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High occurence of liver diseases (toxic, viral hepatitis, liver failure, cirrhosis) requires urgent search of new methods for management of the hepatobiliary diseases. At the present time, the role of immune mechanisms in pathogenesis of diffuse toxic liver damage is not finally clarified. The model of toxic hepatitis induced by carbon tetrachloride (CCl4) is widely known, but this approach allows us to perform complex evaluation and develop the methods for adequate correction of liver disorders in experimental model, which is not always feasible in clinical setting. To design a model of diffuse toxic liver damage, the CCl4 oil solution was used, having been administered intraperitoneally to experimental animals, at a single dose of 50 mg per 100 g body mass. Aiming for correction of toxic liver damage, the injections of aminophthalhydrazide (APH) to experimental animals were carried out intramuscularly at the dose of 2 mg/kg over the terms of experiment. An evaluation of the role of sinusoidal cells (SC) and cytokine production at the local and systemic level were carried out in the model of toxic liver damage caused by CCl4 and its correction by APH treatment. In the course of developing diffuse toxic liver damage induced by CCl4, the production of proinflammatory cytokines TNFα, IL-1α and IL-18 was enhanced at the local level, whereas an increase in TNFα concentration was observed in blood plasma. Following aminophthalhydrazide (APH) administration, the concentrations of proinflammatory cytokines (TNFα and IL-18) decreased at system level, along with locally decreased levels of IL-6 and IFNγ. Changes in the functional state of immunocompetent cells, which include sinusoidal cells (SC), have a significant impact on the development of pathological processes in the liver. The results of our study presume that, over the early periods of toxic impact upon liver tissue, the number of SCs increases both due to influx of blood monocytes and mature macrophages from the pe

The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin-mediated (mTORmediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation-related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis - all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.Copyright © 2019, American Society for Clinical Investigation.

Introduction: Urinary oxygen tension (PO2) may provide a useful estimate of renal medullary PO2 and thus risk of acute kidney injury (AKI). However, the relationship between urinary PO2 and medullary PO2 could be confounded by variations in urine flow and arterial PO2. In the current study we exploited data we had previously generated, from experimental studies in ovine septic AKI and in patients undergoing cardiac surgery requiring cardiopulmonary bypass (CPB), to perform a detailed analysis of the influences of urine flow and arterial PO2 on urinary PO2. We also investigated the potential for simultaneous measurement of urine flow and urinary PO2, during CPB, to improve prediction of AKI over that provided by measurement of each variable on its own. Method(s): In ovine septic AKI urine flow, urinary PO2, arterial PO2, and medullary PO2 were measured. In human patients undergoing cardiac surgery requiring CPB urinary PO2 and arterial PO2 were measured continuously and urine flow was estimated every 5 minutes. Result(s): In 28 conscious septic sheep breathing room air, urinary PO2 was more closely correlated with medullary PO2 (r2=0.29) than with arterial PO2 (r2=0.06) or urine flow (r2=0.03). The difference between medullary PO2 and urinary PO2 varied little with urine flow or arterial PO2. In 20 patients, urine flow increased abruptly from 2.81 +/- 0.24 mL/min to 6.53 +/- 0.30 mL/min upon commencement of CPB, usually coincident with a fall in urinary PO2 (Fig. 1). During hyperoxic CPB high values of urinary PO2 were often observed at low urine flow. In 65 patients, low urinary PO2 during CPB (<10 mmHg at any time during CPB) was associated with greater (4.5-fold) risk of AKI. However, the level of urine flow on bypass was not significantly associated with risk of AKI. [Figure presented] Conclusion(s): We developed a method for virtually continuous measurement of urine flow in the operating theatre. We found that urinary PO2 provides a relatively robust estimate of

Plasma cells can provide lifelong immunity by the continued production of high-affinity, antigen-specific antibody. Low et al. reveal that the transcription factor IRF4 acts within established plasma cells to prevent apoptosis and maintain cellular identity and mitochondrial homeostasis. The transcription factor interferon regulatory factor 4 (IRF4) is critical for the development, maintenance, and function of plasma cells. The mechanism by which IRF4 exerts its action in mature plasma cells has been elusive due to the death of all such cells upon IRF4 loss. While we identify apoptosis as a critical pathway for the death of plasma cells caused by IRF4 loss, we also determine that IRF4 did not regulate the intrinsic apoptotic pathway directly. By using an inducible IRF4 deletion system in the presence of the overexpression of anti-apoptotic BCL2, we identify genes whose expression is coordinated by IRF4 and that in turn specify plasma cell identity and mitochondrial homeostasis.Copyright © 2019 The Authors

Background: Fetal growth restriction (FGR) is a serious pregnancy complication associated with increased risk of adverse neurodevelopment and neuromorbidity. Current imaging techniques, including conventional magnetic resonance imaging (MRI), are not sensitive enough to detect subtle structural abnormalities in the FGR brain. We examined whether advanced MRI analysis techniques have the capacity to detect brain injury (particularly white matter injury) caused by chronic hypoxia-induced fetal growth restriction in newborn preterm lambs. Method(s): Surgery was undertaken in twin bearing pregnant ewes at 88-90 days gestation (term = 150 days) to induce FGR in one fetus. At 127 days gestation (~32 weeks human brain development), FGR and control (appropriate for gestational age, AGA) lambs were delivered by caesarean section, intubated and ventilated. Conventional and advanced brain imaging was conducted within the first two hours of life using a 3T MRI scanner. T1-weighted (T1w) and T2-weighted (T2w) structural imaging, magnetic resonance spectroscopy (MRS), and diffusion MRI (dMRI) data were acquired. Diffusion tensor imaging (DTI) modelling and analysis of dMRI data included the following regions of interest (ROIs): subcortical white matter, periventricular white matter, cerebellum, hippocampus, corpus callosum and thalamus. Fixel-based analysis of 3-tissue constrained spherical deconvolution (CSD) of the dMRI data was performed and compared between FGR and AGA lambs. Lambs were euthanised immediately after the scans and brain histology performed in the regions of interest to correlate with imaging. Result(s): FGR and AGA lamb (body weight, mean (SD): 2.2(0.5) vs. 3.3(0.3) kg, p = .002) MRI brain scans were analysed. There were no statistically significant differences observed between the groups in conventional T1w, T2w or MRS brain data. Mean, axial and radial diffusivity, and fractional anisotropy indices obtained from DTI modelling also did not show any statistical

Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.Copyright © 2019, The Author(s).

A hallmark feature of schizophrenia is altered high frequency neural oscillations, including reduced auditory-evoked gamma oscillatory power, which is underpinned by parvalbumin (PV) interneuron dysfunction. Maternal immune activation (MIA) in rodents models an environmental risk factor for schizophrenia and recapitulates these PV interneuron changes. This study sought to link reduced PV expression in the MIA model with alterations to auditory-evoked gamma oscillations and transcript expression. We further aligned transcriptional findings from the animal model with human genome sequencing data. We show that MIA, induced by the viral mimetic, poly-I:C in C57Bl/6 mice, caused in adult offspring reduced auditory-evoked gamma and theta oscillatory power paralleled by reduced PV protein levels. We then showed the Arx gene, critical to healthy neurodevelopment of PV interneurons, is reduced in the forebrain of MIA exposed mice. Finally, in a whole-genome sequenced patient cohort, we identified a novel missense mutation of ARX in a patient with schizophrenia and in the Psychiatric Genomics Consortium 2 cohort, a nominal association of proximal ARX SNPs with the disorder. This suggests MIA, as a risk factor for schizophrenia, may be influencing Arx expression to induce the GABAergic dysfunction seen in schizophrenia and that the ARX gene may play a role in the prenatal origins of schizophrenia pathophysiology.Copyright © 2019 Elsevier Inc.

Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC. In genetically engineered and xenograft (human cell line and patient-derived) KrasG12D-driven LAC models, the specific blockade of ADAM17, including with a non-toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro-tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL-6R, to release soluble IL-6R that drives IL-6 trans-signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in KrasG12D-driven LAC was independent of bone marrow-derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho-ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS-driven LAC and provide the rationale to employ ADAM17-based therapeutic strategies for targeting KRAS mutant cancers.Copyright © 2019 The Authors. Published under the terms of the CC BY 4.0 license

