Repair of acute respiratory distress syndrome by stromal cell administration (REALIST): a structured study protocol for an open-label dose-escalation phase 1 trial followed by a randomised, triple-blind, allocation concealed, placebo-controlled phase 2 trial.

BACKGROUND: Mesenchymal stromal cells (MSCs) may be of benefit in ARDS due to immunomodulatory and reparative properties. This trial investigates a novel CD362 enriched umbilical cord derived MSC product (REALIST ORBCEL-C), produced to Good Manufacturing Practice standards, in patients with moderate to severe ARDS due to COVID-19 and ARDS due to other causes. METHODS: Phase 1 is a multicentre open-label dose-escalation pilot trial. Patients will receive a single infusion of REALIST ORBCEL-C (100 × 106 cells, 200 × 106 cells or 400 × 106 cells) in a 3 + 3 design. Phase 2 is a multicentre randomised, triple blind, allocation concealed placebo-controlled trial. Two cohorts of patients, with ARDS due to COVID-19 or ARDS due to other causes, will be recruited and randomised 1:1 to receive either a single infusion of REALIST ORBCEL-C (400 × 106 cells or maximal tolerated dose in phase 1) or placebo. Planned recruitment to each cohort is 60 patients. The primary safety outcome is the incidence of serious adverse events. The primary efficacy outcome is oxygenation index at day 7. The trial will be reported according to the Consolidated Standards for Reporting Trials (CONSORT 2010) statement. DISCUSSION: The development and manufacture of an advanced therapy medicinal product to Good Manufacturing Practice standards within NHS infrastructure are discussed, including challenges encountered during the early stages of trial set up. The rationale to include a separate cohort of patients with ARDS due to COVID-19 in phase 2 of the trial is outlined. TRIAL REGISTRATION: ClinicalTrials.gov NCT03042143. Registered on 3 February 2017. EudraCT Number 2017-000584-33.

Repair of Acute Respiratory Distress Syndrome by Stromal Cell Administration in COVID-19 (REALIST-COVID-19): A structured summary of a study protocol for a randomised, controlled trial.

