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Decisions to withdraw life sustaining treatment in the ICU are common, but there is little information about how treatment should be withdrawn. A pilot study showed that doctors withdraw life sustaining treatment in different ways even in identical cases. This variation can cause stress for ICU staff and relatives.  Our study investigated the decisions of doctors working in ICUs in Sweden regarding the withdrawal of life sustaining treatment for two fictitious patients. There was variation in if and how drug treatments should be withdrawn, as well as how ventilatory support should be withdrawn. Less experienced doctors tended to choose to prolong the dying process by weaning, even if it is unclear if that is preferable for the staff or for relatives.  Our study could be used in discussions in ICUs to try to understand how individual doctors make decisions about withdrawing life sustaining treatment.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy., Competing Interests: Declaration of interests D.A.M. holds ownership interest in iOncologi, Inc. E.J.S is a paid consultant for Siren Biotechnology. The manuscript discusses patented technologies from H.R.M.-G., P.C., S.Q., J.M., A.P.G., J.H., W.G.S., M.R., D.A.M., and E.J.S. Patented technologies are under option to license by iOncologi, Inc., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Messenger RNA (mRNA) has emerged as a remarkable tool for COVID-19 prevention but its use for induction of therapeutic cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Herein, we develop a facile approach for substantially enhancing immunogenicity of tumor-derived mRNA in lipid-particle (LP) delivery systems. By using mRNA as a molecular bridge with ultrapure liposomes and foregoing helper lipids, we promote the formation of 'onion-like' multi-lamellar RNA-LP aggregates (LPA). Intravenous administration of RNA-LPAs mimics infectious emboli and elicits massive DC/T cell mobilization into lymphoid tissues provoking cancer immunogenicity and mediating rejection of both early and late-stage murine tumor models. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for toll-like receptor engagement, RNA-LPAs stimulate intracellular pathogen recognition receptors (RIG-I) and reprogram the TME thus enabling therapeutic T cell activity. RNA-LPAs were safe in acute/chronic murine GLP toxicology studies and immunologically active in client-owned canines with terminal gliomas. In an early phase first-in-human trial for patients with glioblastoma, we show that RNA-LPAs encoding for tumor-associated antigens elicit rapid induction of pro-inflammatory cytokines, mobilization/activation of monocytes and lymphocytes, and expansion of antigen-specific T cell immunity. These data support the use of RNA-LPAs as novel tools to elicit and sustain immune responses against poorly immunogenic tumors.