Introduction: Mineralocorticoid receptor antagonists (MRAs) reduce hypertension, inflammation and tissue injury in human and experimental diabetes. Furthermore, inflammation and injury in diabetic kidneys and hearts is dependent on infiltrating macrophages. Therefore, we hypothesized that the tissue protective effects of MRAs in diabetes is partially due to inhibition of MR signaling in macrophages. In this study, we used mice genetically deficient in myeloid MR to evaluate whether macrophage mineralocorticoid receptor signaling contributes to the development of kidney and cardiac disease in hypertensive diabetic mice. Method(s): Transgenic mice with intact myeloid MR (MRflox/flox) or myeloid MR deficiency (MRflox/flox LysMCre) were developed on the hypertensive endothelial nitric oxide synthase deficient (Nos3-/-) mouse strain. Groups of these mice (n=13) were made diabetic with multiple low dose injections of streptozotocin (STZ) and were assessed after 15 weeks for the development of hypertension (systolic blood pressure), nephropathy (albuminuria, renal function impairment and kidney damage) and cardiomyopathy (cardiac dysfunction-echocardiography and cardiac damage). Result(s): MR intact Nos3-/- mice with diabetes developed hypertension, kidney damage (macrophage accumulation, albuminuria, renal function impairment, hypertrophy, tubular injury, fibrosis), and cardiac damage (Left ventricular (LV) fractional shortening, macrophage accumulation, fibrosis), but no neutrophil infiltrate. In comparison, myeloid MR deficient Nos3-/- mice had equivalent diabetes, hypertension and albuminuria, but showed protection against loss of renal function (plasma cystatin-C reduced 48% vs MR intact mice with diabetes, p<0.01) and cardiac dysfunction (LV fractional shortening reduced 26% in MR intact mice with diabetes, p<0.01, but not in myeloid MR deficient mice with diabetes). The cardiac protection resulting from myeloid MR deficiency included the prevention of a diabetes-ind

Introduction: Streptozotocin (STZ)-induced diabetes in male mice lacking endothelial nitric oxide synthase (Nos3-/-) is a widely used model for examining the development of diabetic kidney disease (DKD). Female mice are partially protected from developing STZ-induced diabetes due to their abundance of 17beta-estradiol and lack of testosterone, and are not normally used for studies of DKD. In this study, we sought to identify whether male and female Nos3-/- mice with equivalent diabetes develop comparable renal injury, facilitating the potential to use both sexes in studies of DKD. Method(s): Male and female Nos3-/- mice were made diabetic with 5 or 6 intraperitoneal injections of STZ (55mg/kg/day), respectively. Groups of male and female mice with equivalent diabetes at week 3 after STZ were assessed for diabetic kidney injury (albuminuria, podocyte loss, inflammation, renal function impairment, glomerulosclerosis, tubular injury) at week 8. Result(s): STZ-treated male and female Nos3-/- mice maintained similar elevated blood glucose levels between weeks 3 and 8 and had equivalent levels of glycated hemoglobin (HbA1c) and hypertension. At week 8, Urine albumin/creatinine levels were increased 8-fold in both diabetic males and females compared to non-diabetic, and were accompanied by a similar (17-20%) loss of podocytes. In diabetic males and females, plasma cystatin-C levels were increased by 33% at week 8, demonstrating similar renal function impairment. Glomerular area of collagen IV immunostaining was also similarly increased by 30% in both genders, signifying equal development of glomerulosclerosis. In addition, kidney mRNA expression of proinflammatory markers (CD68, TNF-alpha, CCL2), profibrotic markers (TGF-beta, Collagen I, Collagen IV, Fibronectin) and KIM-1 were equally elevated in males and females, indicating similar renal inflammation, renal fibrosis and tubular injury. Conclusion(s): Our study shows that equivalent diabetes induces a similar onset of D

The cellular responses induced by mitochondrial dysfunction remain elusive. Intrigued by the lack of almost any glomerular phenotype in patients with profound renal ischemia, we comprehensively investigated the primary sources of energy of glomerular podocytes. Combining functional measurements of oxygen consumption rates, glomerular metabolite analysis, and determination of mitochondrial density of podocytes in vivo, we demonstrate that anaerobic glycolysis and fermentation of glucose to lactate represent the key energy source of podocytes. Under physiological conditions, we could detect neither a developmental nor late-onset pathological phenotype in podocytes with impaired mitochondrial biogenesis machinery, defective mitochondrial fusion-fission apparatus, or reduced mtDNA stability and transcription caused by podocyte-specific deletion of Pgc-1alpha, Drp1, or Tfam, respectively. Anaerobic glycolysis represents the predominant metabolic pathway of podocytes. These findings offer a strategy to therapeutically interfere with the enhanced podocyte metabolism in various progressive kidney diseases, such as diabetic nephropathy or focal segmental glomerulosclerosis (FSGS). Glomerular podocytes form the third and most outer layer of the kidney filtration barrier responsible for restricting the passage of proteins into the urine. Brinkkoetter et al. show that podocyte metabolism primarily relies on anaerobic glycolysis and the fermentation of glucose to lactate.Copyright © 2019 The Authors

Pulmonary hypertension secondary to bronchopulmonary dysplasia (BPD-PH) represents a major complication of BPD in extremely preterm infants for which there are currently no safe and effective interventions. The abundance of interleukin-1 (IL-1) is strongly correlated with the severity and long-term outcome of BPD infants and we have previously shown that IL-1 receptor antagonist (IL-1Ra) protects against murine BPD; therefore, we hypothesized that IL-1Ra may also be effective against BPD-PH. We employed daily injections of IL-1Ra in a murine model in which BPD/BPD-PH was induced by antenatal LPS and postnatal hyperoxia of 65% O2. Pups reared in hyperoxia for 28 days exhibited a BPD-PH-like disease accompanied by significant changes in pulmonary vascular morphology: micro-CT revealed an 84% reduction in small vessels (4-5 mum diameter) compared to room air controls; this change was prevented by IL-1Ra. Pulmonary vascular resistance, assessed at day 28 of life by echocardiography using the inversely-related surrogate marker time-to-peak-velocity/right ventricular ejection time (TPV/RVET), increased in hyperoxic mice (0.27 compared to 0.32 in air controls), and fell significantly with daily IL-1Ra treatment (0.31). Importantly, in vivo cine-angiography revealed that this protection afforded by IL-1Ra treatment for 28 days is maintained at day 60 of life. Despite an increased abundance of mediators of pulmonary angiogenesis in day 5 lung lysates, namely vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1), no difference was detected in ex vivo pulmonary vascular reactivity between air and hyperoxia mice as measured in precision cut lung slices, or by immunohistochemistry in alpha-smooth muscle actin (alpha-SMA) and endothelin receptor type-A (ETA) at day 28. Further, on day 28 of life we observed cardiac fibrosis by Sirius Red staining, which was accompanied by an increase in mRNA expression of galectin-3 and CCL2 (chemokine (C-C motif) ligand 2) in whole h

Introduction: Glomerular disease accounts for more than 90% of chronic kidney disease (CKD), which is predicted to cost the Australian health care system AUD $20 billion in 2020. There is increasing evidence that podocytes play a vital role in the development of most glomerular disease. In a landmark study by Wharram et al (2005), it was shown that podocyte depletion <20% resulted in remaining podocytes being able to adapt and repair the glomerular filtration barrier, preventing the development of glomerulosclerosis. However more severe podocyte depletion resulted in focal segmental glomerulosclerosis (FSGS), with sustained proteinuria and reduced renal function. This provided strong support for the podocyte depletion hypothesis, which proposes that if sufficient podocyte depletion occurs, the glomerulus will not be able to recover and thus result in progressive FSGS. To investigate this concept, it is important to develop animal models of varying and predictable podocyte depletion and FSGS. Here we describe a mouse model of diphtheria toxin (DT)-induced podocyte depletion that results in differing degrees of podocyte loss, proteinuria and FSGS. Method(s): PodCreiDTR mice that express human DT receptors only in podocytes were injected with varying doses of DT or with saline. Experimental animals received two injections of DT, 12 days apart. The cumulative total dose varied between 100 - 750 ng/kg DT. Urine was collected at regular time points for analysis of urinary total protein/creatinine (UTP/Cre). Peak UTP/Cre was observed at 21 days following the first DT injection. Mice were killed 9 weeks after the first DT injection, and kidneys were harvested. The left kidney was assessed for FSGS (glomerulosclerotic index; GSI) and the right kidney was used for 3D imaging to count the total number of podocytes in individual whole glomeruli. Result(s): Mice presented with a variable peak UTP/Cre between 1946-9244 g/mol when injected with DT. Saline injected mice had UTP/Cre

HYPOTHESIS/AIMS OF STUDY: The aim of this study was to compare two methods for trans-vaginal delivery of autologous endometrial mesenchymal stem cells (eMSC) on a novel polyamide (PA) synthetic mesh with biomechanical properties designed to match human vaginal tissue using an ovine model of vaginal wall weakness. The second aim was to determine the retention of implanted autologous Iodex-labelled eMSC and to assess their effect on the extracellular matrix and infammatory response to the implanted gelatin-coated (PA/G) and non-coated PA mesh. STUDY DESIGN, MATERIALS AND METHODS: Multiparous sheep (n=30) which underwent quantifcation of pelvic organ prolapse using a modifed POP-Q (Pelvic Organ Prolapse Quantifcation) were divided into 4 experimental and 1 incisional control group. Subtotal hysterectomy was performed via ventral midline laparotomy on 12 sheep to collect endometrial tissue. Ovine autologous eMSC were isolated from hysterectomy tissue utilizing cell sorting by fow cytometry of CD271+ ovine eMSCs, cell culture and eMSC labeling with FITC-labelled IODEX paramagnetic nan-oparticles. Polyamide synthetic grafts were fabricated and used alone or with eMSC or were dip-coated in 12% porcine gelatin and stabilized with 0.5% glutaraldehyde for PA/G constructs; autologous labelled ovine eMSC-seeded PA/G constructs were also prepared. Surgery consisted of a 40 mm, full-thickness midline incision on the posterior vaginal wall and eMSC/PA/G (n=6), PA/G (n=6), PA (n=12) were surgically implanted. A 2 step procedure occurred in the fourth group with eMSC in a gelatin hydrogel (eMSC/G) were applied to the 6 already implanted PA meshes (PA + eMSC/G) and the gelatin crosslinked in situ with blue light. No graft was placed for the incisional controls. After suturing the ewes were allowed to recover and were harvested 30 days post implantation. The analyses of explants included histology, collagen analysis using sirius red birefringence, image analysis of immunofuorescence a