OBJECTIVES: The primary objective of the study is to assess the safety of a single intravenous infusion of Mesenchymal Stromal Cells (MSCs) in patients with Acute Respiratory Distress Syndrome (ARDS) due to COVID-19. Secondary objectives are to determine the effects of MSCs on important clinical outcomes, as described below. TRIAL DESIGN: REALIST COVID 19 is a randomised, placebo-controlled, triple blinded trial. PARTICIPANTS: The study will be conducted in Intensive Care Units in hospitals across the United Kingdom. Patients with moderate to severe ARDS as defined by the Berlin definition, receiving invasive mechanical ventilation and with a diagnosis of COVID-19 based on clinical diagnosis or PCR test will be eligible. Patients will be excluded for the following reasons: more than 72 hours from the onset of ARDS; age < 16 years; patient known to be pregnant; major trauma in previous 5 days; presence of any active malignancy (other than non-melanoma skin cancer); WHO Class III or IV pulmonary hypertension; venous thromboembolism currently receiving anti-coagulation or within the past 3 months; patient receiving extracorporeal life support; severe chronic liver disease (Child-Pugh > 12); Do Not Attempt Resuscitation order in place; treatment withdrawal imminent within 24 hours; prisoners; declined consent; non-English speaking patients or those who do not adequately understand verbal or written information unless an interpreter is available; previously enrolled in the REALIST trial. INTERVENTION AND COMPARATOR: Intervention: Allogeneic donor CD362 enriched human umbilical cord derived mesenchymal stromal cells (REALIST ORBCEL-C) supplied as sterile, single-use cryopreserved cell suspension of a fixed dose of 400 x106 cells in 40ml volume, to be diluted in Plasma-Lyte 148 to a total volume of 200mls for administration. Comparator (placebo): Plasma-Lyte 148 Solution for Infusion (200mls). The cellular product (REALIST ORBCEL-C) was developed and patented by Orbsen Therapeutics. MAIN OUTCOMES: The primary safety outcome is the incidence of serious adverse events. The primary efficacy outcome is Oxygenation Index (OI) at day 7. Secondary outcomes include: OI at days 4 and 14; respiratory compliance, driving pressure and PaO2/FiO2 ratio (PF ratio) at days 4, 7 and 14; Sequential Organ Failure Assessment (SOFA) score at days 4, 7 and 14; extubation and reintubation; ventilation free days at day 28; duration of mechanical ventilation; length of ICU and hospital stay; 28-day and 90-day mortality. RANDOMISATION: After obtaining informed consent, patients will be randomised via a centralised automated 24-hour telephone or web-based randomisation system (CHaRT, Centre for Healthcare Randomised Trials, University of Aberdeen). Randomisation will be stratified by recruitment centre and by vasopressor use and patients will be allocated to REALIST ORBCEL-C or placebo control in a 1:1 ratio. BLINDING (MASKING): The investigator, treating physician, other members of the site research team and participants will be blinded. The cell therapy facility and clinical trials pharmacist will be unblinded to facilitate intervention and placebo preparation. The unblinded individuals will keep the treatment information confidential. The infusion bag will be masked at the time of preparation and will be administered via a masked infusion set. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): A sample size of 60 patients with 30 patients randomised to the intervention and 30 to the control group. If possible, recruitment will continue beyond 60 patients to provide more accurate and definitive trial results. The total number of patients recruited will depend on the pandemic and be guided by the data monitoring and ethics committee (DMEC). TRIAL STATUS: REALIST Phase 1 completed in January 2020 prior to the COVID-19 pandemic. This was an open label dose escalation study of REALIST ORBCEL-C in patients with ARDS. The COVID-19 pandemic emerged as REALIST Phase 2 was planned to commence and the investigator team decided to repurpose the Phase 2 trial as a COVID-19 specific trial. This decision was discussed and approved by the Trial Steering Committee (TSC) and DMEC. Submissions were made to the Research Ethics Committee (REC) and MHRA to amend the protocol to a COVID-19 specific patient population and the protocol amendment was accepted by the REC on 27th March 2020 and MHRA on 30th March 2020 respectively. Other protocol changes in this amendment included an increase in the time of onset of ARDS from 48 to 72 hours, inclusion of clinical outcomes as secondary outcomes, the provision of an option for telephone consent, an indicative sample size and provision to continue recruitment beyond this indicative sample size. The current protocol in use is version 4.0 23.03.2020 (Additional file 1). Urgent Public Health status was awarded by the NIHR on 2 April 2020 and the trial opened to recruitment and recruited the first participant the same day. At the time of publication the trial was open to recruitment at 5 sites across the UK (Belfast Health and Social Care Trust, King's College London, Guys and St Thomas' Hospital London, Birmingham Heartlands Hospital and the Queen Elizabeth Hospital Birmingham) and 12 patients have been recruited across these sites. Additional sites are planned to open and appropriate approvals for these are being obtained. It is estimated recruitment will continue for 6 months. TRIAL REGISTRATION: ClinicalTrials.gov NCT03042143 (Registered 3 Feb 2017). EudraCT 2017-000585-33 (Registered 28 Nov 2017). FULL PROTOCOL: The full protocol (version 4.0 23.03.2020) is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest of expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).

Dynamic evolution of the GnRH receptor gene family in vertebrates.

BACKGROUND: Elucidating the mechanisms underlying coevolution of ligands and receptors is an important challenge in molecular evolutionary biology. Peptide hormones and their receptors are excellent models for such efforts, given the relative ease of examining evolutionary changes in genes encoding for both molecules. Most vertebrates possess multiple genes for both the decapeptide gonadotropin releasing hormone (GnRH) and for the GnRH receptor. The evolutionary history of the receptor family, including ancestral copy number and timing of duplications and deletions, has been the subject of controversy. RESULTS: We report here for the first time sequences of three distinct GnRH receptor genes in salamanders (axolotls, Ambystoma mexicanum), which are orthologous to three GnRH receptors from ranid frogs. To understand the origin of these genes within the larger evolutionary context of the gene family, we performed phylogenetic analyses and probabilistic protein homology searches of GnRH receptor genes in vertebrates and their near relatives. Our analyses revealed four points that alter previous views about the evolution of the GnRH receptor gene family. First, the "mammalian" pituitary type GnRH receptor, which is the sole GnRH receptor in humans and previously presumed to be highly derived because it lacks the cytoplasmic C-terminal domain typical of most G-protein coupled receptors, is actually an ancient gene that originated in the common ancestor of jawed vertebrates (Gnathostomata). Second, unlike previous studies, we classify vertebrate GnRH receptors into five subfamilies. Third, the order of subfamily origins is the inverse of previous proposed models. Fourth, the number of GnRH receptor genes has been dynamic in vertebrates and their ancestors, with multiple duplications and losses. CONCLUSION: Our results provide a novel evolutionary framework for generating hypotheses concerning the functional importance of structural characteristics of vertebrate GnRH receptors. We show that five subfamilies of vertebrate GnRH receptors evolved early in the vertebrate phylogeny, followed by several independent instances of gene loss. Chief among cases of gene loss are humans, best described as degenerate with respect to GnRH receptors because we retain only a single, ancient gene.

