The inhibition of several components of terminal complement pathway results in C3 binding to PNH red blood cells.

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.

Delayed presentation of children to the emergency department during the first wave of COVID-19 pandemic in Italy: Area-based cohort study.

AIM: To ascertain a delay in attendances to the emergency department (ED) during 2020 lockdown. METHODS: Area-based cohort study on paediatric (0-15 years) attendances resulting in hospital admission in Tuscany, Italy, in February-May 2020, and the corresponding periods in 2018-19. We analysed times from symptom onset to arrival, the odds of arriving late (>90th centile of time) and paediatricians' judgements of a late presentation by logistic models. RESULTS: As expected, ED attendance fell sharply (-62%) in 2020 vs 2018-19. As for cases studied (455 in 2020 and 1161 in 2018-19), we documented a delay in arrival to the ED in 2020 versus 2018-19 for several groups of diagnoses, namely gastroenteritis, sepsis, wounds, burns and infections overall. Time to presentation over 90th centile was also higher in 2020 (odds ratio, OR: 1.44; 95% confidence interval: 1.00, 2.06), as were paediatricians' judgements of a late arrival (18.9% of cases in 2020 vs. 13.4% in 2018-19; OR: 1.58; 1.14, 2.19) CONCLUSION: In a population-based cohort, delayed attendances to ED ascertained both subjectively and objectively convey the message to families and to paediatricians to seek hospital care in case of severe or unremitting symptoms and not to wait longer than they normally would.

Peptide inhibitors of C3 activation as a novel strategy of complement inhibition for the treatment of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis due to the lack of CD55 and CD59 on affected erythrocytes. The anti-C5 antibody eculizumab has proven clinically effective, but uncontrolled C3 activation due to CD55 absence may result in opsonization of erythrocytes, possibly leading to clinically meaningful extravascular hemolysis. We investigated the effect of the peptidic C3 inhibitor, compstatin Cp40, and its long-acting form (polyethylene glycol [PEG]-Cp40) on hemolysis and opsonization of PNH erythrocytes in an established in vitro system. Both compounds demonstrated dose-dependent inhibition of hemolysis with IC50 ∼4 µM and full inhibition at 6 µM. Protective levels of either Cp40 or PEG-Cp40 also efficiently prevented deposition of C3 fragments on PNH erythrocytes. We further explored the potential of both inhibitors for systemic administration and performed pharmacokinetic evaluation in nonhuman primates. A single intravenous injection of PEG-Cp40 resulted in a prolonged elimination half-life of >5 days but may potentially affect the plasma levels of C3. Despite faster elimination kinetics, saturating inhibitor concentration could be reached with unmodified Cp40 through repetitive subcutaneous administration. In conclusion, peptide inhibitors of C3 activation effectively prevent hemolysis and C3 opsonization of PNH erythrocytes, and are excellent, and potentially cost-effective, candidates for further clinical investigation.

Glycosylphosphatidylinositol-specific, CD1d-restricted T cells in paroxysmal nocturnal hemoglobinuria.

The mechanism of bone marrow failure (BMF) in paroxysmal nocturnal hemoglobinuria (PNH) is not yet known. Because in PNH the biosynthesis of the glycolipid molecule glycosylphosphatidylinositol (GPI) is disrupted in hematopoietic stem and progenitor cells by a somatic mutation in the PIG-A gene, BMF might result from an autoimmune attack, whereby T cells target GPI in normal cells, whereas PIG-A mutant GPI-negative cells are spared. In a deliberate test of this hypothesis, we have demonstrated in PNH patients the presence of CD8(+) T cells reactive against antigen-presenting cells (APCs) loaded with GPI. These T cells were significantly more abundant in PNH patients than in healthy controls; their reactivity depended on CD1d expression and they increased upon coculture with CD1d-expressing, GPI-positive APCs. In GPI-specific T cells captured by CD1d dimer technology, we identified, through global T-cell receptor α (TCRα) analysis, an invariant TCRVα21 sequence, which was then found at frequencies higher than background in the TCR repertoire of 6 of 11 PNH patients. Thus, a novel, autoreactive, CD1d-restricted, GPI-specific T-cell population, enriched in an invariant TCRα chain, is expanded in PNH patients and may be responsible for BMF in PNH.

The complement receptor 2/factor H fusion protein TT30 protects paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and C3 fragment.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membrane-targeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membrane-targeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients.

Polymorphism of the complement receptor 1 gene correlates with the hematologic response to eculizumab in patients with paroxysmal nocturnal hemoglobinuria.

