Rice endosperm as a production platform for third-generation HIV microbicides

L’objectiu de la meva tesis doctoral és la producció en l’endosperma de l’arròs d’una tercera generació de microbicides contra el virus de la sida. Durant la meva recerca he utilitzat l’anticòs monoclonal 2G12 i dues proteïnes que neutralitzen el virus de la sida CV-N i GRFT, soles o en combinació, per a descobrir noves estratègies de producció. En la primera part he investigat si podia produir 2G12 a l’endosperma de l’arròs i quins eren els factors que influenciaven la seva producció. En la segona part de la meva tesis he introduït GRFT a l’endosperma de l’arròs i he analitzat si la proteïna expressada tenia activitat neutralitzant del virus de la SIDA. La expressió de GRFT en l’endosperma de l’arròs va ser comparable o inclús en algun cas més alta a GRFT expressada de forma transitòria en tabac. A la tercera part de la meva tesis he investigat si l’endosperma d’arròs seria una plataforma funcional per a la producció de CV-N i si aquesta proteïna tindria capacitat neutralitzant del virus de la sida. Finalment he utilitzat una combinació de microbicides 2G12, CV-N i GRFT per a introduir-les simultàniament a l’endosperma de l’arròs i identificar la millor combinació contra el virus de la sida, i si la co-expresió augmentava el seu efecte neutralitzant. Aquesta es la primera vegada que més d’una proteïna que neutralitzen el virus de la sida son produïdes de forma estable en l’endosperma d’un cereal, obrint un camí nou cap a la producció d’un còctel microbicida per a evitar la transmissió del virus de la sida.El objetivo de mi tesis doctoral es la producción en el endospermo de arroz de una tercera generación de microbicidas contra el virus del SIDA. Durante mi trabajo de investigación utilicé el anticuerpo monoclonal 2G12 y dos proteínas que neutralizan el virus, CV-N y GRFT, solas o en combinación, para explorar diferentes estrategias de producción. En la primera parte de mi tesis investigué si podía producir 2G12 en el endospermo de arroz y cuáles eran los factores que afectaban a su producción. En la segunda parte de mi tesis, he introducido GRFT en el endospermo de arroz y he investigado si la proteína producida tenía actividad neutralizante del virus del SIDA. La expresión de GRFT en el endospermo del arroz fue comparable o incluso en algún caso más alta a GRFT producida de forma transitoria en tabaco. En la tercera parte de mi tesis he investigado si el endospermo de arroz sería una plataforma funcional para la producción de CV-N y si esta proteína sería capaz de neutralizar el virus del sida. La proteína Finalmente he utilizado una combinación de microbicidas 2G12, CV-N y GRFT para introducirlos simultáneamente en el endospermo de arroz e identificar la mejor combinación contra el virus del sida, y si la co-expresión potenciaba su efecto neutralizante. La combinación Esta es la primera vez que más de una proteína que neutraliza el virus del sida es producida de forma estable en el endospermo de un cereal, abriendo un nuevo camino hacia la producción de un cóctel microbicida para evitar la transmisión del virus del sida.The focus of this thesis is the production of third-generation HIV microbicides in rice cells. I used one monoclonal antibody (2G12) and two anti-HIV proteins (CV-N and GRFT) alone or in combination, to explore different strategies to produce an efficient microbicide against HIV. The first part of my PhD thesis was to test whether rice endosperm can function as a platform for the production of 2G12 and to gain insight into the factors that affect the production and functional efficacy of neutralizing antibodies in plants. The human mAb2G12 was expressed successfully into rice endosperms as an aglycosylated form. In the second part of the thesis I introduced GRFT into rice endosperm and I tested whether the rice-derived GRFT protein retains its potent HIV-neutralizing activity. GRFT was successfully expressed in rice endosperm resulting in yields comparable or even higher to GRFT produced transiently in tobacco and other recombinant proteins. In the third part of my thesis I explored whether rice endosperm could function as a production platform to produce CV-N and I evaluated whether the rice-derived CV-N could neutralize efficiently the HIV virus. Finally I used a combinatorial approach introducing the 2G12, CV-N and GRFT into rice cells to identify the best combination against HIV, and whether their co-expression enhanced their effect. This is the first time that more than one protein against HIV is stable expressed into endosperm opening a new way for the production of a microbicide cocktail for the prevention of HIV transmission

Οι διοικητικές αποφάσεις της Δ΄ Οικουμενικής Συνόδου κατά τα πρακτικά της. Ιστορικοκανονική προσέγγιση.