The complex evolutionary dynamics of Hsp70s: a genomic and functional perspective.

Hsp70 molecular chaperones are ubiquitous. By preventing aggregation, promoting folding, and regulating degradation, Hsp70s are major factors in the ability of cells to maintain proteostasis. Despite a wealth of functional information, little is understood about the evolutionary dynamics of Hsp70s. We undertook an analysis of Hsp70s in the fungal clade Ascomycota. Using the well-characterized 14 Hsp70s of Saccharomyces cerevisiae, we identified 491 orthologs from 53 genomes. Saccharomyces cerevisiae Hsp70s fall into seven subfamilies: four canonical-type Hsp70 chaperones (SSA, SSB, KAR, and SSC) and three atypical Hsp70s (SSE, SSZ, and LHS) that play regulatory roles, modulating the activity of canonical Hsp70 partners. Each of the 53 surveyed genomes harbored at least one member of each subfamily, and thus establishing these seven Hsp70s as units of function and evolution. Genomes of some species contained only one member of each subfamily that is only seven Hsp70s. Overall, members of each subfamily formed a monophyletic group, suggesting that each diversified from their corresponding ancestral gene present in the common ancestor of all surveyed species. However, the pattern of evolution varied across subfamilies. At one extreme, members of the SSB subfamily evolved under concerted evolution. At the other extreme, SSA and SSC subfamilies exhibited a high degree of copy number dynamics, consistent with a birth-death mode of evolution. KAR, SSE, SSZ, and LHS subfamilies evolved in a simple divergent mode with little copy number dynamics. Together, our data revealed that the evolutionary history of this highly conserved and ubiquitous protein family was surprising complex and dynamic.

Monoarthritis Induced by Bupropion Hydrochloride.

Bupropion hydrochloride is an inhibitor of dopamine and norepinephrine, which is commonly prescribed for major depression, smoking cessation, and bipolar depression. Here we report a highly unusual case of bupropion induced knee monoarthritis in a bipolar depression patient. With bupropion XL 150 mg for 2 weeks, her left knee began to swell; at the third week, this condition was worsening. The aggravation of the left knee effusion stopped after the discontinuation of bupropion XL. The effusion and swelling disappeared after 15 ml of synovial fluid was drawn out and the effusion has never returned. Analysis of the synovial fluid showed noninflammatory effusion. Her left knee swelling was most likely due to angioedema caused by bupropion XL.

Transgenic pigs designed to express human CD59 and H-transferase to avoid humoral xenograft rejection.

Research in pig-to-primate xenotransplantation aims to solve the increasing shortage of organs for human allotransplantation and develop new cell- and tissue-based therapies. Progress towards its clinical application has been hampered by the presence of xenoreactive natural antibodies that bind to the foreign cell surface and activate complement, causing humoral graft rejection. Genetic engineering of donor cells and animals to express human complement inhibitors such as hCD59 significantly prolonged graft survival. Strategies to decrease the deposition of natural antibodies were also developed. Expression of human alpha1,2-fucosyltransferase (H transferase, HT) in pigs modifies the cell-surface carbohydrate phenotype resulting in reduced Galalpha1,3-Gal expression and decreased antibody binding. We have developed transgenic pigs that coexpress hCD59 and HT in various cells and tissues to address both natural antibody binding and complement activation. Functional studies with peripheral blood mononuclear cells and aortic endothelial cells isolated from the double transgenic pigs showed that coexpression of hCD59 and HT markedly increased their resistance to human serum-mediated lysis. This resistance was greater than with cells transgenic for either hCD59 or HT alone. Moreover, transgene expression was enhanced and protection maintained in pig endothelial cells that were exposed for 24 h to pro-inflammatory cytokines. These studies suggest that engineering donor pigs to express multiple molecules that address different humoral components of xenograft rejection represents an important step toward enhancing xenograft survival and improving the prospect of clinical xenotransplantation.