Complement blockade by eculizumab is clinically effective in hemolytic paroxysmal nocturnal hemoglobinuria. However, the response is variable and some patients remain dependent on red blood cell transfusions. In 72 patients with hemolytic paroxysmal nocturnal hemoglobinuria on eculizumab we tested the hypothesis that response may depend on genetic polymorphisms of complement-related genes. We found no correlation between the complement component C3 genotypes and the need for blood transfusions. On the other hand, we found a significant correlation with the HindIII polymorphism of a complement regulatory gene, the complement receptor 1 (CR1) gene. At this locus two co-dominant alleles are known, of which H (common) is associated with high expression, whereas L (rare) is associated with low expression of CR1 on red blood cells. Patients who still needed blood transfusion on eculizumab accounted for 18% of the H/H homozygotes, 33% of the H/L heterozygotes and 68% of the L/L homozygotes (P=0.016). Thus, patients with paroxysmal nocturnal hemoglobinuria who have the L/L genotype are seven times more likely to be sub-optimal responders to eculizumab. Both in vitro and in vivo we found that the CR1 HindIII genotype correlates with the abundance of paroxysmal nocturnal hemoglobinuria red cells that have bound C3, and with the kinetics of C3 binding. These results are consistent with the notion that by affecting C3 binding the CR1 genotype influences the response to eculizumab treatment, and this emerges as a novel example of pharmacogenetics.

Value of Including the Children's Experience for Improving Their Rights During Hospitalization: Protocol for the VoiCEs Project.

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.

Delayed Start of Routine Vaccination in Preterm and Small-for-Gestational-Age Infants: An Area-Based Cohort Study from the Tuscany Region, Italy.

Preterm and small-for-gestational-age (SGA) infants are more susceptible to vaccine-preventable diseases. To evaluate routine vaccination timeliness in these high-risk groups, a full birth cohort of infants (n = 41,502) born in 2017 and 2018 in Tuscany was retrospectively followed up until 24 months of age. Infants were classified by gestational age (GA) and SGA status. The vaccinations included: hexavalent (HEXA), measles-mumps-rubella, varicella, pneumococcal conjugate (PCV), and meningococcal C conjugate. Time-to-event (Kaplan-Meier) analyses were conducted to evaluate the timing of vaccination according to GA; logistic models were performed to evaluate the associations between GA and SGA with vaccination timeliness. Time-to-event analyses show that the rate of delayed vaccine receipt increased with decreasing GA for all the vaccinations, with a less marked gradient in later vaccine doses. Compared to full-term infants, very preterm infants significantly showed an increased odds ratio (OR) for delayed vaccination in all the vaccinations, while moderate/late preterm infants only showed an increased OR for HEXA-1, HEXA-3, PCV-1, and PCV-3. SGA infants had a significantly higher risk of delayed vaccination only for HEXA-1 and PCV-1 compared to non-SGA infants. In conclusion, vaccinations among preterm and SGA infants showed considerable delay. Tailored public health programs to improve vaccination timeliness are required in these high-risk groups.

Immune dysregulation and dyserythropoiesis in the myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) are clonal disorders characterised by ineffective haematopoiesis with high risk of leukaemia progression. The relevance of immune-dysregulation for emergence, dominance and progression of dysplastic clones has been suggested, but valuable criteria to obtain insight into these connections are lacking. This study showed significant increase of CD8 lymphocytes and mature B cells in the bone marrow (BM) compared to peripheral blood (PB) of low risk MDS patients. Different BM levels of Regulatory T cells (Treg) identified two sub-groups in these patients; only the sub-group with lower Treg percentage showed BM recruitment of CD8 lymphocytes. Different levels of CD54 on BM CD8 cells revealed two sub-groups of Intermediate-1 (Int-1) patients. The sub-group with higher CD54 expression on BM CD8 showed high levels of this molecule also on CD4 cells. BM recruitment of CD8 lymphocytes in the low risk group and/or the presence of high CD54 expression on BM CD8 in Int-1 patients were associated with more pronounced dyserythropoiesis and erythropoietin treatment. Our data shed light on the involvement of immune-mediated mechanisms in Low and Int-1 risk MDS patients and suggest that BM versus PB levels of immune effectors could represent useful criteria for a more homogeneous grouping of MDS patients.

Easy genotyping of complement C3 'slow' and 'fast' allotypes by tetra-primer amplification refractory mutation system PCR.

