Publications by authors named "Rosario Notaro"

50 Publications

Danicopan: an oral complement factor D inhibitor for paroxysmal nocturnal hemoglobinuria.

Haematologica 2020 10 29;Online ahead of print. Epub 2020 Oct 29.

University of Auckland, Auckland.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterised by complement-mediated intravascular hemolysis (IVH) due to absence of complement regulators CD55 and CD59 on affected erythrocytes. Danicopan is a first-in-class oral proximal, complement alternative pathway factor D (FD) inhibitor. Therapeutic FD inhibition was designed to control IVH and prevent C3-mediated extravascular hemolysis (EVH). In this open-label, phase 2, dose-finding trial, 10 untreated hemolytic PNH patients received danicopan monotherapy (100-200 mg thrice daily). Endpoints included change in lactate dehydrogenase (LDH) at day 28 (primary) and day 84 and hemoglobin. Safety, pharmacokinetics/pharmacodynamics, and patient-reported outcomes were measured. Ten patients reached the primary endpoint; two later discontinued: one for a serious adverse event (elevated aspartate aminotransferase/alanine aminotransferase coincident with breakthrough hemolysis, resolving without sequelae) and one for personal reasons unrelated to safety. Eight patients completed treatment. IVH was inhibited, demonstrated by mean decreased LDH (5.7 times upper limit of normal [ULN] at baseline vs 1.8 times ULN [day 28] and 2.2 times ULN [day 84]; both p.
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http://dx.doi.org/10.3324/haematol.2020.261826DOI Listing
October 2020

Complement-mediated oxidative damage of red cells impairs response to eculizumab in a G6PD-deficient patient with PNH.

Blood 2020 Dec;136(26):3082-3085

Laboratory of Cancer Genetics and Gene Transfer, Core Research Laboratory-Institute for Cancer Research, Prevention and Clinical Network (ISPRO), Florence, Italy.

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http://dx.doi.org/10.1182/blood.2020007780DOI Listing
December 2020

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

J Hematol Oncol 2020 08 13;13(1):112. Epub 2020 Aug 13.

Laboratory of Cancer Genetics, Core Research Laboratory, Istituto per lo Studio, la Prevenzione e la Rete Oncologica (ISPRO), Florence, 50139, Italy.

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.
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http://dx.doi.org/10.1186/s13045-020-00943-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427297PMC
August 2020

Glucose-6-phosphate dehydrogenase deficiency.

Blood 2020 Sep;136(11):1225-1240

Laboratory of Cancer Genetics and Gene Transfer, Core Research Laboratory, Institute for Cancer Research, Prevention and Clinical Network (ISPRO), Florence, Italy.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is 1 of the commonest human enzymopathies, caused by 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: approximately 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 triggered by ingestion of fava beans, by any of a number of drugs (for example, primaquine, rasburicase), or, more rarely, by infection. Approximately one-half of the G6PD mutations are instead sporadic: rare patients with these mutations present with chronic nonspherocytic hemolytic anemia. Almost all G6PD mutations are missense mutations, causing amino acid replacements that entail deficiency of G6PD enzyme activity: they compromise the stability of the protein, the catalytic activity is decreased, or a combination of both mechanisms occurs. 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 recently introduced analog tafenoquine are required for the elimination of malaria.
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http://dx.doi.org/10.1182/blood.2019000944DOI Listing
September 2020

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

Front Immunol 2019 14;10:1157. Epub 2019 Jun 14.

Severe Aplastic Anemia Working Party of the European Group for Blood and Marrow Transplantation, Leiden, Netherlands.

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.
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http://dx.doi.org/10.3389/fimmu.2019.01157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6587878PMC
November 2020

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

Semin Hematol 2018 07 5;55(3):130-135. Epub 2018 Jun 5.

Laboratory of Cancer Genetics and Gene Transfer, Core Research Laboratory - Istituto per lo Studio, la Prevenzione e la Rete Oncologica (ISPRO), Florence, Italy.