As the comparative pathophysiology of perinatal infection in the fetus and newborn is uncertain, this study contrasted the cerebral effects of endotoxemia in conscious fetal sheep and newborn lambs. Responses to intravenous bacterial endotoxin (lipopolysaccharide, LPS) or normal saline were studied on three consecutive days in fetal sheep (LPS 1 mug/kg, n = 5; normal saline n = 5) and newborn lambs (LPS 2 mug/kg, n = 10; normal saline n = 5). Cerebro-vascular function was assessed by monitoring cerebral blood flow (CBF) and cerebral vascular resistance (CVR) over 12 h each day, and inflammatory responses were assessed by plasma TNF alpha (TNF-alpha), nitrate and nitrite concentrations. Brain injury was quantified by counting both resting and active macrophages in the caudate nucleus and periventricular white matter (PVWM). An acute cerebral vasoconstriction (within 1 h of LPS injection) occurred in both the fetus (DELTACVR +53%) and newborn (DELTACVR +63%); subsequently prolonged cerebral vasodilatation occurred in the fetus (DELTACVR -33%) in association with double plasma nitrate/nitrite concentrations, but not in the newborn. Abundant infiltration of activated macrophages was observed in both CN and PVWM at each age, with the extent being 2-3 times greater in the fetus (P < 0.001). In conclusion, while the fetus and newborn experience a similar acute disruption of the cerebral circulation after LPS, the fetus suffers a more prolonged circulatory disruption, a greater infiltration of activated macrophages, and an exaggerated susceptibility to brain injury.Copyright © 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Host defenses against pathogens are energetically expensive, leading ecological immunologists to postulate that they might participate in energetic trade-offs with other maintenance programs. However, the metabolic costs of immunity and the nature of physiologic trade-offs it engages are largely unknown. We report here that activation of immunity causes an energetic trade-off with the homeothermy (the stable maintenance of core temperature), resulting in hypometabolism and hypothermia. This immunity-induced physiologic trade-off was independent of sickness behaviors but required hematopoietic sensing of lipopolysaccharide (LPS) via the toll-like receptor 4 (TLR4). Metabolomics and genome-wide expression profiling revealed that distinct metabolic programs supported entry and recovery from the energy-conserving hypometabolic state. During bacterial infections, hypometabolic states, which could be elicited by competition for energy between maintenance programs or energy restriction, promoted disease tolerance. Together, our findings suggest that energy-conserving hypometabolic states, such as dormancy, might have evolved as a mechanism of tissue tolerance. Immune activation after infection is metabolically costly, competing for energy with the maintenance of normal body temperature, and this dynamic trade-off leads to preferential use of tolerance as a mechanism of bacterial defense.Copyright © 2019 Elsevier Inc.

Background: Pulmonary hypertension (PH) occurring secondary to bronchopulmonary dysplasia (BPD) contributes to significant morbidity and mortality among extremely preterm infants and currently there are no effective and safe interventions. We have previously shown that prophylactic treatment with interleukin-1 receptor antagonist (IL-1Ra) prevents murine BPD, so we sought to determine if its protection also extends to murine BPD-PH. Method(s): Pregnant C57BL/6J mice received an i.p. injection of LPS (150 ug/kg) at d14 of gestation. Immediately after birth, pups were randomised to hyperoxia (65% O2) or room air (21% O2). Pups were treated with daily s.c. injections of IL-1Ra (10 mg/kg) or saline. On d28, lung parenchyma was assessed by histology, pulmonary blood vessels by synchrotron micro-CT imaging and pulmonary blood pressures by echocardiography. To assess pulmonary vasculature later in life, mice were returned to room air after d28 and then at d60 in vivo synchrotron radiation microangiography was performed. Result(s): IL-1Ra prevented BPD-like lung disease (28% reduction in alveolar size, 34% increase in alveolar number compared to hyperoxia saline). Micro-CT revealed significant pulmonary vascular remodelling in hyperoxia saline-treated pups e.g. 41% decrease in small vessels (4-5mum diameter) and 35-fold increase in large vessels (50-60mum diameter), which was accompanied by PH (TPV/RVET 0.27 for hyperoxia saline vs 0.32 for controls); treatment with IL-1Ra was protective. In vivo microangiography revealed 18-25% fewer vessels across all branching generations in hyperoxia saline mice than air controls, which was rescued by IL-1Ra. Conclusion(s): IL-1Ra protected the pulmonary vasculature of pups from perinatal inflammation and hyperoxia.

Acute kidney injury leading to chronic kidney disease through tubulointerstitial fibrosis is a major challenge in nephropathy. Several signaling pathways promote interstitial fibrosis; however, effective suppression of fibrosis may require blockade of more than one pathway. This study investigated whether blockade of Smad3 and c-Jun N-Terminal kinase (JNK) signaling gives added suppression of interstitial fibrosis in folic acid nephropathy. A single high dose of folic acid (FA) causes acute tubular damage in C57BL/6J mice followed by interstitial fibrosis and chronic renal impairment. Co-Activations of Smad3 and JNK signaling occur in both tubular epithelial cells and myofibroblasts in areas of tubulointerstitial damage and fibrosis in both murine FA-induced nephropathy and human IgA nephropathy. Groups of mice were treated with a Smad3 inhibitor (SIS3), a JNK inhibitor (SP600125), or a combination from day 6 after FA administration until being killed on day 28. Each drug efficiently inhibited its specific target (Smad3 phosphorylation or c-Jun phosphorylation) without affecting the other pathway. Given alone, each drug partially reduced renal fibrosis, whereas the combination therapy gave an additive and profound protection from renal fibrosis and improved renal function. Inhibition of Smad3 and/or JNK signaling activities prevented down-regulation of PGC-1alpha in tubular epithelial cells and up-regulation of PGC-1alpha in myofibroblasts during FA-induced renal fibrosis and inflammation. The expression of PGC-1alpha was upregulated in Smad3-/- NRK52E cells while downregulated in Smad3-/-NRK49F cells, suggesting that Smad3 signaling may regulate expression of PGC-1alpha in renal tubular epithelial cells and fibroblasts in distinct fashion. In vivo and cell culture studies also indicate that Smad3 and JNK signaling cooperate to cause mitochondrial dysfunction and cell damage in tubular epithelial cells via direct actions on the transcription of PGC-1alpha. These pat

Background and Aim: Inflammatory bowel disease (IBD) requires longterm therapy to control symptoms and prevent complications. Despite therapeutic advances, some patients experience side effects or are refractory to conventional treatment. Current therapies are unable to reduce or reverse established intestinal fibrosis. Isolated from placentas, human amnion epithelial cells (hAECs) are ethically sourced pluripotent stem cells that possess anti-inflammatory and antifibrotic properties. With no tumorogenicity and an excellent safety profile, hAECs are a promising novel therapy for IBD. We examined the efficacy of hAECs and their conditioned media (hAEC-CM) in a dextran sulfate sodium (DSS)-induced murine model of chronic colitis. Method(s): C57BL/6J mice received a cycle of 2% DSS (w/v) in normal water for 1 week and then normal water for 2 weeks. This 3-week regimen was repeated twice until cull at week 10. Treatment began at week 6, with one group (Chr-hAEC, n = 14) receiving weekly tail vein injections of 2 x 106 hAECs, and another group (Chr-CM, n = 15) receiving 250-muL hAEC-CM three times weekly. Normal mice (n = 7) and DSS control mice (Chr-DSS, n = 13) received no therapy. Mice were monitored daily. Colitis severity was scored using HE-stained colon sections. Interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-6, transforming growth factor (TGF)-beta, interferon (IFN)-gamma, IL-4, and IL- 10 were measured by ELISA. Fibrosis extent was measured via computer- assisted morphometry of picro-sirius-stained colon sections. Fibroblasts and macrophages were identified by immunohistochemistry (alpha-SMA, F4/80). Result(s): Bodyweight significantly improved with hAECs (10%, P < 0.05) and hAEC-CM (12.5%, P < 0.001) compared with untreated mice on DSS (4.2%). hAECs and hAEC-CM also prevented colon shortening (6.8 cm, P < 0.001; and 7.3 cm, P < 0.0001, respectively) compared with controls (5.9 cm). Splenomegaly was greater in Chr-DSS mice (212 mg) than mice treat

