Search

BackgroundETV4 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.MethodsWe 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.ResultsETV4 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,cdkn1aand 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 theCDKN1Apromoter that was proven, by luciferase assay, to be functionally competent. ETV4 further controlsCDKN1Aexpression by downregulating p53 protein: this reduction of p53 was confirmed in vivo in ETV4 mice.ConclusionsETV4 overexpression results in the development of mPIN but not in progression to cancer. ETV4 increases prostate cell proliferation through multiple mechanisms, including downregulation ofCDKN1Aand its p21 protein product: this in turn is mediated through direct binding of ETV4 to theCDKN1Apromoter 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.

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.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0496-x) contains supplementary material, which is available to authorized users.

Introduction Eculizumab (Ecu) is the standard treatment for paroxysmal nocturnal hemoglobinuria (PNH), as it results in sustained control of intravascular hemolysis and prevention of thrombosis, and significant improvement of long-term survival. However, the hematological benefits remain heterogeneous among patients, and most response analyses are restricted to transfusion independence. In the era of new anti-complement therapies, we found appropriate to investigate in detail the quality of hematological response of Ecu-treated PNH patients. Methods We have recently proposed (Risitano et al, Frontiers in Immunology 2019) a classification of hematological response to anti-complement therapies based on hemoglobin (Hb) levels and laboratory markers of hemolysis. Six distinct categories were determined (Table 1): i. complete response (no transfusion requirement with normal stable Hb and no evidence of hemolysis); ii. major response (no transfusion requirement with normal Hb but evidence of residual intravascular or extravascular hemolysis); iii. good response (no transfusion requirement with persistent chronic mild anemia); iv. partial response (persistent chronic moderate anemia and/or with occasional red blood cell transfusions); v. minor response (3-6 red blood cell transfusions every 6 months); vi. no response (>6 blood cell transfusions every 6 months). Here we have retrospectively exploited this classification to assess hematological response in a large cohort of 93 PNH patients evaluated in six international reference PNH centers (Paris, Naples, London, Florence, São Paulo, and Ribeirão Preto). Results Forty-two patients were male and 51 were female; the median age at diagnosis (treatment starting) was 36 years (range, 12 to 74 years). All the patients have started Ecu treatment for hemolytic PNH; 57% of patients had classic PNH, whereas 30% and 13% had aplastic anemia (AA)/PNH or intermediate PNH (Peffault de Latour et al, Blood 2006), respectively. Two-thirds of patients had been receiving red blood cell (RBC) transfusion before starting Ecu; the mean treatment duration was 97 months (range 6-179). The majority of patients (87%) received Ecu at the standard schedule of 900 mg every 2 weeks; due to chronic, residual, intravascular hemolysis 13% were treated with higher doses (1200 mg) or with shorter administration interval (10-12 days). Hematological responses were evaluated early during the treatment course (at 6 and 12 months), and then later during the last 6 months of treatment (Figure 1). After 6 (n=80) and 12 months (n=79) of Ecu, respectively, rates of hematological response were as follows: complete response 9% and 10%, major response 4% and 6%, good response 34% and 47%, partial response 34% and 28%, minor response 14% and 6% and no response 6% and 3%. In 83 patients treated for more than 18 months late response rates also were evaluated; most patients responded and the rates of hematological response during the last 6 months of treatment were as follows: complete response, 11%; major response, 6%; good response, 41%; partial response, 29%; minor response, 7% and no response, 6%. Breakthrough episodes were recorded in 21% of patients (equally split between pharmacokinetic and pharmacodynamic ones); thromboembolic events and AA were seen in 2% and 3% of patients, respectively. These data suggest that, albeit individual and unpredictable, the extent of the hematological benefit of Ecu in most PNH patient can be determined quite early in the treatment course, after a minimum of 12 months. Conclusion This is the first attempt to classify hematological response to Ecu based on Hb levels and residual hemolysis. The proposed classification allows the identification of distinct patient subgroups with different medical needs, as well as of meaningful clinical goals for future anti-complement therapies. In our cohort, about 60% of patients are well controlled with Ecu, even if less than 20% of patients reach normal Hb values; about 40% of patients remain anemic, and this represents a goal for future therapies in PNH. This classification is helpful to characterize more precisely the clinical response, which should be assessed after 1 year of treatment. Even if it should be validated in independent, larger cohorts, this classification represents a useful tool to design future trials, and to compare the clinical results of the novel anti-complement agents in development for PNH. Disclosures Notaro: Alexion: Membership on an entity's Board of Directors or advisory committees, Other: letture fees. Scheinberg:Pfizer,: Speakers Bureau; Celgene: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kulasekararaj:Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Achilleon: Consultancy; Ra Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Akari Therapeutics: Consultancy. Risitano:Biocryst: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Samsung: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ra Pharma: Research Funding; Alnylam: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Amyndas: Consultancy; Achillion: Research Funding; Amyndas: Consultancy; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees. Peffault de Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