Complement C3 'slow' and 'fast' allotypes are associated with immune-mediated disorders and may affect the outcome of renal transplantation. We report a tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) that provides a rapid, reproducible and cost-effective method to genotype both complement C3 'slow' and 'fast' alleles by a single tube reaction.

ETV4 promotes late development of prostatic intraepithelial neoplasia and cell proliferation through direct and p53-mediated downregulation of p21.

BACKGROUND: ETV4 is one of the ETS proteins overexpressed in prostate cancer (PC) as a result of recurrent chromosomal translocations. In human prostate cell lines, ETV4 promotes migration, invasion, and proliferation; however, its role in PC has been unclear. In this study, we have explored the effects of ETV4 expression in the prostate in a novel transgenic mouse model. METHODS: We have created a mouse model with prostate-specific expression of ETV4 (ETV4 mice). By histochemical and molecular analysis, we have investigated in these engineered mice the expression of p21, p27, and p53. The implications of our in vivo findings have been further investigated in human cells lines by chromatin-immunoprecipitation (ChIP) and luciferase assays. RESULTS: ETV4 mice, from two independent transgenic lines, have increased cell proliferation in their prostate and two-thirds of them, by the age of 10 months, developed mouse prostatic intraepithelial neoplasia (mPIN). In these mice, cdkn1a and its p21 protein product were reduced compared to controls; p27 protein was also reduced. By ChIP assay in human prostate cell lines, we show that ETV4 binds to a specific site (-704/-696 bp upstream of the transcription start) in the CDKN1A promoter that was proven, by luciferase assay, to be functionally competent. ETV4 further controls CDKN1A expression by downregulating p53 protein: this reduction of p53 was confirmed in vivo in ETV4 mice. CONCLUSIONS: ETV4 overexpression results in the development of mPIN but not in progression to cancer. ETV4 increases prostate cell proliferation through multiple mechanisms, including downregulation of CDKN1A and its p21 protein product: this in turn is mediated through direct binding of ETV4 to the CDKN1A promoter and through the ETV4-mediated decrease of p53. This multi-faceted role of ETV4 in prostate cancer makes it a potential target for novel therapeutic approaches that could be explored in this ETV4 transgenic model.

Anti-complement Treatment for Paroxysmal Nocturnal Hemoglobinuria: Time for Proximal Complement Inhibition? A Position Paper From the SAAWP of the EBMT.

The treatment of paroxysmal nocturnal hemoglobinuria has been revolutionized by the introduction of the anti-C5 agent eculizumab; however, eculizumab is not the cure for Paroxysmal nocturnal hemoglobinuria (PNH), and room for improvement remains. Indeed, the hematological benefit during eculizumab treatment for PNH is very heterogeneous among patients, and different response categories can be identified. Complete normalization of hemoglobin (complete and major hematological response), is seen in no more than one third of patients, while the remaining continue to experience some degree of anemia (good and partial hematological responses), in some cases requiring regular red blood cell transfusions (minor hematological response). Different factors contribute to residual anemia during eculizumab treatment: underlying bone marrow dysfunction, residual intravascular hemolysis and the emergence of C3-mediated extravascular hemolysis. These two latter pathogenic mechanisms are the target of novel strategies of anti-complement treatments, which can be split into terminal and proximal complement inhibitors. Many novel terminal complement inhibitors are now in clinical development: they all target C5 (as eculizumab), potentially paralleling the efficacy and safety profile of eculizumab. Possible advantages over eculizumab are long-lasting activity and subcutaneous self-administration. However, novel anti-C5 agents do not improve hematological response to eculizumab, even if some seem associated with a lower risk of breakthrough hemolysis caused by pharmacokinetic reasons (it remains unclear whether more effective inhibition of C5 is possible and clinically beneficial). Indeed, proximal inhibitors are designed to interfere with early phases of complement activation, eventually preventing C3-mediated extravascular hemolysis in addition to intravascular hemolysis. At the moment there are three strategies of proximal complement inhibition: anti-C3 agents, anti-factor D agents and anti-factor B agents. These agents are available either subcutaneously or orally, and have been investigated in monotherapy or in association with eculizumab in PNH patients. Preliminary data clearly demonstrate that proximal complement inhibition is pharmacologically feasible and apparently safe, and may drastically improve the hematological response to complement inhibition in PNH. Indeed, we envision a new scenario of therapeutic complement inhibition, where proximal inhibitors (either anti-C3, anti-FD or anti-FB) may prove effective for the treatment of PNH, either in monotherapy or in combination with anti-C5 agents, eventually leading to drastic improvement of hematological response.