Background: Serially transplantable patient-derived xenografts (PDXs) are invaluable preclinical models for studying tumor biology and evaluating therapeutic agents. As these models are challenging to establish from prostate cancer specimens, the ability to preserve them through cryopreservation has several advantages for ongoing research. Despite this, there is still uncertainty about the ability to cryopreserve PDXs of prostate cancer. This study compared three different cryopreservation protocols to identify a method that can be used to reproducibly cryopreserve a diverse cohort of prostate cancer PDX models. Method(s): One serially transplantable prostate cancer PDX from the Melbourne Urological Research Alliance cohort was used to compare three cryopreservation protocols: slow freezing in fetal calf serum (FCS) with 10% dimethyl sulfoxide (DMSO), FCS with 10% DMSO supplemented with the Rho-associated kinase (ROCK) inhibitor Y-27632 and vitrification. The efficiency of the slow freezing protocols was then assessed in 17 additional prostate cancer PDXs. Following cryopreservation, PDXs were re-established in host mice that were either intact and supplemented with testosterone or castrated. Graft take rate, tumor growth, histological features, and transcriptome profiles before and after cryopreservation were compared. Result(s): Slow freezing maintained the viability and histological features of prostate cancer PDXs, and the addition of a ROCK inhibitor increased their growth following cryopreservation. Using the slow freezing method, we re-established 100% of PDXs grown in either testosterone-supplemented or castrated host mice. Importantly, the long-term tumor growth rate and transcriptome profile were maintained following cryopreservation. Conclusion(s): This study has identified a protocol to reliably cryopreserve and re-establish a diverse cohort of serially transplantable PDXs of prostate cancer. This study has the potential to significantly improve the prac

Schizophrenia is a debilitating disorder characterised by three main symptom categories: positive, negative and cognitive. Cognitive symptoms emerge first, and currently do not have appropriate treatments, despite being a strong predictor of the severity and progress of the illness. Cognitive deficits are strongly associated with the dysfunction of GABAergic parvalbumin interneurons (PV-IN). PV-IN are supported by Brain-Derived Neurotrophic Factor (BDNF) via its receptor Tropomyosin-related Kinase B (TrkB). The main aim of this study was to investigate the cognitive and affective consequences of disrupted BDNF-TrkB signalling at PV-IN. We crossed PV-Cre mice with heterozygous TrkB floxed mice (PV-Cre:Fl+/-) to knock-down TrkB receptors on PV-IN. Male and female mice underwent a battery of tests including: Y-Maze, Cheeseboard Maze, Elevated Plus Maze, and Locomotor activity. Co-expression of PV and TrkB in the hippocampus was assessed by fluorescent immunohistochemistry and detailed stereology. Sex-specific spatial memory impairments were found in the Y-Maze. Only male PV-Cre:Fl+/- mice showed no preference for the novel arm. Furthermore, there was a male specific genotype difference in memory retrieval in the Cheeseboard Maze. Male PV-Cre:Fl+/- mice were more preservative in their learning than male PV-Cre control mice. Overall, the evidence from this study suggests that sex had a developmental influence on this constitutive model. Male spatial memory was altered by the disruption to BDNF-TrkB signalling at PV-IN. This aligns with males showing more severe cognitive dysfunction in schizophrenia.Copyright © 2019 Elsevier B.V.

Background: Myeloperoxidase (MPO)-ANCA-associated GN is a significant cause of renal failure. Manipulating autoimmunity by inducing regulatory T cells is potentially a more specific and safer therapeutic option than conventional immunosuppression. Method(s): To generate MPO-specific regulatory T cells, we used a modified protein-conjugating compound, 1-ethyl-3-(3'dimethylaminopropyl)-carbodiimide (ECDI), to couple the immunodominant MPO peptide (MPO409-428) or a control ovalbumin peptide (OVA323-339) to splenocytes and induced apoptosis in the conjugated cells. We then administered MPO- and OVA-conjugated apoptotic splenocytes (MPO-Sps and OVA-Sps, respectively) to mice and compared their effects on development and severity of anti-MPO GN. We induced autoimmunity to MPO by immunizing mice with MPO in adjuvant; to trigger GN, we used low-dose antiglomerular basement membrane globulin, which transiently recruits neutrophils that deposit MPO in glomeruli. We also compared the effects of transferring CD4+ T cells from mice treated with MPO-Sp or OVA-Sp to recipient mice with established anti-MPO autoimmunity. Result(s): MPO-Sp but not OVA-Sp administration increased MPO-specific, peripherally derived CD4+Foxp3- type 1 regulatory T cells and reduced anti-MPO autoimmunity and GN. However, in mice depleted of regulatory T cells, MPO-Sp administration did not protect from anti-MPO autoimmunity or GN. Mice with established anti-MPO autoimmunity that received CD4+ T cells transferred from mice treated with MPO-Sp (but not CD4+ T cells transferred from mice treated with OVA-Sp) were protected from anti-MPO autoimmunity and GN, confirming the induction of therapeutic antigen-specific regulatory T cells. Conclusion(s): These findings in a mouse model indicate that administering apoptotic splenocytes conjugated with the immunodominant MPO peptide suppresses anti-MPO GN by inducing antigen-specific tolerance.Copyright © 2019 by the American Society of Nephrology.

Psychological distress has long been suspected to influence cancer incidence and mortality. It remains largely unknown whether and how stress affects the efficacy of anticancer therapies. We observed that social defeat caused anxiety-like behaviors in mice and dampened therapeutic responses against carcinogen-induced neoplasias and transplantable tumors. Stress elevated plasma corticosterone and upregulated the expression of glucocorticoid-inducible factor Tsc22d3, which blocked type I interferon (IFN) responses in dendritic cell (DC) and IFN-gamma+ T cell activation. Similarly, close correlations were discovered among plasma cortisol levels, TSC22D3 expression in circulating leukocytes and negative mood in patients with cancer. In murine models, exogenous glucocorticoid injection, or enforced expression of Tsc22d3 in DC was sufficient to abolish therapeutic control of tumors. Administration of a glucocorticoid receptor antagonist or DC-specific Tsc22d3 deletion reversed the negative impact of stress or glucocorticoid supplementation on therapeutic outcomes. Altogether, these results indicate that stress-induced glucocorticoid surge and Tsc22d3 upregulation can subvert therapy-induced anticancer immunosurveillance.Copyright © 2019, The Author(s), under exclusive licence to Springer Nature America, Inc.

Memory regulatory T cells (mTregs) have been demonstrated to persist long-term in hosts after the resolution of primary influenza A virus (IAV) infection. However, whether such IAV infection-experienced (IAV-experienced) mTregs differentiate into a phenotypically and functionally distinct Treg subset and what function they play at the infection site remains poorly defined. In this study, we characterized the phenotype, examined the responsiveness and assessed the suppressive function of IAV-experienced memory Tregs (mTregs). In comparison with inexperienced naive Tregs (nTregs), mTregs exhibited elevated expression of CD39, CD69, CD103, cytotoxic T lymphocyte-associated antigen-4, leukocyte function-associated antigen-1 and programmed cell death-1 and could be activated in an antigen-specific manner in vitro and in vivo. When mTregs and nTregs were adoptively cotransferred into recipient mice, mTregs had a competitive advantage in migrating to the IAV-infected lungs. mTregs were more capable of controlling in vitro proliferation of CD4+ and CD8+ T cells and suppressed CD40 and CD86 upregulation on bone marrow-derived dendritic cells. Adoptively transferred mTregs, but not adoptively transferred nTregs, significantly attenuated body weight loss, lung pathology and immune cell infiltration into the infected lungs after IAV infection. These results suggest that mTregs generated after IAV infection differentiate into a phenotypically distinct and functionally enhanced Treg subset that can be activated in an antigen-specific manner to exert immunosuppression. We propose vaccination to induce such mTregs as a potential novel strategy to protect against severe IAV infection.Copyright © 2019 Australian and New Zealand Society for Immunology Inc.

Fetal growth restriction (FGR) and prematurity are associated with high risk of brain injury and long-term neurological deficits. FGR infants born preterm are commonly exposed to mechanical ventilation, but it is not known whether ventilation differentially induces brain pathology in FGR infants compared with appropriate for gestational age (AGA) infants. We investigated markers of neuropathology in moderate-to late-preterm FGR lambs, compared with AGA lambs, delivered by caesarean birth and ventilated under standard neonatal conditions for 24 h. FGR was induced by single umbilical artery ligation in fetal sheep at 88-day gestation (term, 150 days). At 125-day gestation, FGR and AGA lambs were delivered, dried, intubated, and commenced on noninjurious ventilation, with surfactant administration at 10 min. A group of unventilated FGR and AGA lambs at the same gestation was also examined. Over 24 h, circulating pH, PO2, and lactate levels were similar between groups. Ventilated FGR lambs had lower cerebral blood flow compared with AGA lambs (P = 0.01). The brain of ventilated FGR lambs showed neuropathology compared with unventilated FGR, and unventilated and ventilated AGA lambs, with increased apoptosis (caspase-3), blood-brain barrier dysfunction (albumin extravasation), activated microglia (Iba-1), and increased expression of cellular oxidative stress (4-hydroxynonenal). The neuropathologies seen in the ventilated FGR brain were most pronounced in the periventricular and subcortical white matter but also evident in the subventricular zone, cortical gray matter, and hippocampus. Ventilation of preterm FGR lambs increased brain injury compared with AGA preterm lambs and unventilated FGR lambs, mediated via increased vascular permeability, neuroinflammation and oxidative stress.Copyright © 2018 the American Physiological Society.