Η Δ΄ Οικουμενική Σύνοδος, από την εποχή της σύγκλισής της μέχρι τις μέρες μας, έχει αποτελέσει πολλές φορές αντικείμενο έρευνας και σημείο αναφοράς, τόσο εξαιτίας της σπουδαιότητας των δογματικών της αποφάσεων, όσο και εξαιτίας της καίριας σημασίας των κανονικών της αποφάσεων για την μετέπειτα διοικητική εξέλιξη της Εκκλησίας. Η παρούσα διπλωματική εργασία σκοπό έχει να αναδείξει το κανονικό έργο της Δ΄ Οικουμενικής Συνόδου, όπως αυτό είναι αποθησαυρισμένο στα πρακτικά της. Η μέθοδος που ακολουθήσαμε κατά την επεξεργασία του άφθονου υλικού των πρακτικών, είναι η συστηματική σε συνδυασμό με την ιστορικοκανονική προσέγγιση. Οδηγός της έρευνάς μας είναι το ίδιο το κείμενο των πρακτικών 11 συνεδριών της Συνόδου της Χαλκηδόνας, κατά τη διάρκεια των οποίων συζητήθηκαν διοικητικά θέματα και ελήφθησαν οι διοικητικές αποφάσεις της Συνόδου. Κατά την εξέταση των εν λόγω θεμάτων, παρακολουθούμε με προσοχή τις διεξαχθείσες συζητήσεις, αναψηλαφούμε τις κανονικές τάσεις και την γενικότερη εκκλησιαστική κατάσταση της εποχής, παρεμβαίνουμε για να συμπληρώσουμε ιστορικές πληροφορίες, χωρίς τις οποίες δεν γίνονται κατανοητά τα αναφυέντα ζητήματα και τέλος, επιχειρούμε να διεισδύσουμε στη σκέψη των Πατέρων και να ερμηνεύσουμε, στο μέτρο του δυνατού, τις αποφάσεις τους και τα εκκλησιολογικά ερείσματα που τους οδήγησαν σε αυτές. Συμπερασματικά, θα μπορούσαμε να πούμε ότι ο αγώνας, που διεξήχθη από την εποχή της Β΄ Οικουμενικής Συνόδου, για την υπαγωγή της μητροπολιτικής πολυαρχίας στην υπερμητροπολιτική αυθεντία των επισημοτάτων θρόνων Ρώμης, Κωνσταντινουπόλεως, Αλεξανδρείας, Αντιοχείας και Ιεροσολύμων, έληξε με την Δ΄ Οικουμενική Σύνοδο. Η Δ΄ Οικουμενική Σύνοδος με τις διοικητικές της αποφάσεις συγκρότησε νεοπαγείς γεωεκκλησιαστικές οντότητες, τα Πατριαρχεία και αναγνώρισε de jure την κανονική τάση αναδείξεως των πέντε επισημοτάτων θρόνων. Η μελέτη των πρακτικών της Δ΄ Οικουμενικής Συνόδου μας επιβεβαιώνει ότι οι διαμορφωθείσες τάσεις αναδιάρθρωσης της εκκλησιαστικής διοίκησης υιοθετήθηκαν και περιεβλήθηκαν με κανονικό κύρος από την Σύνοδο της Χαλκηδόνας, επειδή θεμελιώνονταν επί της αυθεντικής κανονικής παράδοσης, υπαγορεύονταν από την εκκλησιαστική συνείδηση και αποσκοπούσαν στην διασφάλιση της ενότητας της Εκκλησίας.The Fourth Ecumenical Synod, since its convergence till present times, has often been the subject of research and a point of reference, thanks to the significance of its dogmatic decisions as well as the crucial importance of its canonical decisions on the subsequent administrative evolution of the Church. This diploma thesis aims to shed light on the canonical work of the Fourth Ecumenical Synod as it is recorded in its minutes. We have followed a combined systematic and historical-canonical approach in our effort to process the abundant material derived from the Acts of the Council of Chalcedon. The very text of the minutes of 11 sessions of the Chalcedon Synod, during which administrative issues were discussed and administrative decisions of the Council were taken, has been the main pivot throughout this study. Throughout the examination of these issues, we meticulously observe the discussions, we look into the canonical tendencies and the general ecclesiastical situation of the time, we intervene to fill in historical information which is essential for the rampant issues to be understood and finally we strive to penetrate into the thoughts of the Fathers, and to interpret, as far as possible, their decisions and the ecclesiological foundations that led to these decisions. In conclusion, the endeavour which took place since the Second Ecumenical Synod, for the subordination of the metropolitan polyarchy to the hypermetropolitan authority of the major sees of Rome, Constantinople, Alexandria, Antioch and Jerusalem, ended with the Fourth Ecumenical Synod. The Fourth Ecumenical Council, with its administrative decisions, set up newly created ecclesiastical entities, the Patriarchates, and recognized de jure the canonical tendency to highlight the five official sees. Through the study of the minutes of the Fourth Ecumenical Synod we were able to confirm that the reformist tendencies of the ecclesiastical administration were indeed adopted and reinforced by the Council of Chalcedon as they were underpinned by the authentic canonical tradition, were dictated by ecclesiastical consciousness and aimed at ensuring the unity of the Church