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.
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http://dx.doi.org/10.1053/j.seminhematol.2018.05.014DOI Listing
July 2018

The "escape" model: a versatile mechanism for clonal expansion.

Br J Haematol 2019 02 24;184(3):465-466. Epub 2018 Jan 24.

Core Research Laboratory - ITT, AOU Careggi, Firenze, Italy.

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http://dx.doi.org/10.1111/bjh.15111DOI Listing
February 2019

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

J Hematol Oncol 2017 06 19;10(1):126. Epub 2017 Jun 19.

Laboratory of Cancer Genetics and Gene Transfer, Core Research Laboratory - Istituto Toscano Tumori (CRL-ITT), AOU Careggi, viale Pieraccini 6, 50139, Florence, Italy.

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.
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http://dx.doi.org/10.1186/s13045-017-0496-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5477256PMC
June 2017

Glycosylphosphatidylinositol-specific T cells, IFN-γ-producing T cells, and pathogenesis of idiopathic aplastic anemia.

Blood 2017 01 30;129(3):388-392. Epub 2016 Nov 30.

Laboratory of Cancer Genetics and Gene Transfer, Core Research Laboratory-Istituto Toscano Tumori, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy.

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http://dx.doi.org/10.1182/blood-2016-09-740845DOI Listing
January 2017

Glucose-6-Phosphate Dehydrogenase Deficiency.

Hematol Oncol Clin North Am 2016 Apr;30(2):373-93

Core Research Laboratory-Istituto Toscano Tumori, Azienda Universitaria-Ospedaliera Careggi, Viale Pieraccini 6, Florence 50139, Italy.

G6PD is a housekeeping gene expressed in all cells. Glucose-6-phosphate dehydrogenase (G6PD) is part of the pentose phosphate pathway, and its main physiologic role is to provide NADPH. G6PD deficiency, one of the commonest inherited enzyme abnormalities in humans, arises through one of many possible mutations, most of which reduce the stability of the enzyme and its level as red cells age. G6PD-deficient persons are mostly asymptomatic, but they can develop severe jaundice during the neonatal period and acute hemolytic anemia when they ingest fava beans or when they are exposed to certain infections or drugs. G6PD deficiency is a global health issue.
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http://dx.doi.org/10.1016/j.hoc.2015.11.006DOI Listing
April 2016

Complement-mediated haemolysis and the role of blood transfusion in paroxysmal nocturnal haemoglobinuria.

Blood Transfus 2015 Jul 2;13(3):363-9. Epub 2015 Feb 2.

Tumour Institute of Tuscany, Florence, Italy.

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http://dx.doi.org/10.2450/2015.0249-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614287PMC
July 2015

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

Blood 2014 Mar 4;123(13):2094-101. Epub 2014 Feb 4.

Hematology, Department of Clinical Medicine and Surgery, Federico II University of Naples, Naples, Italy;

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.
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http://dx.doi.org/10.1182/blood-2013-11-536573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3968392PMC
March 2014

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

Haematologica 2014 Feb 13;99(2):262-6. Epub 2013 Sep 13.

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.
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http://dx.doi.org/10.3324/haematol.2013.090001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3912955PMC
February 2014

Is green tea a potential trigger for autoimmune hepatitis?

Phytomedicine 2013 Oct 6;20(13):1186-9. Epub 2013 Aug 6.

University of Florence, Department of Neuroscience, Psychology, Drug Research and Child Health (NeuroFarBa), Center of Molecular Medicine (CIMMBA), Florence, Italy.

A case of autoimmune liver hepatitis is reported: the onset was triggered by consumption of green tea infusion in a patient taking oral contraceptives and irbesartan. We hypothesize that our patient, carrying genetic variant of hepatic metabolism making her particularly susceptible to oxidative stress, developed an abnormal response to a mild toxic insult, afforded by a combination of agents (oral contraceptives+irbesartan+green tea) that normally would not be able to cause damage. Her particular hepatic metabolism further increased the drugs' concentration, favoring the haptenization of liver proteins, eventually leading to the development of an autoimmune hepatitis.
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http://dx.doi.org/10.1016/j.phymed.2013.07.004DOI Listing
October 2013

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

Blood 2013 Apr 31;121(14):2753-61. Epub 2013 Jan 31.