Background/Purpose: Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease associated with B cell hyperactivity driven by dysregulated type I IFN. Lyn-deficient mice develop lupus-like autoimmunity due to loss of inhibitory regulation of B cell receptor signalling, resulting in hyperactive B cells, excess IL-6 production, and tissue inflammation. Mice deficient in glucocorticoid-induced leucine zipper (GILZ), an intracellular protein involved in glucocorticoid actions, develop lupus-like autoimmunity and excess B cell activation. GILZ is suppressed in human SLE (Jones et al., 2016), but the effects of GILZ in murine models of SLE are unknown. We tested the hypothesis that loss of GILZ exacerbates autoimmunity in the Lyn-deficient murine model of lupus. Method(s): We crossed GILZ-deficient mice onto a Lyn-deficient background (GILZ-Lyn double knockout (DKO)) and compared them to WTand Lyn KO mice. The effects of GILZ deficiency on spleen weight, nephritis, serum autoantibodies and cytokines, and spleen cell expression of Type I interferon-induced genes (ISG) were examined. Result(s): We observed heightened lupus-like autoimmunity in GILZ-Lyn DKO mice, compared to Lyn KO, that include increased spleen weight (p=0.041) and more severe glomerulonephritis, including worse glomerular segmental necrosis (p=0.0051) and crescent formation (p=0.0044) (Fig 1A-C). Despite this, serum levels of serum autoantibodies against ENAs (dsDNA, histone, Sm, Jo-1, U1RNP, Ro52, ribosomes, and Scl-70) and a panel of serum pro-inflammatory cytokines (BAFF, IL-6, IFNgamma, IL-10 and TNFalpha) measured by Luminex assay were unaffected. In contrast, GILZ deficiency in Lyn deficient mice resulted in increased expression of ISG (ifi44, usp18, oas3, isg15, mx1, and irf7) and an overall ISG signature (p=0.0023) (Fig 1D). Conclusion(s): In Lyn KO lupus-prone mice, GILZ deficiency significantly exacerbated disease expression, accompanied by activation of Type I IFN, despite serum aut

Accumulation of excess iron in tissues causes organ damage and dysfunction and may lead to serious clinical consequences including liver cirrhosis, diabetes, growth retardation and heart failure. Iron overload is a major health threat in iron loading anemias, like beta-thalassemia, myelodysplastic syndrome and in hereditary hemochromatosis. In patients with beta-thalassemia major, iron overload develops due to frequent blood transfusions to control the severe anemia. In addition, iron overload also occurs in patients with beta thalassemia intermedia (non-transfusion dependent betathalassemia). In the later cases, iron overload develops through gastrointestinal iron hyperabsorption due to stressed and ineffective erythropoiesis. Importantly, expression of the peptide hormone hepcidin, which is the key modulator in iron homeostasis, is abnormally low and unable to block ferroportin-mediated intestinal iron absorption. In hereditary hemochromatosis, gene defects in the hepcidin-ferroportin axis controlling iron homeostasis, lead to hepatic iron overload. Therefore, in these indications, iron overload is caused by dysregulation or dysfunction of the hepcidinferroportin axis. Hepcidin is predominantly produced by the liver and is induced by activation of the BMP/SMAD signaling pathway. Furthermore, hepcidin is under the negative control of the transmembrane protease matriptase-2, encoded by the TMPRSS6 gene. RNA interference is a natural mechanism and a powerful approach for inhibiting the expression of disease-associated genes. Silence Therapeutics has developed short interfering RNA (siRNA) conjugate technology for the selective inhibition of target gene expression in the liver. GalNAcconjugated siRNAs bind efficiently to the asialoglycoprotein (ASGP) receptor expressed predominantly by hepatocytes thereby providing a highly specific, safe and efficient delivery technology to enable a new class of therapeutic use. Here we present the pharmacological characterization of

Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN-/- mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN-/- mice underwent hindlimb ischemia surgery and 1.5 x 106 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% +/- 0.8 and 70.9% +/- 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovasc

Clinical studies indicate that sex differences exist in susceptibility for developing diabetic kidney disease (DKD), supporting the need to examine both sexes in animal studies of DKD. Streptozotocin (STZ) is commonly used in male mice to induce diabetes and DKD. However, females are not normally included because their sex hormones partially protect them from STZ-induced islet injury and consequent diabetes. To address this issue, we identified a strategy to induce comparable diabetes in male and female mice using STZ and determined whether both sexes develop equivalent renal injury. Male and female mice lacking the gene for endothelial nitric oxide synthase (Nos3-/-) were made diabetic with five or six low-dose STZ injections, respectively. Groups of male and female mice with equivalent hyperglycemia at week 3 after STZ were assessed for DKD at week 8. STZ-treated male and female Nos3-/- mice maintained comparable hyperglycemia between weeks 3 and 8 had an equivalent increase in HbA1c levels and comparable hypertension. Urine albumin/creatinine levels were elevated eightfold in mice of both sexes at week 8, accompanied by an equivalent loss of podocytes. In diabetic males and females, plasma cystatin C levels and glomerular collagen deposition were similarly increased. Kidney mRNA levels of proinflammatory and profibrotic markers and kidney injury molecule-1 (KIM-1) were equally elevated in males and females, indicating comparable kidney injury. This study shows that equivalent diabetes induces a comparable onset of DKD in male and female Nos3-/- mice, demonstrating that it is possible to include males and females together in studies of DKD.Copyright © 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.Copyright © 2019 International Society of Nephrology

Introduction: Patients with chronic kidney disease (CKD) are at high risk of developing acute kidney injury following an episode of renal ischemia/reperfusion (I/R) injury. However, there has been a lack of suitable models in which to investigate the mechanisms of renal I/R injury in CKD and to test potential therapies. Mice lacking the endothelial nitric oxide synthase gene (Nos3-/-) spontaneously develop hypertension, albuminuria, and glomerulosclerosis with age. We hypothesized that aged Nos3-/- mice may be more susceptible to I/R injury and provide a useful model to study I/R in CKD. Method(s): Bilateral renal I/R (or sham) surgery is performed in groups of male Nos3-/- or age-matched wild type (WT) mice (n=6-8) at the age of 3, 6, or 12 months. In addition, 12 month old female mice from both strains were examined. All the animals were killed after 24 hour reperfusion. Result(s): Nos3-/- mice have established hypertension by 3 months of age, and from 6 months show progressive glomerulosclerosis and albuminuria. By 12 months of age, Nos3-/- mice exhibit glomerulosclerosis in 35.4+/-9.2% of glomeruli, and a 35-fold increase in urine albumin excretion and a 2-fold increase in serum creatinine compared to age-matched WT mice. Using the same duration of bilateral renal I/R injury, male Nos3-/- mice exhibit more severe renal failure compared to age-matched WT mice at both 6 months (serum creatinine 62.0+/-24.4 vs 27.3+/-6.4 micromol/L, P<0.01) and 12 months (serum creatinine 139.3+/-8.4 vs 57.75+/-17.3 micromol/L, P<0.001). Tubular damage, as assessed by tubular injury score and KIM-1 mRNA, was also exacerbated in Nos3-/- mice. However, these differences were not seen in the 3 months old mice, indicating that newly established hypertension does not impact upon susceptibility to renal I/R injury. In Nos3-/- mice, serum creatinine levels in I/R injury correlated with the severity of glomerulosclerosis (r=0.8985, p<0.001). Nos3-/- mice developed significantly more tubula

Introduction: Renal ischaemia/reperfusion (I/R) injury is a common cause of acute kidney injury. However, the clinical situation is often complex with patients having co-morbidities such as diabetes that make them more susceptible to I/R-induced acute kidney injury. To address this, we sought to develop a model of renal I/R injury in mice with established diabetes. Activation of the c-Jun amino terminal kinase (JNK) is known to play a role in various types of acute kidney injury; however, whether this is important in diabetic I/R injury is unknown. The aims of this study were to: (i) characterize a model of renal I/R injury in diabetic mice, and; (ii) determine if treatment with the JNK inhibitor, CC-930, can prevent the development of I/R-induced acute kidney injury in diabetic mice. Method(s): Groups of 8 to 10 mice were made diabetic by 5 daily injections of low dose streptozotocin (STZ). Diabetes was monitored weekly by fasting blood glucose levels. After 8 weeks, mice underwent 13min of bilateral renal ischaemia and were killed 24hr after reperfusion (i.p. saline was provided to minimize fluid loss). Groups of mice received either 150mg/kg/BID CC-930 or vehicle alone by oral gavage starting 1hr before surgery. The same 13min ischaemic insult was performed in age-matched non-diabetic mice as a control. Result(s): Diabetic mice had normal renal function at 8 weeks after STZ injections (14.3+/-0.8 vs 12.5+/-1.9umol/L serum creatinine (sCr) in non-diabetic; P=NS). Bilateral ischaemia of 13min caused more severe renal dysfunction in diabetic compared to non-diabetic mice (82.7+/-25.4 vs 31.6+/-11.9umol/L sCr, respectively; P<0.001). The percentage of tubules showing damage in the inner cortex and outer medulla was also greater in diabetic vs non-diabetic I/R injury (61+/-17% vs 26+/-12% damaged tubules, respectively; P<0.001). Activation of JNK signalling, shown by c-Jun phosphorylation, was substantially increased in diabetic versus non-diabetic I/R kidney. Treatme