Although doctor-led RAT has advantages, serial processing (triage - investigation - treatment) still predominates in UK EDs. We have designed a RAT decision-support app to assist ED nurses to select investigations and treatments at initial patient assessment and aid acuity scoring., Methods: Test nurses accompanied triage ('control') nurses in an observational study of 529 adult patients. Investigations, treatments and procedures, selected using the app, were compared with those selected later by ED clinicians. Acuities set by both nurses were re-evaluated blind by a consultant panel., Results: Data capture and decision making using RAT-support took a median of 1.43 min (IQR 1.13-2.07). Odds ratios are reported for matching of test versus control investigation and treatment orders. The ability to predict, within minutes, control investigations which were ordered at median of 50 min (IQR 21-99) is encouraging. Median times to order treatments (analgesia 88 min, IV antibiotics 112 min) were also reduced. Acuity scores versus the consultant panel gave weighted kappa of 0.54 (CI 0.48-0.61) for study nurses and for controls 0.45 (CI 0.36-0.53)., Conclusions: We conclude that nurse-led RAT for use in initial assessment is feasible given decision support. We also identified improvements required for the app., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Pompe disease is a neuromuscular disease caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase leading to lysosomal and cytoplasmic glycogen accumulation in neurons and striated muscle. In the decade since availability of first-generation enzyme replacement therapy (ERT) a better understanding of the clinical spectrum of disease has emerged. The most severe form of early onset disease is typically identified with symptoms in the first year of life, known as infantile-onset Pompe disease (IOPD). Infants are described at floppy babies with cardiac hypertrophy in the first few months of life. A milder form with late onset (LOPD) of symptoms is mostly free of cardiac involvement with slower rate of progression. Glycogen accumulation in the CNS and skeletal muscle is observed in both IOPD and LOPD. In both circumstances, multi-system disease (principally motoneuron and myopathy) leads to progressive weakness with associated respiratory and feeding difficulty. In IOPD the untreated natural history leads to cardiorespiratory failure and death in the first year of life. In the current era of ERT clinical outcomes are improved, yet, many patients have an incomplete response and a substantial unmet need remains. Since the neurological manifestations of the disease are not amenable to peripheral enzyme replacement, we set out to better understand the pathophysiology and potential for treatment of disease manifestations using adeno-associated virus (AAV)-mediated gene transfer, with the first clinical gene therapy studies initiated by our group in 2006. This review focuses on the preclinical studies and clinical study findings which are pertinent to the development of a comprehensive gene therapy strategy for both IOPD and LOPD. Given the advent of newborn screening, a significant focus of our recent work has been to establish the basis for repeat administration of AAV vectors to enhance neuromuscular therapeutic efficacy over the life span., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Intubation and admission to ICU are vital stages in the management of unconscious patients. Treatment records for all patients who died within 5 days of admission to Södersjukhuset during 2015 were obtained. Patients with GCS <9 in the ER were selected. It was noted if the patients had been intubated, had done a CT brain scan and if they had been admitted to ICU. They were divided into one of three diagnosis groups: cardiac arrest, intracerebral hemorrhage/stroke or other. 48 of 51 cardiac arrest patients were intubated and transferred to ICU. Only 17 of 46 ICH/stroke patients were intubated, and 22 ICH/stroke patients did a CT brain scan with an unprotected airway. Possible organ donors were more difficult to detect in the cardiac arrest group (6 of 14 possible) compared with the ICH/stroke group (12 of 14 possible). Our analysis shows that improvements need to be made in the airway management of unconscious patients who have suffered an ICH or stroke, and that identification of possible organ donors amongst victims of cardiac arrest also needs to be improved.

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 10(14) rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 10(5) vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.

A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon-optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e., rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early- and late-onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be readministered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies that may reduce the effectiveness of the vector, especially if readministration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa(-/-) mice, an animal model of Pompe disease. This article describes the IND-enabling preclinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for readministration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with late-onset Pompe disease will be enrolled. The goal of the immune modulation strategy is to ablate B-cells before the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of the active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the evidence generated from gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the three months following each dosing will assess the safety, biochemical, and functional impact of the vector.

Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

This study compared equine and ovine LH secretory responses to GnRH treatment. Dioestrous mares and ewes were challenged with continuous GnRH for 15 h. Mares that received constant GnRH (110 micrograms h-1) had sustained LH secretion (P < 0.01), whereas LH concentrations in ewes treated with continuous GnRH (25 micrograms h-1) initially increased, then declined and remained low, suggesting GnRH receptor desensitization or downregulation. In addition, progesterone-primed, ovariectomized mares and ewes were challenged with pulsatile or continuous GnRH for 5 days. Plasma LH concentrations were increased by day 5 in mares treated with pulsatile (25 micrograms pulse-1 h-1) and continuous (110 micrograms h-1) GnRH (P < 0.01). Furthermore, mean LH concentrations and time-response curves were not different. In contrast, ewes treated with continuous GnRH (2.5 micrograms h-1) demonstrated LH secretory patterns indicative of GnRH receptor downregulation on day 1 of treatment. LH concentrations in ewes treated with pulsatile GnRH (250 ng pulse-1 h-1) did not differ from controls. In conclusion, pony mares responded continuously to GnRH treatment (pulsatile and continuous), whereas ewes treated with continuous GnRH experienced reduced LH secretion. These findings suggest a unique hypothalamic-pituitary axis in pony mares.