Centre for Haematology, Department of Medicine, Hammersmith Hospital, Imperial College London, London, United Kingdom.

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.
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http://dx.doi.org/10.1182/blood-2012-11-469353DOI Listing
April 2013

The frequency of granulocytes with spontaneous somatic mutations: a wide distribution in a normal human population.

PLoS One 2013 14;8(1):e54046. Epub 2013 Jan 14.

Laboratory of Cancer Genetics and Gene Transfer, Core Research Laboratory-CRL, Istituto Toscano Tumori-ITT, Firenze, Italy.

Germ-line mutation rate has been regarded classically as a fundamental biological parameter, as it affects the prevalence of genetic disorders and the rate of evolution. Somatic mutation rate is also an important biological parameter, as it may influence the development and/or the course of acquired diseases, particularly of cancer. Estimates of this parameter have been previously obtained in few instances from dermal fibroblasts and lymphoblastoid cells. However, the methodology required has been laborious and did not lend itself to the analysis of large numbers of samples. We have previously shown that the X-linked gene PIG-A, since its product is required for glycosyl-phosphatidylinositol-anchored proteins to become surface bound, is a good sentinel gene for studying somatic mutations. We now show that by this approach we can accurately measure the proportion of PIG-A mutant peripheral blood granulocytes, which we call mutant frequency, ƒ. We found that the results are reproducible, with a variation coefficient (CV) of 45%. Repeat samples from 32 subjects also had a CV of 44%, indicating that ƒ is a relatively stable individual characteristic. From a study of 142 normal subjects we found that log ƒ is a normally distributed variable; ƒ variability spans a 80-fold range, from less than 1×10⁻⁶ to 37.5×10⁻⁶, with a median of 4.9×10⁻⁶. Unlike other techniques commonly employed in population studies, such as comet assay, this method can detect any kind of mutation, including point mutation, as long as it causes functional inactivation of PIG-A gene. Since the test is rapid and requires only a small sample of peripheral blood, this methodology will lend itself to investigating genetic factors that underlie the variation in the somatic mutation rate, as well as environmental factors that may affect it. It will be also possible to test whether ƒ is a determinant of the risk of cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0054046PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3544671PMC
August 2013

A systematic analysis of bone marrow cells by flow cytometry defines a specific phenotypic profile beyond GPI deficiency in paroxysmal nocturnal hemoglobinuria.

Cytometry B Clin Cytom 2013 Mar 26;84(2):71-81. Epub 2012 Dec 26.

Unità Funzionale di Ematologia, Università degli Studi, AOU Careggi, Florence, Italy.

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a unique disorder caused by a PIG-A gene mutation in a stem cell clone. Its clinical picture can sometimes make challenging the distinction from other disorders, and especially from myelodysplastic syndromes (MDS), since both diseases correlate with cytopenias and morphological abnormalities of bone marrow (BM) cells. Recently, flow cytometry (FC) has been proposed to integrate the morphologic assessment of BM dysplasia, and thus to improve the diagnostics of MDS.

Methods: In the present study, we have analyzed systematically FC data resulting from the study of BM cells from patients with PNH and MDS.

Results: Our data demonstrated abnormalities in PNH beyond the deficiency of glycosylphosphatidylinositol-linked proteins and the application of a systematic approach allowed us to separate effectively MDS and PNH in a cluster analysis and to highlight disease-specific abnormalities. Indeed, the parallel evaluation of some key parameters, i.e. patterns of expression of CD45 and CD10, provided information with practical diagnostic usefulness in the distinction between PNH and MDS. Moreover, the hypo-expression of CD36 that we observed on monocytes might be related to the thrombotic tendency in PNH.