Beside the classical flat parietal epithelial cells (PECs), we investigated proximal tubular epithelial-like cells, a neglected subgroup of PECs. These cells, termed cuboidal PECs, make up the most proximal part of the proximal tubule and may also line parts of Bowman's capsule. Additionally, a third intermediate PEC subgroup was identified at the junction between the flat and cuboidal PEC subgroups at the tubular orifice. The transgenic mouse line PEC-rtTA labeled all three PEC subgroups. Here we show that the inducible Pax8-rtTA mouse line specifically labeled only cuboidal and intermediate PECs, but not flat PECs. In aging Pax8-rtTA mice, cell fate mapping showed no evidence for significant transdifferentiation from flat PECs to cuboidal or intermediate PECs or vice versa. In murine glomerular disease models of crescentic glomerulonephritis, and focal segmental glomerulosclerosis (FSGS), intermediate PECs became more numerous. These intermediate PECs preferentially expressed activation markers CD44 and Ki-67, suggesting that this subgroup of PECs was activated more easily than the classical flat PECs. In mice with FSGS, cuboidal and intermediate PECs formed sclerotic lesions. In patients with FSGS, cells forming the tip lesions expressed markers of intermediate PECs. These novel PEC subgroups form sclerotic lesions and were more prone to cellular activation compared to the classical flat PECs in disease. Thus, colonization of Bowman's capsule by cuboidal PECs may predispose to lesion formation and chronic kidney disease. We propose that tip lesions originate from this novel subgroup of PECs in patients with FSGS.Copyright © 2019 International Society of Nephrology

Aim: Protease-activated receptor 2 (PAR2) has been implicated in the development of renal inflammation and fibrosis. In particular, activation of PAR2 in cultured tubular epithelial cells induces extracellular signal-regulated kinase signalling and secretion of fibronectin, C-C Motif Chemokine Ligand 2 (CCL2) and transforming growth factor-beta1 (TGF-beta1), suggesting a role in tubulointerstitial inflammation and fibrosis. We tested this hypothesis in unilateral ureteric obstruction (UUO) in which ongoing tubular epithelial cell damage drives tubulointerstitial inflammation and fibrosis. Method(s): Unilateral ureteric obstruction surgery was performed in groups (n = 9/10) of Par2-/- and wild type (WT) littermate mice which were killed 7 days later. Non-experimental mice were controls. Result(s): Wild type mice exhibited a 5-fold increase in Par2 messenger RNA (mRNA) levels in the UUO kidney. In situ hybridization localized Par2 mRNA expression to tubular epithelial cells in normal kidney, with a marked increase in Par2 mRNA expression by tubular cells, including damaged tubular cells, in WT UUO kidney. Tubular damage (tubular dilation, increased KIM-1 and decreased alpha-Klotho expression) and tubular signalling (extracellular signal-regulated kinase phosphorylation) seen in WT UUO were not altered in Par2-/- UUO. In addition, macrophage infiltration, up-regulation of M1 (NOS2) and M2 (CD206) macrophage markers, and up-regulation of pro-inflammatory molecules (tumour necrosis factor, CCL2, interleukin-36alpha) in WT UUO kidney were unchanged in Par2-/- UUO. Finally, the accumulation of alpha-SMA+ myofibroblasts, deposition of collagen IV and expression of pro-fibrotic factors (CTGF, TGF-beta1) were not different between WT and Par2-/- UUO mice. Conclusion(s): Protease-activated receptor 2 expression is substantially up-regulated in tubular epithelial cells in the obstructed kidney, but this does not contribute to the development of tubular damage, renal inflammatio

Objective Infants with a congenital diaphragmatic hernia (CDH) are at high risk of developing pulmonary hypertension after birth, but little is known of their physiological transition at birth. We aimed to characterise the changes in cardiopulmonary physiology during the neonatal transition in an ovine model of CDH. Methods A diaphragmatic hernia (DH) was surgically created at 80 days of gestational age (dGA) in 10 fetuses, whereas controls underwent sham surgery (n=6). At 138 dGA, lambs were delivered via caesarean section and ventilated for 2 hours. Physiological and ventilation parameters were continuously recorded, and arterial blood gas values were measured. Results DH lambs had lower wet lung-to-body-weight ratio (0.016+/-0.002vs0.033+/-0.004), reduced dynamic lung compliance (0.4+/-0.1mL/cmH 2 O vs1.2+/-0.1 mL/cmH 2 O) and reduced arterial pH (7.11+/-0.05vs7.26+/-0.05), compared with controls. While measured pulmonary blood flow (PBF) was lower in DH lambs, after correction for lung weight, PBF was not different between groups (4.05+/-0.60mL/min/gvs4.29+/-0.57 mL/min/g). Cerebral tissue oxygen saturation was lower in DH compared with control lambs (55.7+/-3.5vs67.7%+/-3.9%). Conclusions Immediately after birth, DH lambs have small, non-compliant lungs, respiratory acidosis and poor cerebral oxygenation that reflects the clinical phenotype of human CDH. PBF (indexed to lung weight) was similar in DH and control lambs, suggesting that the reduction in PBF associated with CDH is proportional to the degree of lung hypoplasia during the neonatal cardiopulmonary transition.Copyright © Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.

Background. The functional relevance of OX40 ligand (OX40L) in the effector phase of crescentic glomerulonephritis (GN) is unknown. These studies defined the role of endogenous OX40L during the effector stage of murine crescentic GN. Methods. GN was induced by immunization with sheep globulin/adjuvant on Day 0 and injection of sheep anti-mouse glomerular basement membrane immunoglobulin (Ig) on Day 10. Rat IgG or neutralizing anti-OX40L antibody was administered on Days 10-18 and immune responses and renal injury assessed on Day 20. Results. Compared with naive animals, OX40L was upregulated in the lymph nodes (LNs) and on leucocytes and resident non-immune cells in the kidneys of mice with GN. Inhibition of OX40L in GN augmented renal injury, as indicated by increased crescent formation, proteinuria and glomerular leucocyte accumulation. In line with increased injury, anti-OX40L treatment increased proliferation and decreased apoptosis of CD4 T cells in the LNs, without affecting LN CD4 cytokine production and CD8 T-cell responses. Blockade of OX40L decreased LN regulatory T-cell (Treg) proliferation, transforming growth factor beta production and foxp3 expression. OX40L inhibition did not affect B cell expansion or circulating antibody levels. In the kidney, neutralization of OX40L augmented interferon I 3 (IFN I 3) expression by CD4 and CD8 T cells and shifted macrophage polarization towards the pro-inflammatory M1 phenotype. Conclusions. OX40L is protective during the effector phase of murine crescentic GN by reducing the expansion of CD4 T cells and enhancing Treg responses in the LNs, and by locally inhibiting T-cell IFN I 3 production and pro-inflammatory macrophage phenotype in the kidney.Copyright © 2018 The Author(s).

Introduction: Cyclophilins are enzymes that regulate protein folding. Within this enzyme family, cyclophilin A (CypA) promotes leukocyte recruitment and inflammation, while cyclophilin D (CypD) facilitates mitochondrial-dependent cell death. CypD-/- mice are known to be protected from ischaemia/reperfusion injury (IRI). We investigated: (1) whether a novel pan-cyclophilin inhibitor (CYPi), which does not block calcineurin function, could prevent anticipated renal IRI, (2) the contributions of CypA and CypD in renal IRI. Aim(s): To determine if cyclophilin (Cyp) inhibition can prevent renal IRI, and examine the specific contributions of CypA and CypD in promoting renal IRI. Method(s): Groups of 10 mice underwent bilateral renal ischaemia and were killed 24 hours after reperfusion: (1) C57BL/6J mice were treated with 30mg/kg/BID CYPi or vehicle by oral gavage; (2) CypA-/- versus wild type (WT) mice on the 129 background. Controls were sham operated. (3) CypD-/- vs WT cultured proximal tubular epithelial cells (PTEC) were exposed to 0.5 mM of H2O2 for 24hr, with or without CYPi. Result(s): Renal IRI caused acute renal failure in C57BL/6J mice (179.0+/-19.1 vs 12.2+/-1.4umol/L serum creatinine (sCr) in sham; P<0.0001). CYPi protected against renal failure (sCr 36.4+/-6.7umol/L; P<0.0001). CYPi treatment reduced histologic tubular damage (P<0.0001). CYPi treatment also reduced apoptotic tubular cells (TUNEL+ cells), TNF-alpha mRNA levels and neutrophil and macrophage infiltration (all P<0.005 vs vehicle). Renal IRI induced acute renal failure in 129 mice (sCr 41+/-13.76 vs 6.25+/-1.66umol/L in sham; P<0.0001). CypA-/- mice were protected from renal dysfunction (sCr 20.7+/-3.53umol/L; P<0.0001). CypA-/- mice had less histologic tubular damage (P<0.005) and lower KIM-1 mRNA levels (P<0.005). CypA-/- mice also had less tubular cell death (TUNEL+ cells; P<0.001), inflammatory cytokines (TNF-alpha and IL-36-alpha PCR; P<0.05), and reduced neutrophil infiltration (P<0.001). In

Autoimmune diseases resulting from MHC class II-restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1alpha,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA323-339 and calcitriol targeted dLN PD-L1hi DCs of immunized mice and reduced their MHC class II expression. OVA323-339/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3+ and IL-10+ peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/ calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture's vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.Copyright © 2019, American Society for Clinical Investigation.

