Simulated shift work during pregnancy does not impair progeny metabolic outcomes in sheep

Disrupted maternal circadian rhythms, such as those experienced during shift work, are associated with impaired progeny metabolism in rodents. The effects of disrupted maternal circadian rhythms on progeny metabolism have not been assessed in altricial, non-litter bearing species. We therefore assessed postnatal growth from birth to adulthood, and body composition, glucose tolerance, insulin secretion and insulin sensitivity in pre-pubertal and young adult progeny of sheep exposed to control conditions (CON: 10 males, 10 females) or to a simulated shift work (SSW) protocol for the first 1/3 (SSW0-7: 11 males, 9 females), the first 2/3 (SSW0-14: 8 males, 11 females), or all (SSW0-21: 8 males, 13 females) of pregnancy. Progeny growth did not differ between maternal treatments. In pre-pubertal progeny (12-14 weeks of age), adiposity, glucose tolerance and insulin secretion during an intravenous glucose tolerance test and insulin sensitivity did not differ between maternal treatments. Similarly, in young adult progeny (12-14 months of age), food intake, adiposity and glucose tolerance did not differ between maternal treatments. At this age, however, insulin secretion in response to a glucose bolus was 30% lower in female progeny in the combined SSW groups compared to control females (P = 0.031), and insulin sensitivity of SSW0-21 singleton females was 236% that of CON singleton female progeny (P = 0.025). At least in this model, maternal SSW does not impair progeny metabolic health, with some evidence of greater insulin action in female young adult progeny.Kathryn L. Gatford, David J. Kennaway, Hong Liu, Christopher G. Schultz, Amy L. Wooldridge, Timothy R. Kuchel and Tamara J. Varco

High mobility group box protein 1 neutralization therapy in ovine bacteremia: lessons learned from an ovine septic shock model incorporating intensive care support

Sepsis is a highly complex and often fatal syndrome which varies widely in its clinical manifestations, and therapies that target the underlying uncontrolled immune status in sepsis are needed. The failure of preclinical approaches to provide significant sepsis survival benefit in the clinic is often attributed to inappropriate animal disease models. It has been demonstrated that high mobility group box protein 1 (HMGB1) blockade can reduce inflammation, mortality and morbidity in experimental sepsis without promoting immunosuppression. Within this study, we explored the use of ovine anti-HMGB1 antibodies in a model of ovine septic shock incorporating intensive care supports (OSSICS). Results: Septic sheep exhibited elevated levels of HMGB1 within 12 h after the induction of sepsis. In this study, sepsis was induced in six anaesthetized adult Border Leicester × Merino ewes via intravenous instillation of E. coli and sheep monitored according to intensive care unit standard protocols for 26 h, with the requirement for noradrenaline as the primary endpoint. Septic sheep exhibited a hyperdynamic circulation, renal dysfunction, deranged coagulation profile and severe metabolic acidosis. Sheep were assigned a severity of illness score, which increased over time. While a therapeutic effect of intravenous anti-HMGB1 antibody could not be observed in this model due to limited animal numbers, a reduced bacterial dose induced a septic syndrome of much lower severity. With modifications including a reduced bacterial dose, a longer timeframe and broad spectrum antibiotics, the OSSICS model may become a robust tool for preclinical assessment of sepsis therapeutics.Natalie E. Stevens, Coralie H. Nash, Cara K. Fraser, Tim R. Kuchel, Matthew J. Maiden, Marianne J. Chapman, Kerrilyn R. Diener, and John D. Haybal

Longitudinal Magnetic Resonance Spectroscopy and Diffusion Tensor Imaging in Sheep (Ovis aries) With Quinolinic Acid Lesions of the Striatum: Time-Dependent Recovery of N-Acetylaspartate and Fractional Anisotropy.

We created an excitotoxic striatal lesion model of Huntington disease (HD) in sheep, using the N-methyl-D-aspartate receptor agonist, quinolinic acid (QA). Sixteen sheep received a bolus infusion of QA (75 mL, 180mM) or saline, first into the left and then (4 weeks later) into the right striatum. Magnetic resonance spectroscopy (MRS) and diffusion tensor imaging (DTI) of the striata were performed. Metabolite concentrations and fractional anisotropy (FA) were measured at baseline, acutely (1 week after each surgery) and chronically (5 weeks or greater after the surgeries). There was a significant decrease in the neuronal marker N-acetylaspartate (NAA) and in FA in acutely lesioned striata of the QA-lesioned sheep, followed by a recovery of NAA and FA in the chronically lesioned striata. NAA level changes indicate acute death and/or impairment of neurons immediately after surgery, with recovery of reversibly impaired neurons over time. The change in FA values of the QA-lesioned striata is consistent with acute structural disruption, followed by reorganization and glial cell infiltration with time. Our study demonstrates that MRS and DTI changes in QA-sheep are consistent with HD-like pathology shown in other model species and that the MR investigations can be performed in sheep using a clinically relevant human 3T MRI scanner.Adam B. O'Connell, Timothy R. Kuchel, Sunthara R. Perumal, Victoria Sherwood, Daniel Neumann, John W. Finnie, Kim M. Hemsley, and A. Jennifer Morto

Intracutaneous Transplantation of Islets within a Biodegradable Temporizing Matrix (BTM) as an Alternative Site for Islet Transplantation

Intra-hepatic islet transplantation for type-1 diabetes is limited by the need for multiple infusions and poor islet viability post-transplantation. The development of alternative transplantation sites is necessary to improve islet survival, and facilitate monitoring and retrieval. We tested a clinically proven Biodegradable Temporizing Matrix (BTM), a polyurethane-based scaffold, to generate a well vascularized intracutaneous "neo-dermis" within the skin for islet transplantation. In murine models, BTM did not impair syngeneic islet renal-subcapsular transplant viability or function, and facilitated diabetes cure for over 150 days. Further, BTM supported functional neonatal porcine islet transplants into RAG-1-/- mice for 400 days. Hence, BTM is non-toxic for islets. two-photon intravital imaging used to map vessel growth through time identified dense vascular networks, with significant collagen deposition and increases in vessel mass up to 30 days post-BTM implantation. In a pre-clinical porcine skin model, BTM implants created a highly-vascularized intracutaneous site by day 7 post-implantation. When syngeneic neonatal porcine islets were transplanted intracutaneously the islets remained differentiated as insulin producing cells, maintained normal islet architecture, secreted c-peptide, and survived for over 100 days. Here we show that BTM facilitates formation of an islet-supportive intracutaneous "neo-dermis" in a porcine pre-clinical model, as an alternative islet transplant site.Darling Rojas-Canales, Stacey N. Walters, Daniella Penko, Daniele Cultrone, Jacqueline Bailey, Tatyana Chtanova, Jodie Nitschke, Julie Johnston, Svjetlana Kireta, Thomas Loudovaris, Thomas W. Kay, Tim R. Kuchel, Wayne Hawthorne, Philip J. O, Connell, Greg Korbutt, John E. Greenwood, Shane T. Grey, Chris J. Drogemuller, and P. Toby Coate

Mechanism and inhibition of the papain-like protease, PLpro, of SARS-CoV-2

The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.Chemical Immunolog