Conclusions: We investigated systematically the phenotypic profile of BM cells from patients with PNH; our data provide useful antigenic patterns to solve between PNH and MDS, sometimes morphologically overlapping. Moreover, some PNH-related phenotypic changes might be involved in the physiopathology of the disease and further studies addressing this issue are warranted.
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http://dx.doi.org/10.1002/cyto.b.21064DOI Listing
March 2013

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

Blood 2012 Jun 10;119(26):6307-16. Epub 2012 May 10.

Hematology, Department of Biochemistry and Medical Biotechnologies, Federico II University, Naples, Italy.

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.
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http://dx.doi.org/10.1182/blood-2011-12-398792DOI Listing
June 2012

Eculizumab treatment modifies the immune profile of PNH patients.

Immunobiology 2012 Jul 3;217(7):698-703. Epub 2011 Dec 3.

Department of Biochemistry and Biomedical Technologies, University of Naples Federico II, Italy.

Paroxysmal Nocturnal Haemoglobinuria (PNH) is due to pathological expansion of a stem progenitor bearing a somatic mutation of PIG-A gene involved in the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor. Numerous data suggest a role for immune-mediated mechanisms in the selection/expansion of GPI-defective clone. Haemolytic anaemia in PNH is dependent on the effect of complement against GPI-defective red cells. Eculizumab, an anti-C5 monoclonal antibody, is dramatically effective in controlling haemolysis and thrombosis, in reducing fatigue and in improving quality of life of patients. However, this therapy presents new challenges that need to be properly faced. Here, we report the decrease in B, Natural Killer (NK) and regulatory T cells (Treg), an altered cytokine profile of invariant-NKT cells (NKTi) and the increasing of C-X-C chemokine receptor type 4 (CXCR4) receptor in PNH patients before the Eculizumab therapy. Treatment significantly affects some of these alterations: after Eculizumab, the number of B lymphocytes, the cytokine secretion of NKTi and CXCR4 expression on CD8 T cells became similar to healthy donors. No effects were observed on NK and Treg. The amplitude of the GPI-defective compartment remained unchanged.
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http://dx.doi.org/10.1016/j.imbio.2011.11.009DOI Listing
July 2012

Thrombolytic therapy is effective in paroxysmal nocturnal hemoglobinuria: a series of nine patients and a review of the literature.

Haematologica 2012 Mar 1;97(3):344-52. Epub 2011 Dec 1.

Division of Hematology, NYU School of Medicine, NYU Langone Cancer Center, New York, NY 10016, USA.

Background: Thrombosis is the major risk factor for death in patients with paroxysmal nocturnal hemoglobinuria. Previous case reports indicate that venous thrombosis in patients with paroxysmal nocturnal hemoglobinuria is amenable to thrombolysis.

Design And Methods: We reviewed the outcome of thrombolytic therapy for patients with paroxysmal nocturnal hemoglobinuria who had thromboses refractory to anticoagulation at our institutions.

Results: In this study of 41 patients who had at least one thrombotic event, we confirmed a very high incidence of recurrence despite anticoagulation. Nine patients with thrombosis were regarded as eligible for administration of intravenous tissue plasminogen activator, which was effective in reversing thrombi in all of 15 occasions in which it was given. Serious hemorrhagic complications developed in three cases. At last follow-up visit, of the nine patients treated, three had died, and six were in very good to excellent condition in terms of clinical outcome and radiological findings. The only patient in whom thrombolysis may have contributed to a fatal outcome also had complications of "heparin induced thrombocytopenia with thrombosis", which we diagnosed in three additional patients. In our review of the literature, nine out of 15 patients treated with thrombolysis have had a good outcome.

