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Biomedical subjects

Lucio Luzzatto

Publications and source records attributed to Lucio Luzzatto.

At least 19 recordsLinked to original sources
PubMed2006

The complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria.

BACKGROUND: We tested the safety and efficacy of eculizumab, a humanized monoclonal antibody against terminal complement protein C5 that inhibits terminal complement activation, in patients with paroxysmal nocturnal hemoglobinuria (PNH). METHODS: We conducted a double-blind, randomized, placebo-controlled, multicenter, phase 3 trial. Patients received either placebo or eculizumab intravenously; eculizumab was given at a dose of 600 mg weekly for 4 weeks, followed 1 week later by a 900-mg dose and then 900 mg every other week through week 26. The two primary end points were the stabilization of hemoglobin levels and the number of units of packed red cells transfused. Biochemical indicators of intravascular hemolysis and the patients' quality of life were also assessed. RESULTS: Eighty-seven patients underwent randomization. Stabilization of hemoglobin levels in the absence of transfusions was achieved in 49% (21 of 43) of the patients assigned to eculizumab and none (0 of 44) of those assigned to placebo (P<0.001). During the study, a median of 0 units of packed red cells was administered in the eculizumab group, as compared with 10 units in the placebo group (P<0.001). Eculizumab reduced intravascular hemolysis, as shown by the 85.8% lower median area under the curve for lactate dehydrogenase plotted against time (in days) in the eculizumab group, as compared with the placebo group (58,587 vs. 411,822 U per liter; P<0.001). Clinically significant improvements were also found in the quality of life, as measured by scores on the Functional Assessment of Chronic Illness Therapy-Fatigue instrument (P<0.001) and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire. Of the 87 patients, 4 in the eculizumab group and 9 in the placebo group had serious adverse events, none of which were considered to be treatment-related; all these patients recovered without sequelae. CONCLUSIONS: Eculizumab is an effective therapy for PNH.

Adolescent↗
PubMed2006

CD157 plays a pivotal role in neutrophil transendothelial migration.

Paracellular diapedesis, a key step in leukocyte recruitment to the site of inflammation, occurs at endothelial junctions and is regulated by highly coordinated interactions between leukocytes and endothelium. We found that CD157, a glycosylphosphatidylinositol-anchored ectoenzyme belonging to the NADase/ADP-ribosyl cyclase family, plays a crucial role for neutrophil diapedesis, because its ligation with specific monoclonal antibodies (both on neutrophils or endothelial cells) results in altered neutrophil movement on the apical surface of endothelium and, ultimately, in loss of diapedesis. Real-time microscopy revealed that CD157 behaves as a sort of compass during the interaction between neutrophils and endothelial cells; indeed, following CD157 ligation, neutrophils appear disoriented, meandering toward junctions where they eventually stop without transmigrating. These findings are relevant in vivo because CD157-deficient neutrophils obtained from patients with paroxysmal nocturnal hemoglobinuria are characterized by a severely impaired diapedesis.

ADP-ribosyl Cyclase↗
PubMed2006

Paroxysmal nocturnal hemoglobinuria: an acquired X-linked genetic disease with somatic-cell mosaicism.

Paroxysmal nocturnal hemoglobinuria (PNH) is a severe hemolytic anemia caused by an intrinsic abnormality of the red blood cells that makes them exceedingly susceptible to the lytic action of activated complement (C). This abnormality results from a mutation in the PIG-A gene on Xp22. Given that the mutation is not inherited but is somatically acquired by a hematopoietic stem cell, it creates two populations of blood cells: normal cells and PNH cells. The clinical expression of PNH depends on the relative and absolute expansion of the PNH cell population, which probably depends, in turn, on a paradoxical growth advantage conferred to it by the existence in the patients of an autoimmune process that exerts negative selection against the 'normal' hematopoietic stem cells.

Animals↗
PubMed2006

The mutation rate in PIG-A is normal in patients with paroxysmal nocturnal hemoglobinuria (PNH).

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the presence in the patient's hematopoietic system of a large cell population with a mutation in the X-linked PIG-A gene. Although this abnormal cell population is often found to be monoclonal, it is not unusual that 2 or even several PIG-A mutant clones coexist in the same patient. Therefore, it has been suggested that the PIG-A gene may be hypermutable in PNH. By a method we have recently developed for measuring the intrinsic rate of somatic mutations (mu) in humans, in which PIG-A itself is used as a sentinel gene, we have found that in 5 patients with PNH, mu ranged from 1.24 x 10(-7) to 11.2 x 10(-7), against a normal range of 2.4 x 10(-7) to 29.6 x 10(-7) mutations per cell division. We conclude that genetic instability of the PIG-A gene is not a factor in the pathogenesis of PNH.

Adult↗
PubMed2006

Recent advances in biological and clinical aspects of paroxysmal nocturnal hemoglobinuria.

