Analysis of leukocyte membrane protein interactions using protein microarrays

BACKGROUND: Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R. RESULTS: Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1–40 μg ml(-1 )corresponding to a limit of sensitivity of 0.01–0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads. CONCLUSION: We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays. We also detected CD200 binding to its receptor, a low affinity interaction, using beads presenting multivalent ligands. Our results demonstrate the quantitative aspects of protein arrays and their potential use in detecting simultaneously multiple protein-protein interactions and in particular the weak interactions found between leukocyte membrane proteins

Uncertainty management in regulatory and health technology assessment decision-making on drugs: guidance of the HTAi-DIA Working Group

ObjectivesUncertainty is a fundamental component of decision making regarding access to and pricing and reimbursement of drugs. The context-specific interpretation and mitigation of uncertainty remain major challenges for decision makers. Following the 2021 HTAi Global Policy Forum, a cross-sectoral, interdisciplinary HTAi-DIA Working Group (WG) was initiated to develop guidance to support stakeholder deliberation on the systematic identification and mitigation of uncertainties in the regulatory-HTA interface. MethodsSix online discussions among WG members (Dec 2021-Sep 2022) who examined the output of a scoping review, two literature-based case studies and a survey; application of the initial guidance to a real-world case study; and two international conference panel discussions. ResultsThe WG identified key concepts, clustered into twelve building blocks that were collectively perceived to define uncertainty: "unavailable," "inaccurate," "conflicting," "not understandable," "random variation," "information," "prediction," "impact," "risk," "relevance," "context," and "judgment." These were converted into a checklist to explain and define whether any issue constitutes a decision-relevant uncertainty. A taxonomy of domains in which uncertainty may exist within the regulatory-HTA interface was developed to facilitate categorization. The real-world case study was used to demonstrate how the guidance may facilitate deliberation between stakeholders and where additional guidance development may be needed. ConclusionsThe systematic approach taken for the identification of uncertainties in this guidance has the potential to facilitate understanding of uncertainty and its management across different stakeholders involved in drug development and evaluation. This can improve consistency and transparency throughout decision processes. To further support uncertainty management, linkage to suitable mitigation strategies is necessary