Conclusions: Although it is associated with a significant but manageable risk of bleeding, systemic thrombolysis is a highly effective treatment for reversing venous thromboses in patients with paroxysmal nocturnal hemoglobinuria.
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http://dx.doi.org/10.3324/haematol.2011.049767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3291587PMC
March 2012

Management of paroxysmal nocturnal haemoglobinuria: a personal view.

Br J Haematol 2011 Jun 22;153(6):709-20. Epub 2011 Apr 22.

Istituto Toscano Tumori, Florence, Italy.

Paroxysmal nocturnal haemoglobinuria (PNH) is a serious form of acquired haemolytic anaemia with several features that make it unique, including the fact that it is caused by clonal expansion, in the context of bone marrow failure, of a haematopoietic stem cell that has a somatic mutation in a gene crucial for the synthesis of glycosylphosphatidylinositol anchors; and that this also produces a life-threatening acquired thrombophilic state. Until recently, the two only main options for patients with PNH were either allogeneic bone marrow transplantation or supportive management, including blood transfusion: both options require some skill and good patient-doctor collaboration. Since the start of this millennium a major advance has been the introduction of eculizumab, a monoclonal antibody that targets the C5 protein of the complement system: blockade of C5 prevents activation of the complement distal pathway, and thus abrogates the complement-mediated intravascular haemolysis that severely plagues patients with PNH. This review outlines an approach to the management of all three major components of the clinical picture of PNH--namely haemolysis, thrombosis and bone marrow failure--based on the literature and on personal experience. We consider specifically how the use of eculizumab has modified other aspects of the management of PNH, and even the pathophysiology itself of this disease. Finally, we develop a treatment algorithm which others might find helpful.
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http://dx.doi.org/10.1111/j.1365-2141.2011.08690.xDOI Listing
June 2011

Synthesis of the essential core of the human glycosylphosphatidylinositol (GPI) anchor.

Bioorg Chem 2011 Apr 17;39(2):88-93. Epub 2010 Dec 17.

Dipartimento di Chimica, Universita' di Firenze, Italy.

The biological role of GPI anchors is of paramount importance; however, we are still far from fully understanding the structure-function relationship of these molecules. One major limiting factor has been the tiny quantities available from natural sources; obtaining homogeneous and well-defined GPI structures by synthesis, is both a challenge and an attractive goal. We report here the convergent synthesis of the essential core of the human GPI anchor 1, exploiting a common precursor to obtain the trisaccharidic donor 2 and a novel protecting groups sequence. The final product, prepared for the first time, is biologically active.
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http://dx.doi.org/10.1016/j.bioorg.2010.12.002DOI Listing
April 2011

Paroxysmal nocturnal hemoglobinuria--hemolysis before and after eculizumab.

N Engl J Med 2010 Dec;363(23):2270-2

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http://dx.doi.org/10.1056/NEJMc1010351DOI Listing
December 2010

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

Mol Cell Probes 2010 Dec 22;24(6):401-2. Epub 2010 Jul 22.

Laboratory of Genetics and Gene Transfer, Core Research Laboratory, Istituto Toscano Tumori (ITT), AOU Careggi, Firenze, Italy.

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.
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http://dx.doi.org/10.1016/j.mcp.2010.07.002DOI Listing
December 2010

Pregnancy in PNH: another eculizumab baby.

Br J Haematol 2010 Sep;150(6):707-8

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http://dx.doi.org/10.1111/j.1365-2141.2010.08258.xDOI Listing
September 2010

Paroxysmal nocturnal hemoglobinuria and eculizumab.

Haematologica 2010 Apr;95(4):523-6

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http://dx.doi.org/10.3324/haematol.2009.017848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857178PMC
April 2010

The use of PIG-A as a sentinel gene for the study of the somatic mutation rate and of mutagenic agents in vivo.

Mutat Res 2010 Jul-Sep;705(1):3-10. Epub 2009 Dec 24.

Unit of Genetics and Gene Transfer, Core Research Laboratory, Istituto Toscano Tumori, Firenze, Italy.