The unique feature of paroxysmal nocturnal hemoglobinuria (PNH), a chronic disease with severe hemolytic anemia, is the presence of a population of blood cells that, being deficient in surface proteins tethered to the membrane through a glycosylphosphatidylinositol molecule, are said to have the PNH phenotype. Therefore, the diagnosis of PNH is based on the demonstration that a substantial proportion of red cells and granulocytes have this phenotype. Diagnosis is currently best done by flow cytometry analysis, most appropriately by using anti-CD59 and anti-CD55 antibodies. Flow cytometry can also quantitate these cells and monitor their numbers as a function of time, thereby aiding clinical management. The most important advance in management has been the introduction of a human monoclonal antibody (eculizumab) that is directed against the C5 component of complement. Because hemolysis in PNH is mostly intravascular and complement dependent, periodic administration of anti-C5 produces complement blockade. This agent is the first to substantially reduce the rate of hemolysis in patients with PNH. Because very small PNH clones have been known for some years to exist in healthy people, it is clear that a crucial factor in causing PNH as a clinical disease is a marked expansion of the PNH clones themselves. Several lines of evidence from studies of mouse models and patients suggest that the process of expansion is probably the result of 2 phenomena: (1) damage to normal hematopoietic stem cells and (2) the sparing of PNH hematopoietic stem cells. This process of somatic cell selection may have an autoimmune basis, and the most likely agents are cells belonging to the natural killer-like subset of T-cells.

Animals↗
PubMed2005

A quantitative measurement of the human somatic mutation rate.

The mutation rate (mu) is a key biological feature of somatic cells that determines risk for malignant transformation, and it has been exceedingly difficult to measure in human cells. For this purpose, a potential sentinel is the X-linked PIG-A gene, because its inactivation causes lack of glycosylphosphatidylinositol-linked membrane proteins. We previously found that the frequency (f) of PIG-A mutant cells can be measured accurately by flow cytometry, even when f is very low. Here we measure both f and mu by culturing B-lymphoblastoid cell lines and first eliminating preexisting PIG-A mutants by flow sorting. After expansion in culture, the frequency of new mutants is determined by flow cytometry using antibodies specific for glycosylphosphatidylinositol-linked proteins (e.g., CD48, CD55, and CD59). The mutation rate is then calculated by the formula mu = f/d, where d is the number of cell divisions occurring in culture. The mean mu in cells from normal donors was 10.6 x 10(-7) mutations per cell division (range 2.4 to 29.6 x 10(-7)). The mean mu was elevated >30-fold in cells from patients with Fanconi anemia (P < 0.0001), and mu varied widely in ataxia-telangiectasia with a mean 4-fold elevation (P = 0.002). In contrast, mu was not significantly different from normal in cells from patients with Nijmegen breakage syndrome. Differences in mu could not be attributed to variations in plating efficiency. The mutation rate in man can now be measured routinely in B-lymphoblastoid cell lines, and it is elevated in cancer predisposition syndromes. This system should be useful in evaluating cancer risk and in the design of preventive strategies.

B-Lymphocytes↗
PubMed2005

Patients with paroxysmal nocturnal hemoglobinuria have a high frequency of peripheral-blood T cells expressing activating isoforms of inhibiting superfamily receptors.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a large clonal population of blood cells deriving from hematopoietic stem cells (HSCs) deficient in glycosylphosphatidylinositol (GPI)-anchored surface molecules. A current model postulates that PNH arises through negative selection against normal HSCs exerted by autoreactive T cells, whereas PNH HSCs escape damage. We have investigated the inhibitory receptor superfamily (IRS) system in 13 patients with PNH. We found a slight increase in the proportion of T cells expressing IRS. In contrast to what applies to healthy donors, the engagement of IRS molecules on T cells from patients with PNH elicited a powerful cytolytic activity in a redirected killing assay, indicating that these IRSs belong to the activating type. This was confirmed by clonal analysis: 50% of IRS+ T-cell clones in patients with PNH were of the activating type, while only 5% were of the activating type in healthy donors. Moreover, the ligation of IRS induces (1) production of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) and (2) brisk cytolytic activity against cells bearing appropriate IRS counter-ligands. In addition, these IRS+ T cells show natural killer (NK)-like cytolytic activity to which GPI- cells were less sensitive than GPI+ cells. Thus, T cells with NK-like features, expressing the activating isoforms of IRS, may include effector cells involved in the pathogenesis of PNH.

Adult↗
PubMed2005

High incidence of thrombosis in African-American and Latin-American patients with Paroxysmal Nocturnal Haemoglobinuria.