Paroxysmal nocturnal hemoglobinuria: significant association with specific HLA-A, -B, -C, and -DR alleles in an Italian population.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the expansion of a PIG-A mutated hematopoietic stem cell. An immune-mediated origin has been suggested for this disease. Because HLA genes represent a susceptibility factor for autoimmunity, we investigated HLA genotype in 42 Italian PNH patients compared with 301 control subjects of the same ethnic origin. A significantly increased frequency of the HLA class I alleles A*0201 (p < 0.05), B*1402 (p < 0.001), and Cw*0802 (p < 0.005), and of the HLA class II DRB1*1501 (p < 0.01) with the linked DQB1*0602 (p

C3-mediated extravascular hemolysis in PNH on eculizumab: Mechanism and clinical implications.

The introduction of eculizumab, a human monoclonal antibody against the C5 component of complement, has changed radically the management of paroxysmal nocturnal hemoglobinuria (PNH). The blockade of the terminal complement pathway by eculizumab abrogates intravascular hemolysis, reduces the transfusion requirement and the risk of thrombosis in most of hemolytic PNH patients. However, in almost all PNH patients on eculizumab arises a fraction of PNH red cells that bind fragments of C3 and become a potential target of phagocytosis by macrophages. Eventually, this phagocytosis results in a variable degree of extravascular hemolysis that may reduce clinical benefits of eculizumab and, in fact, about one-fourth of patients remain transfusion-dependent. The treatment of the few PNH patients in which this de novo extravascular hemolysis become clinically relevant is still unsatisfactory. Nevertheless, the investigations of the mechanisms responsible of the extravascular hemolysis on eculizumab have resulted in the development of novel strategies for complement blockade that could overcome this condition.

Eculizumab treatment: stochastic occurrence of C3 binding to individual PNH erythrocytes.

BACKGROUND: C5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH). However, C3-bound PNH red blood cells (RBCs), arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+) and without (C3-) C3 binding. METHODS: To investigate this paradox, the phenomenon has been modeled in vitro by incubating RBCs from eculizumab untreated PNH patients with compatible sera containing eculizumab, and by assessing the C3 binding after activation of complement alternative pathway. RESULTS: When RBCs from untreated patients were exposed in vitro to activated complement in the context of C5-blockade, there was the prompt appearance of a distinct C3+ PNH RBC population whose size increased with time and also with the rate of complement activation. Eventually, all PNH RBCs become C3+ to the same extent, without differences between old and young (reticulocytes) PNH RBCs. CONCLUSIONS: This study indicates that the distinct (C3+ and C3-) PNH RBC populations are not intrinsically different; rather, they result from a stochastic all-or-nothing phenomenon linked to the time-dependent cumulative probability of each individual PNH red cell to be exposed to levels of complement activation able to trigger C3 binding. These findings may envision novel approaches to reduce C3 opsonization and the subsequent extravascular hemolysis in PNH patients on eculizumab.

T cells from paroxysmal nocturnal haemoglobinuria (PNH) patients show an altered CD40-dependent pathway.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoiesis disorder characterized by the expansion of a stem cell bearing a somatic mutation in the phosphatidylinositol glycan-A (PIG-A) gene, which is involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor. A number of data suggest the inability of the PIG-A mutation to account alone for the clonal dominance of the GPI-defective clone and for the development of PNH. In this context, additional immune-mediated mechanisms have been hypothesized. We focused on the analysis of T lymphocytes in three PNH patients bearing a mixed GPI(+) and GPI(-) T cell population and showing a marked cytopenia. To analyze the biological mechanisms underlying the control of T cell homeostasis in PNH, we addressed the study of CD40-dependent pathways, suggested to be of crucial relevance for the control of autoreactive T cell clones. Our data revealed significant, functional alterations in GPI(+) and GPI(-) T cell compartments. In the GPI(-) T cells, severe defects in T cell receptor-dependent proliferation, interferon-gamma production, CD25, CD54, and human leukocyte antigen-DR surface expression were observed. By contrast, GPI(+) T lymphocytes showed a significant increase of all these parameters, and the analysis of CD40-dependent pathways revealed a functional persistence of CD154 expression on the CD48(+)CD4(+) lymphocytes. The alterations of the GPI(+) T cell subset could be involved in the biological mechanisms underlying PNH pathogenesis.

GPI-defective monocytes from paroxysmal nocturnal hemoglobinuria patients show impaired in vitro dendritic cell differentiation.

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 alpha and IL-12 production. Finally, GPI-defective DCs showed a severe impairment in delivering accessory signals for T cell receptor-dependent T cell proliferation.