Michela Sica

C3 binding on PNH red blood cells after in vitro complement activation in normal and terminal complement depleted (∆5, ∆6, ∆9) sera: C3 binding, and the subsequent extravascular hemolysis, happens anytime there is a block of a component of the terminal complement pathway.

Background: Users’ feedback is a key asset for organizations that want to improve their services. Studying how organizations are enabling their users to participate in evaluation activities is particularly important, especially when there are vulnerable or disadvantaged people, and the services to be evaluated can be life-changing. This is the case in the coassessment by pediatric patients experiencing hospital stay. The international literature reports a few attempts and several challenges in systematically collecting and using the pediatric patient experience with respect to hospitalization, to undertake quality improvement actions. Objective: This paper describes the research protocol of a European project intended to develop and implement a systematic pediatric patient-reported experience measures (PREMs) observatory that will be shared by 4 European children’s hospitals in Finland, Italy, Latvia, and the Netherlands. Methods: The VoiCEs (Value of including the Children’s Experience for improving their rightS during hospitalization) project uses a participatory action research approach, based on a mixture of qualitative and quantitative methods. It consists of 6 different phases, including a literature review, an analysis of the previous experiences of pediatric PREMs reported by project partners, a Delphi process, a cycle of focus groups or in-depth interviews with children and their caregivers, a series of workshops with interactive working groups, and a cross-sectional observational survey. The project guarantees the direct participation of children and adolescents in the development and implementation phases of the project. Results: The expected results are (1) a deeper knowledge of published methodologies and tools on collecting and reporting pediatric patients’ voice; (2) lessons learnt from the analysis of previous experiences of pediatric PREMs; a consensus reached through a participatory process (3) among experts, (4) pediatric patients and caregivers about a standard set of measures for the evaluation of hospitalization by patients; (5) the implementation of a European observatory on pediatric PREMs; and (6) the collection and comparative reporting of the pediatric patients’ voice. In addition, the project is aimed at studying and proposing innovative methodologies and tools for capturing the pediatric patients’ feedback directly, avoiding the intermediation of parents/guardians. Conclusions: Over the last decade, the collection and use of PREMs have gained importance as a research field. Children and adolescents’ perspectives have also been increasingly taken into consideration. However, to date, there are limited experiences regarding the continuous and systematic collection and use of pediatric PREMs data for implementing timely improvement actions. In this perspective, the VoiCEs project provides room for innovation, by contributing to the creation of an international, continuous, and systematic pediatric PREMs observatory that can be joined by other children’s hospitals or hospitals with pediatric patients, and foresees the return of usable and actionable data in benchmarking. International Registered Report Identifier (IRRID): DERR1-10.2196/42804

BACKGROUND Users’ feedback is a key asset for organizations that want to improve their services. Studying how organizations are enabling their users to participate in evaluation activities is particularly important, especially when there are vulnerable or disadvantaged people, and the services to be evaluated can be life-changing. This is the case in the coassessment by pediatric patients experiencing hospital stay. The international literature reports a few attempts and several challenges in systematically collecting and using the pediatric patient experience with respect to hospitalization, to undertake quality improvement actions. OBJECTIVE This paper describes the research protocol of a European project intended to develop and implement a systematic pediatric patient-reported experience measures (PREMs) observatory that will be shared by 4 European children’s hospitals in Finland, Italy, Latvia, and the Netherlands. METHODS The VoiCEs (Value of including the Children’s Experience for improving their rightS during hospitalization) project uses a participatory action research approach, based on a mixture of qualitative and quantitative methods. It consists of 6 different phases, including a literature review, an analysis of the previous experiences of pediatric PREMs reported by project partners, a Delphi process, a cycle of focus groups or in-depth interviews with children and their caregivers, a series of workshops with interactive working groups, and a cross-sectional observational survey. The project guarantees the direct participation of children and adolescents in the development and implementation phases of the project. RESULTS The expected results are (1) a deeper knowledge of published methodologies and tools on collecting and reporting pediatric patients’ voice; (2) lessons learnt from the analysis of previous experiences of pediatric PREMs; a consensus reached through a participatory process (3) among experts, (4) pediatric patients and caregivers about a standard set of measures for the evaluation of hospitalization by patients; (5) the implementation of a European observatory on pediatric PREMs; and (6) the collection and comparative reporting of the pediatric patients’ voice. In addition, the project is aimed at studying and proposing innovative methodologies and tools for capturing the pediatric patients’ feedback directly, avoiding the intermediation of parents/guardians. CONCLUSIONS Over the last decade, the collection and use of PREMs have gained importance as a research field. Children and adolescents’ perspectives have also been increasingly taken into consideration. However, to date, there are limited experiences regarding the continuous and systematic collection and use of pediatric PREMs data for implementing timely improvement actions. In this perspective, the VoiCEs project provides room for innovation, by contributing to the creation of an international, continuous, and systematic pediatric PREMs observatory that can be joined by other children’s hospitals or hospitals with pediatric patients, and foresees the return of usable and actionable data in benchmarking. INTERNATIONAL REGISTERED REPORT DERR1-10.2196/42804