Paroxysmal Nocturnal Haemoglobinuria (PNH) results in a marked thrombophilic state by unknown mechanisms. Geographic differences in thrombosis incidence in PNH have been observed. We have reviewed 64 patients with "Classic PNH" from a single institution in order to determine the rate of thrombosis in different ethnic groups. When we compared African-Americans (n=11) and Latin-Americans (n=8) with other patients (n=45), we found that African-American and Latin-American patients are at increased risk [Hazard ratio 3.66 (p=0.005) and 3.52, (p=0.035) respectively by Cox regression]. Our data also suggest that this difference in the rate of thrombosis has an impact on length of survival. These findings demonstrate that ethnicity is a risk factor for thrombosis in PNH and have implications for decision-making regarding the management of these patients, including the prevention of thrombosis.

Adolescent↗
PubMed2004

CD157 is an important mediator of neutrophil adhesion and migration.

CD157, a glycosylphosphatidylinositol (GPI)-anchored protein encoded by a member of the CD38 NADase/ADP-ribosyl cyclase gene family, is expressed on the surface of most human circulating neutrophils. This work demonstrates that CD157 is a receptor that induces reorganization of the cytoskeleton and significant changes in cell shape, and that signals mediated by CD157 act through modulation of cytosolic Ca(2+) concentration. These signals are independent of the products of CD157's enzymatic activities (ie, cyclic adenosine diphosphate [ADP]-ribose and ADP-ribose). Indeed, the enzymatic activities of CD157 in circulating neutrophils as well as in dimethyl sulfoxide (DMSO)-differentiated (CD157(+)/CD38(-)) HL-60 cells, are hardly detectable. This work also shows that the receptorial activity relies on cross-talk between CD157 and beta(2) integrin. CD157 localizes in GM1-enriched lipid rafts and, upon activation, it migrates to the uropod, a structure specialized in motility and adhesive functions. Indeed, CD157 is involved in adhesion to extracellular matrix proteins and in chemotaxis induced in vitro by formyl-methionyl-leucyl-phenylalanine (fMLP). These findings were consistent with the results obtained in neutrophils from patients with paroxysmal nocturnal hemoglobinuria (PNH), in which CD157 is deficient. These neutrophils showed constant defects in adhesion and migration. Our data attribute specific and crucial roles to CD157 in the regulation of innate immunity during inflammation.

ADP-ribosyl Cyclase↗
PubMed2004

G6PD is indispensable for erythropoiesis after the embryonic-adult hemoglobin switch.

Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.42) is an essential enzyme for the rapid production of NADPH, as required on exposure to oxidative stress. Mouse embryonic stem (ES) cells can produce all embryonic and fetal/adult cell types. By studying the in vitro differentiation of embryoid bodies produced from G6pdDelta ES cells that are totally unable to produce G6PD protein, we found that these cells are able to differentiate into mesodermal cells, cardiomyocytes, hepatocytes, and primitive erythroid cells. However, we show here that, after the hemoglobin switch has taken place, definitive erythrocytes die by apoptosis. This apoptotic death is delayed by reducing agents and by a caspase inhibitor, but it is prevented only by the restoration of G6PD activity. Thus, G6PD proves indispensable for definitive erythropoiesis.

Animals↗
PubMed2004

In vivo gene marking of rhesus macaque long-term repopulating hematopoietic cells using a VSV-G pseudotyped versus amphotropic oncoretroviral vector.

BACKGROUND: Gene transfer efficiency into primitive hematopoietic cells may be limited by their expression of surface receptors allowing vector entry. Vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope do not need receptors to enter cells, and therefore may provide superior transduction efficiency. METHODS: Using a competitive repopulation model in the rhesus macaque, we examined in vivo gene marking levels of blood cells transduced with two vectors: (i) a VSV-G pseudotyped retrovirus and (ii) a conventional amphotropic retrovirus. The VSV-G vector, containing the human glucose-6-phosphate dehydrogenase (G6PD) gene, was constructed for treatment of severe hemolytic anemia caused by G6PD deficiency. Three myeloablated animals were transplanted with peripheral blood CD34+ cells, half of which were transduced with the VSV-G vector and the other half with the amphotropic vector. RESULTS: In all animals post-transplantation, levels of in vivo marking in circulating granulocytes and mononuclear cells were similar: 1% or less with both vectors. In one animal, the human G6PD enzyme transferred by the VSV-G vector was expressed in erythrocytes, early after transplantation, at a level of 45% of the endogenous rhesus G6PD protein. CONCLUSIONS: In a clinically relevant animal model, we found similar in vivo marking with a VSV-G pseudotyped and a standard amphotropic oncoretroviral vector. Amphotropic receptor expression may not be a limiting factor in transduction efficiency, but VSV-G pseudotypes possess other practical advantages that may make them advantageous for clinical use.

Animals↗
PubMed2003

Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2.