ChemoPROphyLaxIs with hydroxychloroquine For covId-19 infeCtious disease (PROLIFIC) to prevent covid-19 infection in frontline healthcare workers: A structured summary of a study protocol for a randomised controlled trial

Abstract: Objectives: Primary objective: To determine whether chemoprophylaxis with hydroxychloroquine versus placebo increases time to contracting coronavirus disease 2019 (COVID-19) in frontline healthcare workers. Secondary objectives: To determine whether chemoprophylaxis with daily versus weekly dosing of hydroxychloroquine increases time to contracting COVID-19 disease in frontline healthcare workers. To compare the number of COVID-19 cases between each trial arm on the basis of positive tests (as per current clinical testing methods and/or serology) To compare the percentage of COVID-19 positive individuals with current testing methods versus serologically-proven COVID-19 in each trial arm To compare COVID-19 disease severity in each trial arm To compare recovery time from COVID-19 infection in each trial arm Exploratory objectives: To determine compliance (as measured by trough pharmacokinetic hydroxychloroquine levels) on COVID-19 positive tests To determine if genetic factors determine susceptibility to COVID-19 disease or response to treatment To determine if blood group determines susceptibility to COVID-19 disease To compare serum biomarkers of COVID-19 disease in each arm Trial design: Double-blind, multi-centre, 2-arm (3:3:2 ratio) randomised placebo-controlled trial Participants: National Health Service (NHS) workers who have direct patient contact delivering care to patients with COVID-19. Participants in the trial will be recruited from a number of NHS hospitals directly caring for patients with COVID-19. Inclusion criteria: To be included in the trial the participant MUST: Have given written informed consent to participate Be aged 18 years to 70 years Not previously have been diagnosed with COVID-19 Work in a high-risk secondary or tertiary healthcare setting (hospitals accepting COVID-19 patients) with direct patient-facing care Exclusion criteria: The presence of any of the following will mean participants are ineligible: Known COVID-19 positive test at baseline (if available) Symptomatic for possible COVID-19 at baseline Known hypersensitivity reaction to hydroxychloroquine, chloroquine or 4-aminoquinolines Known retinal disease Known porphyria Known chronic kidney disease (CKD; eGFR<30ml/min) Known epilepsy Known heart failure or conduction problems Known significant liver disease (Gilbert’s syndrome is permitted) Known glucose-6-phosphate dehydrogenase (G6PD) deficiency Currently taking any of the following contraindicated medications: Digoxin, Chloroquine, Halofantrine, Amiodarone, Moxifloxacin, Cyclosporin, Mefloquine, Praziquantel, Ciprofloxacin, Clarithromycin, Prochlorperazine, Fluconazole Currently taking hydroxychloroquine or having a clinical indication for taking hydroxychloroquine Currently breastfeeding Unable to be followed-up during the trial Current or future involvement in the active treatment phase of other interventional research studies (excluding observational/non-interventional studies) before study follow-up visit Not able to use or have access to a modern phone device/web-based technology Any other clinical reason which may preclude entry in the opinion of the investigator Intervention and comparator: Interventions being evaluated are: Daily hydroxychloroquine or Weekly hydroxychloroquine or Placebo The maximum treatment period is approximately 13 weeks per participant. Hydroxychloroquine-identical matched placebo tablets will ensure that all participants are taking the same number and dosing regimen of tablets across the three trial arms. There is no variation in the dose of hydroxychloroquine by weight. The dosing regimen for the three arms of the study (A, B, C) are described in further detail below. Arm A: Active Hydroxychloroquine (– daily dosing and placebo-matched hydroxychloroquine - weekly dosing). Form: Tablets Route: Oral. Dose and Frequency: Active hydroxychloroquine:Days 1-2: Loading phase - 400mg (2 x 200mg tablets) taken twice a day for 2 days Days 3 onwards: Maintenance Phase - 200mg (1 x 200mg tablet) taken once daily, every day for 90 days (~3 months) Matched Placebo hydroxychloroquine: Days 3 onwards: Maintenance Phase - 2 tablets taken once a week on the same day each week (every 7th day) for 90 days (~3 months) Arm B: Active Hydroxychloroquine (- weekly dosing and placebo matched hydroxychloroquine – daily dosing.) Form: Tablets Route: Oral. Dose and Frequency: Active hydroxychloroquine: Days 1-2: Loading Phase - 400mg (2 x 200mg tablets) taken twice daily for 2 days Days 3 onwards: Maintenance Phase - 400mg (2 x 200mg tablets) taken once a week on the same day each week (every 7th day) for 90 days (~3 months) Matched Placebo hydroxychloroquine: Days 3 onwards: Maintenance Phase - 1 tablet taken once daily for 90 days (~3 months) Arm C: Matched placebo Hydroxychloroquine (- daily dosing and matched placebo hydroxychloroquine - weekly dosing.) Form: Table. Route: Oral. Frequency: Matched placebo hydroxychloroquine - daily dosing: Days 1-2: Loading Phase - 2 tablets taken twice daily for 2 days Days 3 onwards: Maintenance Phase - 1 tablet taken once daily for 90 days (~3 months) Matched placebo hydroxychloroquine – weekly dosing: Days 3 onwards: Maintenance Phase - 2 tablets taken once a week on the same day each week (every 7th day) for 90 days (~3 months) A schematic of the dosing schedule can be found in the full study protocol (Additional File 1). Main outcomes: Time to diagnosis of positive COVID-19 disease (defined by record of date of symptoms onset and confirmed by laboratory test) Randomisation: Participants will be randomised to either hydroxychloroquine dosed daily with weekly placebo, HCQ dosed weekly with daily placebo, or placebo dosed daily and weekly. Randomisation will be in a 3:3:2 ratio [hydroxychloroquine-(daily), hydroxychloroquine-(weekly), placebo], using stratified block randomisation. Random block sizes will be used, and stratification will be by study site. Blinding (masking): Participants and trial investigators consenting participants, delivering trial assessments and procedures will be blinded to intervention. Numbers to be randomised (sample size): A sufficient number of participants will be enrolled so that approximately 1000 participants in total will have data suitable for the primary statistical analysis. It is anticipated that approximately 1,200 participants will need to be enrolled in total, to allow for a 20% dropout over the period of the trial. This would result in approximately 450:450:300 participants randomised to hydroxychloroquine daily, hydroxychloroquine weekly+daily matched placebo or matched-placebo daily and weekly. Trial Status: V 1.0, 7th April 2020 EU Clinical Trials Register EudraCT Number: 2020-001331-26 Date of registration: 14th April 2020 Trial registered before first participant enrolment. Trial site is Cambridge University Hospitals NHS Foundation Trust. Recruitment started on 11th May 2020. It is anticipated that the trial will run for 12 months. The recruitment end date cannot yet be accurately predicted. Full protocol: The full protocol 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)