The equine embryonic capsule, an acellular covering that envelops the conceptus during the second and third weeks of pregnancy, is composed of mucin-like glycoproteins. Its structure is consistent with a dual role during early pregnancy: protection of the conceptus, and communication between the embryo and the mother. Loss of sialic acid from the capsular glycoproteins at day 16 correlates with the time of "fixation," or loss of conceptus mobility throughout the uterine horns. This study investigated how the structure of the capsule is linked to the maintenance of pregnancy. Six pregnancies, confirmed by ultrasound, were terminated by prostaglandin injection on day 14, prior to the time of embryo fixation. These "defective" conceptuses were collected at day 17, and the structure and molecular properties of their capsules were compared to those of day 17 conceptuses collected from 5 normal pregnancies. Defective capsules were not significantly different from normal capsules in terms of dry weight, amino acid composition, and content of neutral and amino sugars. However, defective capsules failed to show the loss of sialic acid normally occurring around the time of embryo fixation. Analysis of the capsular mucins following trypsin digestion was carried out by radioactive labeling with 3H on sialyl-oligosaccharides and 125I on tyrosine residues, followed by fast protein liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Differences in the trypsin fragmentation patterns indicated increased susceptibility of the defective capsules to proteolysis. We conclude that there is a temporal association between desialylation of the equine capsule and embryonic survival, and that failure to desialylate alters the properties of the capsule.

A family of estrogen-dependent porcine oviductal secretory glycoproteins (POSPs) that exhibit structural similarities are synthesized and secreted into the oviductal lumen at proestrus, estrus, and metestrus. The objectives of this study were to clone the POSP cDNA, obtain the full-length cDNA and protein sequence, examine tissue specificity and species distribution, characterize its regulation, and establish its identity by comparison to other known protein, RNA, or DNA sequences. A full-length cDNA of 2022 base pairs was obtained with an open reading frame of 1581 nucleotides, coding for a deduced protein of 527 amino acids (57 970 M(r)). The deduced protein contained three potential N-glycosylation sites, a consensus heparin-binding site, and potential O-glycosylation sites. Amino acid analysis of POSP-E3 confirmed the presence of a 21-amino acid signal sequence. Northern blot analysis revealed an oviduct-specific mRNA species of 2.25 kb in the infundibulum (INF), ampulla (A), and isthmus (I). An mRNA of similar size was detected in the oviduct of the sheep, cow, and rabbit, and one of slightly greater size (2.8 kb) in the mouse and hamster oviduct but not in the horse or alligator oviduct. Dot blot analysis indicated that steady-state levels of POSP mRNA were significantly greater (p = 0.0001) in the A than in the INF or I regardless of day of the estrous cycle and were greater on Day 0 (estrus; p = 0.0001) regardless of location. Further, steady-state mRNA levels were significantly increased (p = 0.02) on Days 0 and 1, declining rapidly to Day 2 through Day 15 of the estrous cycle. Steady-state POSP mRNA levels were significantly greater (p < 0.003) in ovariectomized gilts treated with estradiol valerate than those treated with other steroid regimens, vehicle, or no treatment (Control), consistent with estrogen control of mRNA expression. The POSP protein exhibited significant identity to oviductal glycoproteins from the baboon, cow, hamster, human, mouse, and sheep, to several mammalian nonoviductal glycoproteins; and to several chitinases. POSP joins a growing subfamily of the chitinase gene family that lacks chitinase enzymatic activity.

Melatonin is thought to play a role in relaying photic information to the central nervous system as part of the seasonal reproductive cycle of the mare. However, the mechanisms by which melatonin may act are unknown. Therefore, this study was designed to determine whether exposure to constant light would, by reducing circulating melatonin concentrations, have any effect on hypothalamic gonadotrophin-releasing hormone (GnRH) content and circulating levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Blood samples were collected for 12 h at 15-min intervals from 8 ovariectomized (OVX) pony mares under ambient light conditions (12 h light, 12 h dark, October 6). Animals were then placed under representative ambient light (12 h light, 12 h dark; control, n = 4) or constant light (24 h light, 0 h dark; treatment, n = 4) in light-controlled rooms. Blood samples were collected daily and on experimental Days 14, 21 and 28 samples were collected at 15-min intervals for 12 h for analysis of circulating LH and melatonin (Bleed 2, 3 and 4, respectively). All animals were killed on Day 28 (following Bleed 4) and the hypothalami were collected. Mares exposed to constant light had significantly higher (P less than 0.05) LH concentrations in daily blood samples and showed significantly (P less than 0.05) higher LH concentrations during frequent sampling periods on Days 14 and 21 (Bleeds 2 and 3) compared with control mares. FSH did not differ significantly among groups in the daily samples. GnRH content was 1.5994 +/- 0.325 and 0.9457 +/- 0.193 pg/mg protein (treatment and control respectively).(ABSTRACT TRUNCATED AT 250 WORDS)