Bone marrow stromal cells, obtained from postnatal bone marrow, contain progenitors able to differentiate into several mesenchymal lineages. Their use in gene and cell therapy requires their in vitro expansion and calls for the investigation of the culture conditions required to preserve these cells as a stem compartment with high differentiative potential during their life span. Here we report that fibroblast growth factor 2 (FGF-2)-supplemented bone marrow stromal cell primary cultures display an early increase in telomere size followed by a gradual decrease, whereas in control cultures telomere length steadily decreases with increasing population doublings. Together with clonogenic culture conditions, FGF-2 supplementation prolongs the life span of bone marrow stromal cells to more than 70 doublings and maintains their differentiation potential until 50 doublings. These results suggest that FGF-2 in vitro selects for the survival of a particular subset of cells enriched in pluripotent mesenchymal precursors and is useful in obtaining a large number of cells with preserved differentiation potential for mesenchymal tissue repair.

Adolescent↗
PubMed2003

Severe telomere shortening in patients with paroxysmal nocturnal hemoglobinuria affects both GPI- and GPI+ hematopoiesis.

A most distinctive feature of paroxysmal nocturnal hemoglobinuria (PNH) is that in each patient glycosylphosphatidylinositol-negative (GPI-) and GPI+ hematopoietic stem cells (HSCs) coexist, and both contribute to hematopoiesis. Telomere size correlates inversely with the cell division history of HSCs. In 10 patients with hemolytic PNH the telomeres in sorted GPI- granulocytes were shorter than in sorted GPI+ granulocytes in 4 cases, comparable in 2 cases, and longer in the remaining 4 cases. Furthermore, the telomeres of both GPI- and GPI+ hematopoietic cells were markedly shortened compared with age-matched controls. The short telomeres in the GPI- cells probably reflect the large number of cell divisions required for the progeny of a single cell to contribute a large proportion of hematopoiesis. The short telomeres of the GPI+ cells indicate that the residual hematopoiesis contributed by these cells is not normal. This epigenetic change is an additional feature shared by PNH and aplastic anemia.

Adolescent↗
PubMed2003

Expression of Plasmodium falciparum G6PD-6PGL in laboratory parasites and in patient isolates in G6PD-deficient and normal Nigerian children.

As the production of NADPH in the pentose phosphate pathway is the main antioxidant defence mechanism available to the Plasmodium falciparum, we have studied the expression of P. falciparum glucose 6-phosphate dehydrogenase-6-phosphogluconolactonase (PfG6PD-6PGL) in G6PD-deficient and normal erythrocyte host cells. Both erythrocytes infected in vitro with a laboratory isolate and erythrocytes from natural human infections were used. Total RNA was prepared from parasites collected from five G6PD-deficient and nine G6PD-normal children in Ibadan, Nigeria, selected after screening 189 rural schoolchildren and 68 clinical malaria patients, and was subjected to Northern blot analysis. The probe was a cDNA fragment of the G6PD domain of the PfG6PD-6PGL gene, with an internal control probe of P. falciparum 18S ribosomal RNA. Quantification was performed using a phosphoimager. Relative to internal control, the abundance of PfG6PD-6PGL mRNA (mean +/- standard deviation) was lower in parasites from G6PD-deficient children (0.29 +/- 0.27) than in G6PD-normal control subjects (0.74 +/- 0.26) (P = 0.014, Mann-Whitney U-test). Although confirmation in a larger study is required, our results suggest a lower relative abundance of PfG6PD-6PGL, and presumably antioxidant activity, in malaria parasites from G6PD-deficient hosts, thus extending the current knowledge of the mechanism of G6PD-deficiency related host protection.

Animals↗
PubMed2002

Maternally transmitted severe glucose 6-phosphate dehydrogenase deficiency is an embryonic lethal.

Mouse chimeras from embryonic stem cells in which the X-linked glucose 6-phosphate dehydrogenase (G6PD) gene had been targeted were crossed with normal females. First-generation (F(1)) G6PD(+/-) heterozygotes born from this cross were essentially normal; analysis of their tissues demonstrated strong selection for cells with the targeted G6PD allele on the inactive X chromosome. When these F(1) G6PD(+/-) females were bred to normal males, only normal G6PD mice were born, because: (i) hemizygous G6PD(-) male embryos died by E10.5 and their development was arrested from E7.5, the time of onset of blood circulation; (ii) heterozygous G6PD(+/-) females showed abnormalities from E8.5, and died by E11.5; and (iii) severe pathological changes were present in the placenta of both G6PD(-) and G6PD(+/-) embryos. Thus, G6PD is not indispensable for early embryo development; however, severe G6PD deficiency in the extraembryonic tissues (consequent on selective inactivation of the normal paternal G6PD allele) impairs the development of the placenta and causes death of the embryo. Most importantly, G6PD is indispensable for survival when the embryo is exposed to oxygen through its blood supply.

Alleles↗