Απλαστική Αναιμία

Απλαστική αναιμία ή μυελική απλασία αναφέρονται στη βιβλιογραφία οι ποσοτικές ανεπάρκειες του μυελού των οστών που εκδηλώνονται με εμφάνιση πανκυτταροπενίας. Ο ορισμός αυτός δεν περιλαμβάνει την μεμονωμένη εξαφάνιση μιας μυελικής σειράς, που η πορεία της διαφέρει τόσο όσον αναφορά την παθοφυσιολογία, την θεραπευτική αντιμετώπιση και την πορεία, όσο και η εξέλιξη σε σχέση με την απλαστική αναιμία. Η απλαστική αναιμία θα πρέπει να διακρίνεται από τις πανκυτταροπενίες που οφείλονται σε έντονη δυσμυελοποίηση με παρουσία υπερπλαστικού μυελού που έχουν εντελώς διαφορετική εξέλιξη και αντιμετώπιση. Σήμερα σαν απλάστική αναιμία ή μυελική απλασία ονομάζεται ένα σύνδρομο που χαρακτηρίζεται από πανκυτταροπενία του περιφερικού αίματος και από μείωση ή εξαφάνιση των κυτταρικών στοιχείων στον μυελό που οφείλεται σε μία ποσοτική ή ποιοτική διαταραχή των αρχέγονων αιμοποιητικών κυττάρων, χωρίς την παρουσία στον μυελό κακοήθους νόσου ή ίνωσης. ΣΚΟΠΟΙ- ΣΤΟΧΟΙ Σκοπός και στόχος της εργασίας είναι η έρευνα, η κατανόηση και η ανάλυση της συχνότητας της νόσου, των αιτιολογικών παραγόντων που σχετίζονται με αυτή, καθώς και τους πιθανούς παθογεννετικούς μηχανισμούς που εμπλέκονται με την απλαστική αναιμία. Επίσης θα μελετήσουμε και θα παρουσιάσουμε την διάγνωση, την πορεία και πρόγνωση της απλαστικής αναιμίας και τέλος την θεραπεία και την εξέλιξη της νόσου. ΥΛΙΚΟ Το υλικό που θα χρησιμοποιηθεί για την εκπόνηση της διπλωματικής εργασίας, θα αναζητηθεί μέσω βιβλιογραφικής ανασκόπησης. ( ελληνικής και ξένης βιβλιογραφίας). ΑΠΟΤΕΛΕΣΜΑΤΑ Πιο αναλυτικά θα εξετάσουμε: Τη συχνότητα. Τα αίτια ( Τοξικά –Λοιμώδη-Κληρονομικά). Τους μηχανισμούς (Διαταραχές αρχέγονων κυττάρων- Διαταραχές στρώματος (μικροπεριβάλλοντος)- Παθολογικά ρυθμιστικά κύτταρα). Τη διάγνωση και τη διαφορική διάγνωση. Την πορεία και πρόγνωση της απλαστικής αναιμίας. Την εφαρμογή θεραπείας (Γενικά μέτρα-υποστηρικτική θεραπεία,- Ανδρογόνα- Ανοσοκατασταλτικά- Μεταμόσχευση μυελού) και τέλος την εξέλιξη της πορείας της νόσου.Aplastic anemia is a syndrome of bone marrow failure characterized by peripheral pancytopenia and marrow hypoplasia (see the image below). Although the anemia is often normocytic, mild macrocytosis can also be observed in association with stress erythropoiesis and elevated fetal hemoglobin levels. The clinical presentation of patients with aplastic anemia includes symptoms related to the decrease in bone marrow production of hematopoietic cells. The onset is insidious, and the initial symptom is frequently related to anemia or bleeding, although fever or infections may be noted at presentation. Severe or very severe aplastic anemia is a hematologic emergency, and care should be instituted promptly. Clinicians must stress the need for patient compliance with therapy. The specific medications administered depend on the choice of therapy and whether it is supportive care only, immunosuppressive therapy, or hematopoietic cell transplantatio

Diversity in Health Informatics: Mentoring and Leadership

International audienceDiversity, inclusion and interdisciplinary collaboration are drivers for healthcare innovation and adoption of new, technology-mediated services. The importance of diversity has been highlighted by the United Nations' Sustainable Development Goal 5 (SDG5) "Achieve gender equality and empower all women and girls", their active engagement and contribution to the adoption of social and digital innovation has been highlighted. Women play an instrumental role in health care and are in position to bring about significant changes to support ongoing digitalization and transformation. At the same time women are underrepresented in Science, Technology, Engineering and Mathematics (STEM). To some extent, the same holds for health care informatics. This paper sums up input to strategies for peer mentoring to ensure diversity in health informatics, to target systemic inequalities and build sustainable, intergenerational communities, improve digital health literacy and build capacity in digital health without losing the human touch

Diversity in Health Informatics: Mentoring and Leadership

Diversity, inclusion and interdisciplinary collaboration are drivers for healthcare innovation and adoption of new, technology-mediated services. The importance of diversity has been highlighted by the United Nations’ in SDG5 “Achieve gender equality and empower all women and girls”, to drive adoption of social and digital innovation. Women play an instrumental role in health care and are in position to bring about significant changes to support ongoing digitalization and transformation. At the same time, women are underrepresented in Science, Technology, Engineering and Mathematics (STEM). To some extent, the same holds for health care informatics. This paper sums up input to strategies for peer mentoring to ensure diversity in health informatics, to target systemic inequalities and build sustainable, intergenerational communities, improve digital health literacy and build capacity in digital health without losing the human touch

