in ALD. In early ALD, collagen types IV and VI are important components of the perisinusoidal and pericellular fibrosis. Immunohistochemistry is a valuable additional tool to characterize the ECM changes in ALD. Further research is needed to explore the functional role of CD271 and the stage-dependent expression of other collagen subtypes in ALD.

Synaptic location is a determinant of the detrimental effects of α-synuclein pathology to glutamatergic transmission in the basolateral amygdala.

The presynaptic protein α-synuclein (αSyn) has been suggested to be involved in the pathogenesis of Parkinson's disease (PD). In PD, the amygdala is prone to develop insoluble αSyn aggregates, and it has been suggested that circuit dysfunction involving the amygdala contributes to the psychiatric symptoms. Yet, how αSyn aggregates affect amygdala function is unknown. In this study, we examined αSyn in glutamatergic axon terminals and the impact of its aggregation on glutamatergic transmission in the basolateral amygdala (BLA). We found that αSyn is primarily present in the vesicular glutamate transporter 1-expressing (vGluT1+) terminals in the mouse BLA, which is consistent with higher levels of αSyn expression in vGluT1+ glutamatergic neurons in the cerebral cortex relative to the vGluT2+ glutamatergic neurons in the thalamus. We found that αSyn aggregation selectively decreased the cortico-BLA, but not the thalamo-BLA, transmission; and that cortico-BLA synapses displayed enhanced short-term depression upon repetitive stimulation. In addition, using confocal microscopy, we found that vGluT1+ axon terminals exhibited decreased levels of soluble αSyn, which suggests that lower levels of soluble αSyn might underlie the enhanced short-term depression of cortico-BLA synapses. In agreement with this idea, we found that cortico-BLA synaptic depression was also enhanced in αSyn knockout mice. In conclusion, both basal and dynamic cortico-BLA transmission were disrupted by abnormal aggregation of αSyn and these changes might be relevant to the perturbed cortical control of the amygdala that has been suggested to play a role in psychiatric symptoms in PD.

Review article: the signalling and functional role of the extracellular matrix in the development of liver fibrosis.

BACKGROUND: Patients with liver fibrosis show a large heterogeneity, and for that reason effective treatments are still lacking. Emerging data suggest that there is more to fibrosis than previously understood. Opposed to earlier belief of being a passive scaffold for cells to reside in, the extracellular matrix (ECM) is now known to hold both signalling and functional properties important for the development of fibrosis. The interaction between the ECM and the collagen-producing cells determines the course of the disease but is still poorly understood. Exploring the dynamics of this interplay will aid in the development of effective treatments. AIM: To summarise and discuss the latest advances in the pathogenesis of liver fibrosis as well as key mediators of early disease progression. METHODS: Through literature search using databases including PubMed and Google Scholar, manuscripts published between 1961 and 2019 were included to assess both well-established and recent theories of fibrosis development. Both pre-clinical and clinical studies were included. RESULTS: Fibrosis alters the structure of the ECM releasing signalling fragments with the potential to escalate disease severity. In a diseased liver, hepatic stellate cells and other fibroblasts, together with hepatocytes and sinusoidal cells, produce an excessive amount of collagens. The cell-to-collagen interactions are unique in the different liver aetiologies, generating ECM profiles with considerable patient-monitoring potential. CONCLUSIONS: The local milieu in the injured area affects the course of fibrosis development in a site-specific manner. Future research should focus on the dissimilarities in the ECM profile between different aetiologies of liver fibrosis.

Biochemical and histological characterisation of an experimental rodent model of non-alcoholic steatohepatitis - Effects of a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist and a glucagon-like peptide-1 analogue.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a prevalent disease that is highly associated with the metabolic syndrome and type II diabetes. The development of in vivo models that reflect all nuances of the human NASH pathology is essential for drug discovery and development. We aimed to further characterise a dietary induced model of NASH both biochemically and histologically. In addition, we also investigated whether pioglitazone and liraglutide, drugs that have both been investigated as potential NASH treatments, could modulate the pathological changes induced by the NASH diet. Furthermore, to aid the translation of data from pre-clinical in vivo models, we aimed to adapt the NASH Clinical Research Network (CRN) histological score system for use in rodent studies. METHODS: Sprague Dawley rats were fed a high-fat diet (HFD) for 9 weeks, after which they were switched to a high fat, high cholesterol and cholate diet (HFCC) for 12 weeks. The rats were divided into treatment groups, receiving either 30 mg/kg pioglitazone p.o. SID or liraglutide s.c. 200 µg/kg BID or the respective vehicles. Serum levels of triglycerides (TG), cholesterol (Chol), LDL, HDL, AST and ALT, as well as body weight were assessed in all subjects. Upon termination, the liver was weighed and evaluated histologically using modified NASH-CRN criteria. RESULTS: HFCC feeding induced severe hepatic injury and hepatomegaly as indicated by significant increases in AST, ALT and an increased liver weight. Additionally, HFCC feeding induced dyslipidaemia, significant increases in circulating cholesterol and LDL were observed. No obesogenic effect of the HFCC diet was observed, though the diet did induce insulin resistance. Histological analysis showed that the HFCC diet induced several NASH like features, though it did not induce the development of severe fibrosis. However, microgranulomas were often prevalent in addition to lobular inflammatory foci. Pioglitazone showed little efficacy upon both biochemical and histological features. However, liraglutide induced weight loss, improved glycaemic control, reduced ALT and AST and showed some beneficial effects upon steatosis and lobular inflammation. CONCLUSION: Similar to previous reports we have shown that the atherogenic diet, HFCC, induces a phenotype akin to that seen in human NASH patients. Despite inducing all histological features of NASH, HFCC feeding does not promote the development of significant fibrosis within rodents. Pioglitazone and liraglutide have been investigated as potential NASH treatments. Within this model of NASH we have shown that pioglitazone has little efficacy, whereas liraglutide reduced the levels of circulating aminotransferases and had some beneficial effects upon NASH histological parameters.