Correction to: ChemoPROphyLaxIs with hydroxychloroquine For covId-19 infeCtious disease (PROLIFIC) to prevent covid-19 infection in frontline healthcare workers: A structured summary of a study protocol for a randomised controlled trial.

An amendment to this paper has been published and can be accessed via the original article

Investigating the Lowest Threshold of Vascular Benefits from LDL Cholesterol Lowering with a PCSK9 mAb Inhibitor (Alirocumab) in Patients with Stable Cardiovascular Disease (INTENSITY-HIGH): protocol and study rationale for a randomised, open label, parallel group, mechanistic study

Introduction Elevated low-density lipoprotein cholesterol (LDL-C) is a strong independent risk predictor of cardiovascular (CV) events, while interventions to reduce it remain the only evidence-based approach to reduce CV morbidity and mortality. Secondary prevention statin trials in combination with ezetimibe and/or proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors showed that there is no ‘J shaped curve’ in LDL-C levels with regard to CV outcomes. The lowest threshold beyond which reduction of LDL-C confers no further CV benefits has not been identified.The INTENSITY-HIGH study seeks to explore physiological mechanisms mediating CV benefits of LDL-C lowering by PCSK9 inhibition in patients with established cardiovascular disease (CVD). The study examines the changes in measures of endothelial function and vascular inflammation imaging following intervention with PCSK9 and against standard of care.Methods and analysis This is a single-centre, randomised, open label, parallel group, mechanistic physiological study. It will include approximately 60 subjects with established CVD, with LDL-C of &lt;4.1 mmol/L on high-intensity statins. All eligible participants will undergo 18-fluorodeoxyglucose positron emission tomography/CT (FDG-PET/CT) scanning of the aorta and carotid arteries, as well as baseline endothelial function assessment. Subsequently, they will be randomised on a 1:1 basis to either alirocumab 150 mg or ezetimibe 10 mg/day. Repeat FDG-PET/CT scan and vascular assessments will be undertaken after 8 weeks of treatment. Any changes in these parameters will be correlated with changes in lipid levels and systemic inflammation biomarkers.Ethics and dissemination The study received a favourable opinion from the Wales Research Ethics Committee 4, was registered on clinicaltrials.gov and conformed to International Conference for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Good Clinical Practice. The results of this study will be reported through peer-reviewed journals and conference presentations.Trial registration number NCT03355027