Outcomes After Drug-Coated Balloon Treatment of Femoropopliteal Lesions in Patients With Critical Limb Ischemia: A Post Hoc Analysis From the IN.PACT Global Study

Purpose: To report a post hoc analysis performed to evaluate 1-year safety and efficacy of the IN.PACT Admiral drug-coated balloon (DCB) for the treatment of femoropopliteal lesions in subjects with critical limb ischemia (CLI) enrolled in the IN.PACT Global study (ClinicalTrials.gov identifier NCT01609296). Materials and Methods: Of 1535 subjects enrolled in the study, 156 participants (mean age 71.8±10.4; 87 men) with CLI (Rutherford categories 4,5) were treated with DCB angioplasty in 194 femoropopliteal lesions. This cohort was compared to the 1246 subjects (mean age 68.2±10.0 years; 864 men) with intermittent claudication (IC) treated for 1573 lesions. The CLI cohort had longer lesions (13.9±10.6 vs 11.9±9.4 cm, p=0.009) and a higher calcification rate (76.8% vs 67.7%, p=0.011). Major adverse events [MAE; composite of all-cause mortality, clinically-driven target lesion revascularization (CD-TLR), major (above-ankle) target limb amputation, and thrombosis at the target lesion site], lesion and vessel revascularization rates, and EuroQol-5D were assessed through 1 year. The Kaplan-Meier method was used to estimate survival, CD-TLR, and amputation events; estimates are presented with the 95% confidence intervals (CI). Results: Estimates of 12-month freedom from major target limb amputation were 98.6% (95% CI 96.7% to 100.0%) in subjects with CLI and 99.9% (95% CI 99.8% to 100.0%) in subjects with IC (p=0.002). Freedom from CD-TLR through 12 months was 86.3% (95% CI 80.6% to 91.9%) in CLI subjects and 93.4% (95% CI 91.9% to 94.8%) in IC subjects (p<0.001). The MAE rate through 12 months was higher in CLI subjects (22.5% vs 10.7%, p<0.001), and CLI patients had poorer overall survival (93.0%, 95% CI 88.9% to 97.2%) than IC subjects (97.0%, 95% CI 96.0% to 97.9%, p=0.011). Health status significantly improved in all domains at 6 and 12 months in both groups. Conclusion: Treatment of femoropopliteal disease with DCB in CLI patients is safe through 12-month follow-up, with a low major amputation rate of 1.4%. The rates of MAE and CD-TLR were higher in CLI subjects and reinterventions were required sooner. Additional research is needed to evaluate long-term outcomes of DCB treatment for femoropopliteal lesions in CLI patients

Multivalent recombinant proteins for probing functions of leucocyte surface proteins such as the CD200 receptor

CD200 (OX2) is a membrane glycoprotein that interacts with a structurally related receptor (CD200R) involved in the regulation of macrophage function. The interaction is of low affinity (K(D) ∼ 1 μm) but can be detected using CD200 displayed in a multivalent form on beads or with dimeric fusion proteins consisting of the extracellular region of CD200 and immunoglobulin Fc regions. We prepared putative pentamers and trimers of mouse CD200 with sequences from cartilage oligomeric matrix protein (COMP) and surfactant protein D (SP-D), respectively. The COMP protein gave high-avidity binding and was a valuable tool for showing the interaction whilst the SP-D protein gave weak binding. In vivo experiments showed that an agonistic CD200R monoclonal antibody caused some amelioration in a model of experimental autoimmune encephalomyelitis but the COMP protein was cleared rapidly and had minimal effect. Pentameric constructs also allowed detection of the rat CD48/CD2 interaction, which is of much lower affinity (K(D) ∼ 70 μm). These reagents may have an advantage over Fc-bearing hybrid molecules for probing cell surface proteins without side-effects due to the Fc regions. The CD200-COMP gave strong signals in protein microarrays, suggesting that such reagents may be valuable in high throughput detection of weak interactions