Serological Assessment of Activated Fibroblasts by alpha-Smooth Muscle Actin (α-SMA): A Noninvasive Biomarker of Activated Fibroblasts in Lung Disorders.

OBJECTIVES: Remodeling of the extracellular matrix (ECM) is a key event in different lung disorders, such as fibrosis and cancer. The most common cell type in the connective tissue is fibroblasts, which transdifferentiate into myofibroblasts upon activation. All myofibroblasts express α-SMA, which has been found to be upregulated in lung fibrosis and cancer. We evaluated the potential of α-SMA as a noninvasive biomarker of activated fibroblasts in lung fibrosis and cancer. METHODS: A monoclonal antibody was raised against the N-terminal of α-SMA, and a novel competitive enzyme-linked immunosorbent assay (ELISA) measuring α-SMA was developed and technically characterized. Levels of α-SMA were measured in the fibroblast model, "scar-in-a-jar", and in serum from patients with idiopathic pulmonary fibrosis (IPF), chronic obstructive lung disorder (COPD) and non-small cell lung cancer (NSCLC) belonging to two different cohorts. RESULTS: The novel α-SMA assay was developed and validated as technically robust. Based on the scar-in-a-jar results, α-SMA was only present in the fibroblasts activated by TGF-ß. In cohort 1, levels of α-SMA were significantly higher in IPF, COPD and NSCLC patients compared to healthy controls (P = 0.04, P = 0.001 and P <0.0001, respectively). The area under the receiver operating characteristics (AUROC) for separation of healthy controls from IPF patients was 0.865, healthy controls from COPD patients was 0.892 and healthy controls from NSCLC patients was 0.983. In cohort 2, levels of α-SMA were also significantly higher in NSCLC patients compared to healthy controls (P = 0) and the AUROC for separating NSCLC and healthy controls was 0.715. CONCLUSIONS: In this study we developed and validated a robust competitive ELISA assay targeting the N-terminal of α-SMA. The level of α-SMA was upregulated when adding TGF-ß, indicating that α-SMA is increased in activated fibroblasts. The level of α-SMA in circulation was significantly higher in patients with IPF, COPD and NSCLC compared to healthy controls. This assay could potentially be used as a novel noninvasive serological biomarker for lung disorders by providing a surrogate measure of activated fibroblasts.

Obstetric and perinatal outcomes for twin pregnancies in adolescent girls.

This was a nine-year retrospective cohort study to investigate obstetric and perinatal outcomes in a cohort of adolescent girls with twin pregnancies from a major Australian tertiary centre in Brisbane, Australia. The adolescent cohort was aged <19 years and the control group was aged 20-24 years. The total study cohort comprised of 183 women. Of these, the adolescent cohort contained 29 girls (15.8%) and the control group comprised of 154 women (84.2%). Adolescent girls were less likely to delivery via an elective caesarean section compared to women in the control group (10.3% vs. 25.7%, p < 0.001). There were no differences in duration of labour, post-partum haemorrhage or perineal trauma rates. After controlling for the confounding effects of parity, chronicity and birth weight, birth <28 weeks remained significant (aOR 11.20, 95% CI 2.97-42.18, p < 0.001) for the adolescent cohort. There was a higher proportion of adolescents whose babies had an adverse composite perinatal outcome (87.9% vs. 69.5%, OR 3.20 95% CI: 1.40-7.31, p = 0.01) however significance was lost after adjusting for parity, chorionicity, birthweight and gestation at birth (aOR 3.27 95% CI: 0.95-11.31, p = 0.06). Our results show that obstetric and perinatal outcomes for twin pregnancies in teenagers were broadly similar compared to controls although the risk of extreme preterm birth was increased after controlling for confounders.

Genetic Modulation at the Neural Microelectrode Interface: Methods and Applications.

The use of implanted microelectrode arrays (MEAs), in the brain, has enabled a greater understanding of neural function, and new treatments for neurodegenerative diseases and psychiatric disorders. Glial encapsulation of the device and the loss of neurons at the device-tissue interface are widely believed to reduce recording quality and limit the functional device-lifetime. The integration of microfluidic channels within MEAs enables the perturbation of the cellular pathways, through defined vector delivery. This provides new approaches to shed light on the underlying mechanisms of the reactive response and its contribution to device performance. In chronic settings, however, tissue ingrowth and biofouling can obstruct or damage the channel, preventing vector delivery. In this study, we describe methods of delivering vectors through chronically implanted, single-shank, "Michigan"-style microfluidic devices, 1⁻3 weeks, post-implantation. We explored and validated three different approaches for modifying gene expression at the device-tissue interface: viral-mediated overexpression, siRNA-enabled knockdown, and cre-dependent conditional expression. We observed a successful delivery of the vectors along the length of the MEA, where the observed expression varied, depending on the depth of the injury. The methods described are intended to enable vector delivery through microfluidic devices for a variety of potential applications; likewise, future design considerations are suggested for further improvements on the approach.