Abstract 370 Paroxysmal nocturnal hemoglobinuria (PNH) is a complex hematological disorder characterized by the expansion of blood cells deficient in the surface complement inhibitors CD55 and CD59; affected erythrocytes suffer from uncontrolled complement activation on their surface, and subsequent membrane attack complex (MAC)-mediated intravascular hemolysis. The anti-C5 antibody eculizumab has proven effective in controlling intravascular hemolysis in vivo, leading to remarkable clinical benefit in almost all PNH patients. Yet, we have demonstrated that persistent C3 activation occurring during eculizumab treatment may lead to progressive C3 deposition on affected erythrocyte and subsequent C3-mediated extravascular hemolysis, possibly limiting the hematological benefit of anti-C5 treatment (Risitano et al, Blood 2009). Thus, upstream inhibition of the complement cascade seems an appropriate strategy to improve the results of current anti-complement treatment; indeed, we have recently documented that the CD21/factor H (FH) fusion protein TT30 efficiently prevents both hemolysis and C3 deposition of PNH erythrocytes (Risitano et al, Blood 2012). Here we used the same in vitro model to evaluate two novel complement inhibitors that both act at the level of C3 convertases. Cp30 is an analog of the peptidic inhibitor compstatin, which is a 13-residue disulphide-bridged peptide that selectively binds to C3 and its activate fragment C3b. Compstatin and its analogues thereby prevent the initiation, amplification and terminal damage of the complement cascade via all its major pathways (classical, alternative, and mannose/lectin). Cp30 is one of the analogues developed to increase potency and stability of compstatin. Mini-FH, on the other hand, is an engineered 43kDa protein that combines the regulatory and surface-recognition activities of FH while showing increased affinity for the opsonins C3b, iC3b and C3d. Indeed, mini-FH retained both convertase decay acceleration and cofactor activities typical of endogenous human FH, resulting in a potent and selective inhibition of activation and amplification of the complement alternative pathway, without affecting the classical and the mannose/lectin pathway. Erythrocytes from PNH patients were washed and incubated in ABO-matched sera and exposed to pH-lowering to activate the alternative pathway, both in absence and presence of Cp30, mini-FH, and appropriate controls. Assessment of hemolysis and of C3 activation and deposition on PNH erythrocytes was performed by flow cytometry analyses of erythrocytes using anti-C3 and anti-CD59 antibodies, as previously described (Risitano et al Blood 2012). In absence of inhibitors, >90% of PNH erythrocytes lysed within 24 hours of incubation. Cp30 demonstrated a dose-dependent inhibition of hemolysis, with an IC₅₀ of 4 μM and full inhibition at 8 μM. Cp30 also prevented deposition of any C3 fragment on the surface of surviving PNH erythrocyte. Similarly, mini-FH also showed dose-dependent inhibition of hemolysis, with an IC₅₀ of 0.05 μM and full inhibition at 0.1 μM. Notably, both full-length fH and fH SCR1-4 were much less efficient in preventing hemolysis and C3 deposition (IC₅₀ ∼ 0.5 μM; full inhibition >1 μM), supporting the higher potency of the engineered protein mini-FH. As expected, mini-FH also prevented surface deposition of C3 fragments on PNH erythrocytes. In conclusion, we confirm that inhibition of early phases of complement activation efficiently prevents hemolysis of PNH erythrocytes and their opsonization with C3 fragments in vitro. This effect may be obtained using either broad or pathway-specific inhibitors of C3 convertase, namely Cp30 and mini-FH, respectively. Thus, both strategies promise to prevent in vivo both MAC-mediated intravascular and C3-mediated extravascular hemolysis; however, according to their effect on specific complement pathways, they likely entail distinct patterns of potential risks. Our study provides the rationale for future translational plans to investigate the risk-to-benefit of these novel complement modulators in PNH.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal, acquired hematopoietic disorder characterized by a phosphatidylinositol (PI) glycan-A gene mutation, which impairs the synthesis of the glycosyl-PI (GPI) anchor, thus causing the absence of all GPI-linked proteins on the membrane of the clonal-defective cells. The presence of a consistent GPI-defective monocyte compartment is a common feature in PNH patients. To investigate the functional behavior of this population, we analyzed its in vitro differentiation ability toward functional dendritic cells (DCs). Our data indicate that GPI-defective monocytes from PNH patients are unable to undergo full DC differentiation in vitro after granulocyte macrophage-colony stimulating factor and recombinant interleukin (IL)-4 treatment. In this context, the GPI-defective DC population shows mannose receptor expression, high levels of the CD86 molecule, and impaired CD1a up-regulation. The analysis of lipopolysaccharide and CD40-dependent, functional pathways in these DCs revealed a strong decrease in tumor necrosis factor α and IL-12 production. Finally, GPI-defective DCs showed a severe impairment in delivering accessory signals for T cell receptor-dependent T cell proliferation.