Objective Fetoscopic endoluminal tracheal occlusion (FETO) aims to reverse pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) and mitigate the associated respiratory insufficiency and pulmonary hypertension after birth. We aimed to determine whether FETO improves the cardiopulmonary transition at birth in an ovine model of CDH. Methods In 12 ovine fetuses with surgically induced diaphragmatic hernia (DH; 80 dGA), an endotracheal balloon was placed tracheoscopically at =110 dGA and removed at =131 dGA (DH+FETO), while 10 were left untreated (DH). At =138 dGA, all lambs (survival at delivery: 67% [DH+FETO], 70% [DH]) were delivered via caesarean section and ventilated for 2 hours. Physiological and ventilation parameters were continuously recorded, and arterial blood-gas values were measured. Results Compared with DH, DH+FETO lambs had increased wet lung-to-body-weight ratio (0.031+/-0.004 vs 0.016+/-0.002) and dynamic lung compliance (0.7+/-0.1 vs 0.4+/-0.1 mL/cmH 2 O). Pulmonary vascular resistance was lower in DH+FETO lambs (0.44+/-0.11 vs 1.06+/-0.17 mm Hg/[mL/min]). However, after correction for lung weight, pulmonary blood flow was not significantly different between the groups (4.19+/-0.57 vs 4.05+/-0.60 mL/min/g). Alveolar-arterial difference in oxygen tension was not significantly different between DH+FETO and DH (402+/-41mm Hg vs 401+/-45 mm Hg). Conclusions FETO accelerated lung growth in fetuses with CDH and improved neonatal respiratory function during the cardiopulmonary transition at birth. However, despite improved lung compliance and reduced pulmonary vascular resistance, there were less pronounced benefits for gas exchange during the first 2 hours of life.Copyright © Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.

The widespread use of synthetic transvaginal polypropylene mesh for treating Pelvic Organ Prolapse (POP) has been curtailed due to serious adverse effects highlighted in 2008 and 2011 FDA warnings and subsequent legal action. We are developing new synthetic mesh to deliver endometrial mesenchymal stem cells (eMSC) to improve mesh biocompatibility and restore strength to prolapsed vaginal tissue. Here we evaluated knitted polyamide (PA) mesh in an ovine multiparous model using transvaginal implantation and matched for the degree of POP. Polyamide mesh dip-coated in gelatin and stabilised with 0.5% glutaraldehyde (PA/G) were used either alone or seeded with autologous ovine eMSC (eMSC/PA/G), which resulted in substantial mesh folding, poor tissue integration and 42% mesh exposure in the ovine model. In contrast, a two-step insertion protocol, whereby the uncoated PA mesh was inserted transvaginally followed by application of autologous eMSC in a gelatin hydrogel onto the mesh and crosslinked with blue light (PA + eMSC/G), integrated well with little folding and no mesh exposure. The autologous ovine eMSC survived 30 days in vivo but had no effect on mesh integration. The stiff PA/G constructs provoked greater myofibroblast and inflammatory responses in the vaginal wall, disrupted the muscularis layer and reduced elastin fibres compared to PA + eMSC/G constructs. This study identified the superiority of a two-step protocol for implanting synthetic mesh in cellular compatible composite constructs and simpler surgical application, providing additional translational value.Copyright © 2019 Elsevier Ltd

Background: Seizures in neonates are relatively common, and are strong predictors of long-term cognitive and developmental impairment like cerebral palsy. Current anti-seizure therapies (phenobarbitone (pheno)) can be neurotoxic. We propose that ganaxolone (ganax), a GABAA agonist, will reduce seizures, and reduce markers of brain injury following hypoxia ischemia (HI). We investigated the effects of ganax or pheno on seizure burden and neuropathology in HI lambs. Method(s): HI was induced via umbilical cord occlusion in term lambs (n = 21; sham n = 7). HI lambs were treated with either ganax (5 mg/kg/bolus followed by 5 mg/kg/d for 2 days) or pheno (20mg/kg/bolus followed by 5 mg/kg/day for 2 days) at 6 h. Seizure burden was assessed using continuous amplitudeintegrated electroencephalogram (aEEG) recording. At 48 h, lambs were euthanized for brain collection and analysis of neuropathology. Result(s): At birth, HI lambs had a pH <7.0 and blood pressure was reduced to 16.4 +/- 1.2 mmHg, indicating severe birth asphyxia. All HI lambs demonstrated electrographic seizures; mean number of seizures/h (6-12 h) was 4.8 +/- 2.2. Ganax treatment reduced the number of seizures to 1.0 +/- 0.9/h compared to pheno (3.2 +/- 1.6/ h) (P = 0.02). Brain histology revealed improved neuronal survival (NeuN+ cell number) and reduced cell death (TUNEL+) in the cortex and basal ganglia after ganax treatment, compared to pheno (P = 0.048 and P = 0.03), respectively. Conclusion(s): Ganax treatment in lambs with HI decreased seizure activity, improved neuronal survival and reduced cell death. Ganax has a high margin of safety and proved to be a better therapy.

Aim: Urinary oxygen tension (uPO2) may provide an estimate of renal medullary PO2 (mPO2) and thus risk of acute kidney injury (AKI). We assessed the potential for variations in urine flow and arterial PO2 (aPO2) to confound these estimates. Method(s): In 28 sheep urine flow, uPO2, aPO2 and mPO2 were measured during development of septic AKI. In 65 human patients undergoing cardiac surgery requiring cardiopulmonary bypass (CPB) uPO2 and aPO2 were measured continuously during CPB, and in a subset of 20 patients, urine flow was estimated every 5 minutes. Result(s): In conscious sheep breathing room air, uPO2 was more closely correlated with mPO2 than with aPO2 or urine flow. The difference between mPO2 and uPO2 varied little with urine flow or aPO2. In patients, urine flow increased abruptly from 3.42 +/- 0.29 mL min-1 to 6.94 +/- 0.26 mL min-1 upon commencement of CPB, usually coincident with reduced uPO2. During hyperoxic CPB high values of uPO2 were often observed at low urine flow. Low urinary PO2 during CPB (<10 mm Hg at any time during CPB) was associated with greater (4.5-fold) risk of AKI. However, low urine flow during CPB was not significantly associated with risk of AKI. Conclusion(s): uPO2 provides a robust estimate of mPO2, but this relationship is confounded by the simultaneous presence of systemic hyperoxia and low urine flow. Urine flow increases and uPO2 decreases during CPB. Thus, CPB is probably the best time to use uPO2 to detect renal medullary hypoxia and risk of post-operative AKI.Copyright © 2019 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd

Endometrial mesenchymal stem/stromal cells (eMSCs) exhibit excellent regenerative capacity in the endometrial lining of the uterus following menstruation and high proliferative capacity in vitro. Bioprinting eMSCs onto a mesh could be a potential therapy for Pelvic Organ Prolapse (POP). This study reports an alternative treatment strategy targeting vaginal wall repair using bioprinting of eMSCs encapsulated in a hydrogel and 3D melt electrospun mesh to generate a tissue engineering construct. Following a CAD, 3D printed poly epsilon-caprolactone (PCL) meshes were fabricated using melt electrospinning (MES) at different temperatures using a GMP clinical grade GESIM Bioscaffolder. Electron and atomic force microscopies revealed that MES meshes fabricated at 100 degreeC and with a speed 20 mm/s had the largest open pore diameter (47.2 +/- 11.4 mum) and the lowest strand thickness (121.4 +/- 46 mum) that promoted optimal eMSC attachment. An Aloe Vera-Sodium Alginate (AV-ALG) composite based hydrogel was optimised to a 1:1 mixture (1%AV-1%ALG) and eMSCs, purified from human endometrial biopsies, were then bioprinted in this hydrogel onto the MES printed meshes. Acute in vivo foreign body response assessment in NSG mice revealed that eMSC printed on MES constructs promoted tissue integration, eMSC retention and an anti-inflammatory M2 macrophage phenotype characterised by F4/80+CD206+ colocalization. Our results address an unmet medical need highlighting the potential of 3D bioprinted eMSC-MES meshes as an alternative approach to overcome the current challenges with non-degradable knitted meshes in POP treatment. Statement of Significance: This study presents the first report of bioprinting mesenchymal stem cells derived from woman endometrium (eMSCs) to boost Pelvic Organ Prolapse (POP) treatment. It impacts over 50% of elderly women with no optimal treatment at present. The overall study is conducted in three stages as fabricating a melt electrospun (MES) mesh, bioprint