Mutations are an inherent risk of cell duplication. On one hand, inheritable mutations are the driving force of biological evolution; on the other hand, their accumulation in somatic cells plays a key role in the development of cancer. The frequency of mutants (f) and the rate of mutation (mu) are biological features of any cell population: their measurement could provide important information about the risk of oncogenesis and the exposure to carcinogenic agents. However, the measurement of these parameters is not trivial. To measure f and mu, a potential sentinel gene is the PIG-A gene, which encodes one of the subunits of an enzyme essential in the biosynthesis of glycosylphosphatidylinositol (GPI). Since PIG-A is X-linked, mutational inactivation of the one single copy active in somatic cells entails absence from the cell surface of all the proteins that require GPI for attachment to the membrane: thus, mutant cells display a GPI-negative surface phenotype that can be easily detected by flow cytometry. The measurement of PIG-A mutants by counting cells with the GPI-negative phenotype has proved to be effective to measure mutant frequency in peripheral blood cells of humans and of others animals. Up to now, mu has been exceedingly difficult to measure in human cells; however, by using as a sentinel the PIG-A gene in lymphoblastoid cell lines we now have a test that makes it practical to measure mu in human cells.
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http://dx.doi.org/10.1016/j.mrrev.2009.12.004DOI Listing
August 2010

CTLA-4 +49A>G polymorphism of recipients of HLA-matched sibling allogeneic stem cell transplantation is associated with survival and relapse incidence.

Ann Hematol 2010 Jun 18;89(6):613-8. Epub 2009 Dec 18.

Medical Oncology C, National Cancer Research Institute, Genoa, Italy.

Conflicting observations have been reported about the role of CTLA-4 gene polymorphisms in the clinical outcome of allogeneic hematopoietic stem cell transplantation (HSCT). We have investigated three polymorphisms of the CTLA-4 gene (-318C>T, +49A>G, CT60G>A) in 133 donor/recipient pairs who underwent HLA-matched sibling donor HSCT for hematological malignancies. We found no association of the clinical outcome of the HSCT with either recipient or donor -318C>T and CT60G>A polymorphisms. At variance, we found a significant association of donor +49A>G G/G genotype with longer overall survival (OS; log-rank test, P = 0.04), and the number of +49A>G G-alleles in the recipient with longer OS (P = 0.027), longer disease-free survival (P = 0.036) and reduced relapse rate (P = 0.042). However, only recipient +49A>G polymorphism was retained as independent prognostic factor in a multivariate analysis, suggesting that the expression of CTLA-4 on the cells of recipient may be relevant for the clinical outcome of HSCT.
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http://dx.doi.org/10.1007/s00277-009-0885-5DOI Listing
June 2010

Complement fraction 3 binding on erythrocytes as additional mechanism of disease in paroxysmal nocturnal hemoglobinuria patients treated by eculizumab.

Blood 2009 Apr 29;113(17):4094-100. Epub 2009 Jan 29.

Hematology, Department of Biochemistry and Medical Biotechnologies, Federico II University, Naples, Italy.

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 investigated by flow cytometry the binding of complement fraction 3 (C3) on RBCs from PNH patients before and during eculizumab treatment. There was no evidence of C3 on RBCs of untreated PNH patients; by contrast, in all patients on eculizumab (n = 41) a substantial fraction of RBCs had C3 bound on their surface, and this was entirely restricted to RBCs with the PNH phenotype (CD59(-)). The proportion of C3(+) RBCs correlated significantly with the reticulocyte count and with the hematologic response to eculizumab. In 3 patients in whom (51)Cr labeling of RBCs was carried out while on eculizumab, we have demonstrated reduced RBC half-life in vivo, with excess (51)Cr uptake in spleen and in liver. Binding of C3 by PNH RBCs may constitute an additional disease mechanism in PNH, strongly enhanced by eculizumab treatment and producing a variable degree of extravascular hemolysis.
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http://dx.doi.org/10.1182/blood-2008-11-189944DOI Listing
April 2009