In paroxysmal nocturnal hemoglobinuria (PNH) hemolytic anemia is due mainly to deficiency of the complement regulator CD59 on the surface of red blood cells (RBCs). Eculizumab, an antibody that targets complement fraction 5 (C5), has proven highly effective in abolishing complement-mediated intravascular hemolysis in PNH; however, the hematologic benefit varies considerably among patients. In the aim to understand the basis for this variable response, we have IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a hematologic disorder characterized by the clonal expansion of one or a few hematopoietic stem cells that are incapable of glycosylphosphatidylinositol (GPI)-anchor biosynthesis, due to an acquired somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene. [1][2][3][4][5][6] Affected progeny cells are deficient in all GPI-anchored surface proteins, including the complement regulators CD55 and CD59. 7-9 Thus, PNH red blood cells (RBCs) are exquisitely vulnerable to activated complement, and particularly to the membrane attack complex (MAC), 10,11 resulting in chronic intravascular hemolysis with recurrent exacerbations, and consequent anemia.Eculizumab (Soliris; Alexion Pharmaceuticals, Cheshire, CT) is a humanized monoclonal antibody against complement fraction 5 (C5), which inhibits MAC formation. 12 Eculizumab has proven highly beneficial in the treatment of transfusion-dependent PNH patients. [13][14][15] In a placebo-controlled phase 3 trial, eculizumab led to a marked decrease in transfusion requirement, and improvement in anemia, fatigue, pain, shortness of breath, and QoL measures. 15 These data were confirmed in 2 subsequent studies, 16,17 the last one also suggesting that eculizumab may reduce the occurrence of thromboembolic events. 17 In the face of such gratifying clinical results, it is clear that not all patients respond equally to the treatment. In some patients there is only little improvement of anemia, and some still require blood transfusion at times, with signs of persistent hemolysis (reticulocytosis, elevated unconjugated bilirubin). 15,16 In this work, we have investigated the notion that in patients with suboptimal hematologic response to eculizumab there may be extravascular hemolysis mediated by complement effector mechanisms other than MAC. 15 Based on flow cytometry analysis of complement fraction 3 (C3) on RBCs, we provide evidence of selective C3 opsonization of GPI-negative red cells, the extent of which tends to correlate with the clinical response to eculizumab, and may be the manifestation of a novel phenomenon in the pathophysiology of PNH. Methods PatientsThe study was conducted in 56 Italian PNH patients (Table 1); biologic samples were collected by venipuncture according to standard procedures, after informed consent was obtained in accordance with the Declaration of Helsinki as approved within the study protocol by the Institutional Review Board at the Federico II University of Naples. Twenty-eight patients were studied at diagnosis, before any t...

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Glucose 6-phosphate dehydrogenase (G6PD) deficiency is one of the commonest human enzymopathies: it is due to inherited mutations of the X-linked gene G6PD. G6PD deficiency makes red cells highly vulnerable to oxidative damage, and therefore susceptible to hemolysis. Over 200 G6PD mutations are known: about one-half are polymorphic and therefore common in various populations. Some 500 million persons with any of these mutations are mostly asymptomatic throughout their lifetime; however, any of them may develop acute and sometimes very severe hemolytic anemia when this is triggered by ingestion of fava beans, or by any of a number of drugs (e.g. primaquine, rasburicase), or more rarely by infection. About one-half of the G6PD mutations are instead sporadic: rare patients with these mutations present with chronic non-spherocytic hemolytic anemia. Almost all G6PD mutations are missense mutations, causing amino acid replacements that entail deficiency of G6PD enzyme activity because they compromise the stability of the protein, or because the catalytic activity is decreased, or through a combination of both mechanisms: thus, genotype-phenotype correlations have been reasonably well clarified in many cases. G6PD deficiency correlates remarkably, in its geographic distribution, with past/present malaria endemicity: indeed, it is a unique example of an X-linked human polymorphism balanced through protection of heterozygotes from malaria mortality. Acute hemolytic anemia can be managed effectively provided it is promptly diagnosed. Reliable diagnostic procedures are available, with point of care tests becoming increasingly important where primaquine and its analogue tafenoquine, recently introduced, are required for the elimination of malaria.

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.

Aging in vivo and cell division in vitro are associated with telomere shortening. Several lines of evidence suggest that telomere length may be a good predictor of the long term replicative capacity of cells. To investigate the natural fate of chromosome telomeres of hematopoietic stem cells in vivo, we measured the telomere length of peripheral blood granulocytes from 11 fully engrafted bone marrow transplant recipients and from their respective donors. In 10 of 11 donor-recipient pairs, the telomere length was significantly reduced in the recipient and the extent of reduction correlated inversely with the number of nucleated cells infused. These data provide internally controlled in vivo evidence that, concomitantly with their proliferation, hematopoietic stem cells lose telomere length; it is possible that, as a result, their proliferative potential is reduced. These findings must be taken into account when developing new protocols in which few stem cells are used for bone marrow transplantation or for gene therapy.

• Peptidic C3 inhibitors of the compstatin family (Cp40) efficiently prevent hemolysis and opsonization of PNH erythrocytes in vitro.• Pharmacokinetic studies show that sustained therapeutic concentrations can be achieved with both Cp40 and its PEGylated derivative, PEG-Cp40.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 IC 50 ∼4 mM and full inhibition at 6 mM. 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. (Blood. 2014;123(13):2094-2101 IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is a complex hematologic disorder characterized by the expansion of hematopoietic cells deficient in glycophosphatidylinositol-anchored surface proteins, including the complement regulators CD55 and CD59. 1 Affected erythrocytes suffer from uncontrolled complement activation on their surface, and subsequent membrane attack complex (MAC)-mediated intravascular hemolysis.2 The therapeutic anti-C5 antibody eculizumab (Soliris, Alexion) has proven effective in controlling intravascular hemolysis in vivo, leading to remarkable clinical benefit in a majority of PNH patients.3,4 Yet, persistent C3 activation occurring during eculizumab treatment may lead to progressive deposition of C3 fragments on affected erythrocytes and subsequent C3-mediated extravascular hemolysis, possibly limiting the hematologic benefit of anti-C5 treatment. 5,6 Thus, upstream inhibition of the complement cascade seems an appropriate strategy to improve the results of current complement-targeted treatment. 7,8 Indeed, it has been recently documented that protein inhibitors of the alternative pathway (AP) of complement activation, such as the CD21/factor H (FH) fusion protein TT30 (Alexion) or the engin...

Bone marrow stromal cells (BMSCs) can be easily isolated from adult marrow and contain a population of pluripotent progenitors that can give rise to different mesenchymal lineages both in vitro and in vivo. These properties make BMSCs an attractive target for cell-based therapeutic strategies for a variety of disorders. However, because of their low frequency in vivo, to obtain a sufficient number of cells for tissue engineering a step of extensive in vitro expansion is required, which could significantly alter BMSC properties. Therefore, effective therapeutic use of BMSCs requires the design of appropriate approaches for in vitro cell expansion. In this study we have investigated the biological effects of in vitro expansion on BMSC proliferative ability and on their spontaneous differentiation. Telomerase activity and telomere shortening kinetics were evaluated together with variations in osteogenic, chondrogenic, and adipogenic gene expression throughout the BMSC life span. In culture BMSCs never displayed telomerase activity and during in vitro expansion telomere length decreased. Furthermore, gene expression patterns spontaneously varied during expansion, indicating a progressive commitment of the population toward the osteogenic lineage. In conclusion, BMSCs in culture undergo progressive replicative aging and osteogenic differentiation. These observations are relevant to their successful use in clinics and should be considered when designing novel therapeutic strategies.