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.

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 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.

Forty-four dogs naturally infected by Leishmania infantum were divided into two groups: 20 thrombocytopenic dogs with fewer than 150 x 10(9) platelets/l, and 24 non-thrombocytopenic dogs with more than 200 x 10(9) platelets/l. Ten clinically healthy dogs were used as controls. A haematological profile was obtained and the dogs' serum was used to assess the presence of platelet-binding IgM and IgG antibodies using a flow cytometry technique. Nineteen of the 20 thrombocytopenic dogs, and 13 of the 24 non-thrombocytopenic dogs had detectable levels of platelet-binding immunoglobulins, but none of the control dogs did so. The differences were significantly different for both IgM and IgG platelet-binding antibodies.

We describe an MDS patient showing neutropenia, CD14+CD56+ monocytosis and polyclonal expansion of NK cells. A defective HLA-E expression on myeloid lineage, the constitutive presence of CD178 on the CD14+CD56+ monocytes combined with expansion of CD94/NKG2A+ CD94/NKG2C+ NK cells are proposed to be relevant for the pathogenesis of this haematopoietic disorder

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the expansion of a P/G-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

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the emergence of a GPI-defective clonal hematopoiesis. Its clinical features are hemolytic anemia, cytopenia, and thrombosis. Circulating monocytes and granulocytes are largely GPI-defective in PNH patients. This study aims to investigate the granulocyte functional properties in PNH. We analyzed bacterial-dependent intracellular ingestion and the consequent activation of oxidative burst in GPI-defective granulocytes from four neutropenic PNH patients. Our data show a significant increase in the ability of GPI-defective granulocytes to ingest opsonized bacteria. In addition, an impaired respiratory burst effectiveness in response to two independent bacterial stimuli, the N-formyl-MetLeuPhe (fMLP) synthetic bacterial peptide and E. coli, was revealed. The occurrence of neutropenia and the severe impairment of oxidative burst, occurring in chronic granulomatosis disease, were unable to significantly affect phagocytosis. Thus, additional mechanisms, able to differentially affect ingestion ability and respiratory burst effectiveness, have to be hypothesized. The reduced burst effectiveness of GPI-defective granulocytes was maintained after treatment with phorbol 12-myristate 13-acetate, a pharmacological stimulus able to extensively recruit and to trigger intracellular protein kinase C (PKC). Moreover, blocking of PKC has been observed to severely affect granulocyte respiratory burst with a mild effect on the phagocytosis. These data suggest a role for a modulation of intracellular PKC in the pathogenesis of the impaired granulocyte oxidative burst. (c) 2006 Wiley-Liss, Inc.

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.