Advancing age is accompanied by a reduction in bone formation and remodeling imbalance, which produces microstructural deterioration. This may be partly caused by a diversion of mesenchymal cells towards adipocytes rather than osteoblast lineage cells. We hypothesized that microstructural deterioration would be associated with an increased marrow adiposity, and each of these traits would be independently associated with nonvertebral fractures and improve discrimination of women with fractures from controls over that achieved by femoral neck (FN) areal bone mineral density (aBMD) alone. The marrow adiposity and bone microstructure were quantified from HR-pQCT images of the distal tibia and distal radius in 77 women aged 40 to 70 years with a recent nonvertebral fracture and 226 controls in Melbourne, Australia. Marrow fat measurement from HR-pQCT images was validated using direct histologic measurement as the gold standard, at the distal radius of 15 sheep, with an agreement (R2 = 0.86, p < 0.0001). Each SD higher distal tibia marrow adiposity was associated with 0.33 SD higher cortical porosity, and 0.60 SD fewer, 0.24 SD thinner, and 0.72 SD more-separated trabeculae (all p < 0.05). Adjusted for age and FN aBMD, odds ratios (ORs) (95% CI) for fracture per SD higher marrow adiposity and cortical porosity were OR, 3.39 (95% CI, 2.14 to 5.38) and OR, 1.79 (95% CI, 1.14 to 2.80), respectively. Discrimination of women with fracture from controls improved when cortical porosity was added to FN aBMD and age (area under the receiver-operating characteristic curve [AUC] 0.778 versus 0.751, p = 0.006) or marrow adiposity was added to FN aBMD and age (AUC 0.825 versus 0.751, p = 0.002). The model including FN aBMD, age, cortical porosity, trabecular thickness, and marrow adiposity had an AUC = 0.888. Results were similar for the distal radius. Whether marrow adiposity and cortical porosity indices improve the identification of women at risk for fractures requires validation i

Rats were exposed to nickel oxide nanoparticles (NiO-NP) inhalation at 0.23 ± 0.01 mg/m3 for 4 h a day 5 times a week for up to 10 months. The rat organism responded to this impact with changes in cytological and some biochemical characteristics of the bronchoalveolar lavage fluid along with a paradoxically little pronounced pulmonary pathology associated with a rather low chronic retention of nanoparticles in the lungs. There were various manifestations of systemic toxicity, including damage to the liver and kidneys; a likely allergic syndrome as indicated by some cytological signs; transient stimulation of erythropoiesis; and penetration of nickel into the brain from the nasal mucous membrane along the olfactory pathway. Against a picture of mild to moderate chronic toxicity of nickel, its in vivo genotoxic effect assessed by the degree of DNA fragmentation in nucleated blood cells (the RAPD test) was pronounced, tending to increasing with the length of the exposure period. When rats were given orally, in parallel with the toxic exposure, a set of innocuous substances with differing mechanisms of expected bioprotective action, the genotoxic effect of NiO-NPs was found to be substantially attenuated. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

Citrus aurantifolia (Christm.) Swingle (syn. C. MEDICA var. ACIDA Brandis) (family: Rutaceae) essential oil is one of the cheapest oils found in local markets. Although, it is generally accepted as non-toxic to vital organs and cells, majority of people are cynical about it usage. Herein, the present study reports the chemical composition and in vivo oral toxicity study of unripe C. aurantifolia essential oil found in Ghana. The toxicity of C. aurantifolia essential oil extract was investigated via oral administration using two methods: The acute toxicity single dose study (SDS) and the repeated dose method. The oil exhibited no acute toxicity but in the sub-chronic studies, the effects was dose and time-dependent. Chemical profile investigation of the oil showed 9 constituent of phytochemicals (Germacrene isomers (61.2%), Pineen (14%), Linalool dimmer (2.9%), Bornane (11%), Citral (2.9%), Anethole (1.5%), Anisole (1.1%), Safrole (0.3%) and Demitol (0.6%)). Histopathological studies revealed conditions such as necrosis, edema and inflammatory reaction in the liver, spleen and kidneys. Marginal upsurge of biochemical parameters above normal and elevated levels of lymphocytes (35.20–46.40 g/dL) demonstrated mild toxicity among the 100 mg/kg and 500 mg/kg dose groups at the sub-chronic stage. Low levels of hemoglobin (13.60 to 12.70 g/dL), MCV (34.20–24.0 fL), MCH (40.20–36.40 g/dL) along with high levels of liver enzymes confirmed the mild toxicity of the oil at sub-chronic stage. These results demonstrate that, despite consideration of lime essential oil as safe, it can have mild hematotoxic, nephrotoxic and hepatotoxic effects.

Introduction/Aim: Transforming growth factor-beta (TGFbeta) is a pleiotropic cytokine involved in a multitude of processes including wound healing and fibrosis. It is central to chronic lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). Bronchoconstriction results in the release of TGFbeta, but the potential autocrine effects of this increased TGFbeta on airway contraction remains unknown. This study hypothesised that TGFbeta leads to increased airway reactivity to methacholine (MCh). Method(s): We utilised a transgenic mouse model that over-expresses active TGFbeta specifically in the lungs following doxycycline (Dox) administration. Dox was added to the drinking water for 8 weeks prior to experimentation, with normal water given to control mice. Lung plethysmography was used to measure airway resistance in vivo, before serum and BAL were collected for TGFbeta ELISA. Formalin-fixed paraffinembedded sections were also prepared, dewaxed and scanned using the Histoindex for analysis of collagen using Fibroindex. Separate control and Dox mice were used to prepare Precision Cut Lung Slice (PCLS) for in vitro methacholine challenge. Result(s): Dox treatment to induce TGFbeta over-expression increased TGFbeta levels in BAL, but not serum. This was associated with ~25% higher lung collagen deposition, predominantly localised around the airways. TGFbeta over-expression increased in vitro contraction of intrapulmonary airways to MCh (maximum %reduction in airway area: control 41 +/- 5%; Dox 75 +/- 9%; n = 5/group, P < 0.05). Conclusion(s): Elevated TGFbeta levels in the lung results in greater airway contraction to MCh, despite increased airway fibrosis. This model can now be used to provide insights into the mechanisms underlying the potential contribution of TGFbeta to airway hyperresponsiveness, and may identify novel therapeutic targets to oppose excessive airway contraction in chronic lung diseases.

The BCL6 Corepressor (BCOR) is a component of a variant Polycomb repressive complex 1 (PRC1) that is essential for normal development. Recurrent mutations in the BCOR gene have been identified in acute myeloid leukaemia and myelodysplastic syndrome among other cancers; however, its function remains poorly understood. Here we examine the role of BCOR in haematopoiesis in vivo using a conditional mouse model that mimics the mutations observed in haematological malignancies. Inactivation of Bcor in haematopoietic stem cells (HSCs) results in expansion of myeloid progenitors and co-operates with oncogenic KrasG12D in the initiation of an aggressive and fully transplantable acute leukaemia. Gene expression analysis and chromatin immunoprecipitation sequencing reveals differential regulation of a subset of PRC1-target genes including HSC-associated transcription factors such as Hoxa7/9. This study provides mechanistic understanding of how BCOR regulates cell fate decisions and how loss of function contributes to the development of leukaemia.Copyright © 2019, The Author(s).

Patients diagnosed with triple negative breast cancer (TNBC) have an increased risk of rapid metastasis compared to other subtypes. Predicting long-term survival post-chemotherapy in patients with TNBC is difficult, yet enhanced infiltration of tumor infiltrating lymphocytes (TILs) has been associated with therapeutic response and reduced risk of metastatic relapse. Immune biomarkers that predict the immune state of a tumor and risk of metastatic relapse pre- or mid-neoadjuvant chemotherapy are urgently needed to allow earlier implementation of alternate therapies that may reduce TNBC patient mortality. Utilizing a neoadjuvant chemotherapy trial where TNBC patients had sequential biopsies taken, we demonstrate that measurement of T-cell subsets and effector function, specifically CD45RO expression, throughout chemotherapy predicts risk of metastatic relapse. Furthermore, we identified the tumor inherent interferon regulatory factor IRF9 as a marker of active intratumoral type I and II interferon (IFN) signaling and reduced risk of distant relapse. Functional implications of tumor intrinsic IFN signaling were demonstrated using an immunocompetent mouse model of TNBC, where enhanced type I IFN signaling increased anti-tumor immunity and metastasis-free survival post-chemotherapy. Using two independent adjuvant cohorts we were able to validate loss of IRF9 as a poor prognostic biomarker pre-chemotherapy. Thus, IRF9 expression may offer early insight into TNBC patient prognosis and tumor heat, allowing for identification of patients that are unlikely to respond to chemotherapy alone and could benefit from further immune-based therapeutic intervention.Copyright © 2019, The Author(s).