PNH is a rare acquired clonal disorder of the hematopoietic stem cell, characterized by a somatic mutation that inactivates the X-linked PIGA gene: this in turn results in deficiency on the cell surface of all proteins anchored by the glycosylphosphatidylinositol (GPI) molecule. Two of these proteins,CD55 andCD59, are complement regulators and their deficiency is responsible for the susceptibility of red cells (RBCs) from the mutant clone to lysis by activated complement. Since oxidative damage is another well-known mechanism of hemolysis (as in G6PD deficient red cells), we have investigated whether this plays a role also in PNH. To this end, we have carried out experiments on RBCs from healthy donors and on PNH-like RBCs (obtained in vitro from the same donors through the use of anti-CD55 and anti-CD59 blocking moAb). After exposure to AB0-compatible serum (in which thecomplement alternative pathway was activated by mild acidification) all PNH-like (but not normal) RBCs were lysed. In parallel experiments in which complement was blocked by eculizumab (ECU) - a moAb that binds to the complement component C5 and controls intravascular hemolysis in PNH patients - we measured the levels of reactive oxygen species (ROS) by the dichlorofluorescin diacetate assay. We found no significant difference of ROS levels between normal RBCs and PNH-like RBCs. We next tested in a similar way G6PD-deficient RBCs, because these are known to be exquisitely sensitive to oxidative damage. We found that ROS levels were significantly higher in the G6PD deficient RBCs that have been made PNH-like (Fig. 1). Thus, complement activation on the surface of PNH-like RBCs results in the production of ROS that can be demonstrated when C5-blockade prevents complement-mediated lysis of RBCs. The notion that G6PD deficiency can interact with PNH was strongly corroborated by the clinical observation of a 40yo woman from Sardinia (Italy) with a 2 years history of pancytopenia, who then developed florid hemolytic PNH: she had anemia with normal granulocyte and platelet counts, dark urine, high reticulocytosis, LDH up to 5x upper normal level, 95% GPI-negative granulocytes. When the patient was started on ECU. LDH levels promptly returned to normal, PNH RBCs rose from 20% (before ECU) to 42%, but reticulocyte count (~250x109/L) and blood transfusion requirement remained high (10 units in the last year). 39% of the GPI-negative RBCs had bound C3 fragments The peripheral blood smear revealed marked macro-anisocytosis, poikilocytosis, spherocytes, and hemighosts: a picture consistent with oxidative damage as seen in G6PD deficient patients during a hemolytic attack. The RBC G6PD activity was about one-half of normal (5 IU/g Hb), and DNA analysis revealed heterozygosity for the G6PD Mediterranean (Med) mutation. By mRNA sequence analysis we found that the GPI-negative clone expressed only the G6PD Med allele, suggesting that the PIG-A mutation took place in a stem cell in which the normal G6PD gene was on the inactive X-chromosome (G6PD, like PIG-A, is on the X chromosome); therefore, all the patients' PNH RBCs were also all G6PD deficient. We have previously shown that the clinical expression of PNH can be influenced by inherited factors: specifically, a polymorphism of the complement receptor 1 (CR1) gene correlates with the blood transfusion requirement of patients on ECU (Rondelli et al, Haematologica 2014). However, the patient here reported was homozygous for the more favorable allele ofCR1. Instead, in keeping with our experimental data, the poor response to ECU seen in this patient results probably from a unique interaction, within the same population of RBCs, between the acquired PNH abnormality and her inherited G6PD deficiency This type of interaction is novel and it seems to have pharmacogenetic implications. Indeed, on its own G6PD deficiency affects mildly the clinical expression of PNH, because complement activation causes RBC lysis regardless; however, paradoxically, when the lysis of PNH RBCs is prevented by C5 blockade, complement activation results in oxidative damage, with which PNH G6PD deficient RBCs are unable to cope. Except for one case previously reported by Oni et al (Blood 1970), this is the first detailed study of PNH associated with G6PD deficiency. Since in some parts of the world the frequency of G6PD deficiency can be as high as 30% or more, we expect that more cases of this association will be discovered in the future. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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.

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.

C5-blockade with eculizumab prevents complement-mediated intravascular hemolysis in PNH patients and its clinical consequences. However, a distinct population of PNH red blood cells bound with C3 fragments appears in almost all treated patients. This C3 binding results in extravascular hemolysis that in some patients reduces the clinical benefit from eculizumab. In each PNH patients on eculizumab there are always two distinct populations of PNH red blood cells, one with (C3+) and one without (C3-) C3 binding. This phenomenon is somehow paradoxical since the glycosylphosphatidylinositol (GPI)-linked complement regulators, CD55 and CD59, are uniformly deficient on the surface of PNH red cells. To investigate this phenomenon, we have modeled in vitro the C3 binding in the context of C5 blockade by incubating red blood cells from PNH patients with AB0-matched sera from patients on eculizumab. Complement alternative pathway has been activated by mild acidification (in presence of Mg/EGTA to prevent the activation of complement classical pathway) and C3 binding has been assessed by flow cytometry at serial time points. In these experimental conditions a fraction of PNH red blood cells, similar to what happens in vivo, become promptly C3+ and its size increases with the time: from 9.4±2.7% after 5 minutes to 21.2±9.5% after 24 hours. The membrane defects of PNH cells suggested that the deficiency of CD55, which regulates the formation and accelerates the dissociation of C3 convertases, should be responsible for C3 binding to PNH red blood cells in presence of eculizumab (Parker CJ. Hematology Am Soc Hematol Educ Program. 2011;2011:21-29). In order to verify experimentally this hypothesis we have inactivated CD55 or CD59 on normal red blood cells by using blocking monoclonal antibodies (moAb - listed in the figure legend), and we have tested them in vitro upon activation of complement alternative pathway by mild acidification in presence or absence of C5 blockade. We found that CD55 inactivation on normal red blood cells results neither in hemolysis (without C5 blockade) nor in any C3 binding (with C5 blockade). As expected without C5 blockade CD59-inactivated normal red blood cells undergo hemolysis but, surprisingly, we found that in presence of C5 blockade they become bound with C3 fragments (Figure 1), just as it occurs in vivo in PNH patients on eculizumab. The simultaneous inactivation of both CD55 and CD59 further increased the level of C3 binding. Thus, at variance with the starting hypothesis, the deficiency of CD59, not that of CD55, plays the major role in C3 binding to PNH red cells of patients on eculizumab. Therapeutic C5 blockade in PNH patients has unmasked a novel function of CD59: in addition to prevent MAC formation, it plays a central role also in the regulation of C3 activation on cell surface through molecular mechanisms not elucidated yet. It remains to be established the physiological role, if any, of this novel function of CD59 and whether it play a role in determining the pleomorphic clinical features of the congenital CD59 deficiency. Finally, these findings may lead to investigate innovative approaches to reduce C3 binding and extravascular hemolysis in PNH patients on eculizumab and, in a broader context, to modulate complement activity. Figure 1 Figure 1. Disclosures Risitano: Novartis: Research Funding; Alexion Pharmaceuticals: Other: lecture fees, Research Funding; Rapharma: Research Funding; Alnylam: Research Funding.

Thirty naturally infected dogs were divided into three groups (10 animals each): group I (canine leishmaniosis), group II (canine ehrlichiosis) and group III (dogs co-infected by both infectious agents). Ten clinically healthy dogs were used as controls. A haematological profile was obtained and dog sera were assessed for the presence of platelet-bound IgM and IgG antibodies (PBIgM, PBIgG) using flow cytometry. Eight of the naturally co-infected dogs (80%), five of the leishmaniotic dogs (50%) and six of the ehrlichiotic dogs (60%) had detectable levels of platelet-bound antibodies (PBIg). No PBIg were observed in the control group. The hypothesis that Leishmania and Ehrlichia co-infection could more severely affect the immune response of infected dogs is discussed. Copyright © 2010 Elsevier Ltd. All rights reserved.

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. Disclosures: Risitano: Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder characterized by the emergence and expansion of a GPI-defective clonal haematopoiesis. Its clinical features are haemolytic anaemia, cytopenia and thrombosis. Several data suggests the pathogenetic relevance for auto-immune-mediated mechanisms in the expansion of the mutated clone. Since HLA genes represent a susceptibility factor for autoimmunity, we have investigated HLA genotypes in 42 Italian patients with PNH. This study analyzed the HLA class I and class II genes in a cohort of 42 Italian PNH patients. Data indicate the occurrence of a significant increase of A*33 (9.5% in PNH patients versus 2.3% in controls; p