Biomedical subjects

C Brugnara

Publications and source records attributed to C Brugnara.

At least 19 recordsLinked to original sources

PubMed2006

Reticulocyte hemoglobin equivalent (Ret He) and assessment of iron-deficient states.

Direct measurement of the reticulocyte hemoglobin content provides useful information for the diagnosis and treatment of iron-deficient states. We have examined direct measurements of reticulocyte and red cell hemoglobin content on the Sysmex XE 2100 (Ret He and RBC He respectively) and the Bayer ADVIA 2120 (CHr and CH respectively) analyzers. Good agreement was found between Ret He and CHr (Y = 1.04X - 1.06; r2= 0.88) and between the RBC He and CH parameters (Y = 0.93X + 1; r2= 0.84 n = 200) in pediatric patients and in normal adults (Ret He and CHr; Y = 1.06X - 0.43; r2= 0.83; n = 126; RBC He and CH; Y = 0.94X + 1; r2= 0.87; n = 126). In 1500 blood samples from patients on chronic dialysis, Ret He was compared with traditional parameters for iron deficiency (serum iron <40 microg/dl, Tsat <20%, ferritin <100 ng/ml, hemoglobin <11 g/dl) for identifying iron-deficient states. Receiver operator characteristic (ROC) curve analysis revealed values of the area under the curve for Ret He of 0.913 (P < 0.0001). With a Ret He cutoff level of 27.2 pg, iron deficiency could be diagnosed with a sensitivity of 93.3%, and a specificity of 83.2%. Ret He is a reliable marker of cellular hemoglobin content and can be used to identify the presence of iron-deficient states.

Aged↗

PubMed2001

Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.

Erythropoietin (Epo) controls red cell production in the basal state and during stress. Epo binding to its receptor, EpoR, on erythroid progenitors leads to rapid activation of the transcription factor Stat5. Previously, fetal anemia and increased apoptosis of fetal liver erythroid progenitors were found in Stat5a(-/-)5b(-/-) mice. However, the role of Stat5 in adult erythropoiesis was not clear. The present study shows that some adult Stat5a(-/-)5b(-/-) mice have a near-normal hematocrit but are deficient in generating high erythropoietic rates in response to stress. Further, many adult Stat5a(-/-)5b(-/-) mice have persistent anemia despite a marked compensatory expansion in their erythropoietic tissue. Analysis of erythroblast maturation in Stat5a(-/-)5b(-/-) hematopoietic tissue shows a dramatic increase in early erythroblast numbers, but these fail to progress in differentiation. Decreased expression of bcl-x(L) and increased apoptosis in Stat5a(-/-)5b(-/-) early erythroblasts correlate with the degree of anemia. Hence, Stat5 controls a rate-determining step regulating early erythroblast survival.

Anemia↗

PubMed2001

A dominant negative mutant of the KCC1 K-Cl cotransporter: both N- and C-terminal cytoplasmic domains are required for K-Cl cotransport activity.

K-Cl cotransport regulates cell volume and chloride equilibrium potential. Inhibition of erythroid K-Cl cotransport has emerged as an important adjunct strategy for the treatment of sickle cell anemia. However, structure-function relationships among the polypeptide products of the four K-Cl cotransporter (KCC) genes are little understood. We have investigated the importance of the N- and C-terminal cytoplasmic domains of mouse KCC1 to its K-Cl cotransport function expressed in Xenopus oocytes. Truncation of as few as eight C-terminal amino acids (aa) abolished function despite continued polypeptide accumulation and surface expression. These C-terminal loss-of-function mutants lacked a dominant negative phenotype. Truncation of the N-terminal 46 aa diminished function. Removal of 89 or 117 aa (Delta(N)117) abolished function despite continued polypeptide accumulation and surface expression and exhibited dominant negative phenotypes that required the presence of the C-terminal cytoplasmic domain. The dominant negative loss-of-function mutant Delta(N)117 was co-immunoprecipitated with wild type KCC1 polypeptide, and its co-expression did not reduce wild type KCC1 at the oocyte surface. Delta(N)117 also exhibited dominant negative inhibition of human KCC1 and KCC3 and, with lower potency, mouse KCC4 and rat KCC2.

Animals↗

PubMed2001

Treatment with NS3623, a novel Cl-conductance blocker, ameliorates erythrocyte dehydration in transgenic SAD mice: a possible new therapeutic approach for sickle cell disease.

The dehydration of sickle red blood cells (RBCs) through the Ca-activated K channel depends on the parallel movement of Cl ions. To study whether Cl-conductance block might prevent dehydration of sickle RBCs, a novel Cl-conductance inhibitor (NS3623) was characterized in vitro using RBCs from healthy donors and sickle cell patients and in vivo using normal mice and a transgenic mouse model of sickle cell disease (SAD mice). In vitro, NS3623 reversibly blocked human RBC Cl-conductance (g(Cl)) with an IC(50) value of 210 nmol/L and a maximal block of 95%. In vivo, NS3623 inhibited RBC g(Cl) after oral administration to normal mice (ED(50) = 25 mg/kg). Although g(Cl), at a single dose of 100 mg/kg, was still 70% inhibited 5 hours after dosing, the inhibition disappeared after 24 hours. Repeated administration of 100 mg/kg twice a day for 10 days caused no adverse effects; therefore, this regimen was chosen as the highest dosing for the SAD mice. SAD mice were treated for 3 weeks with 2 daily administrations of 10, 35, and 100 mg/kg NS3623, respectively. The hematocrit increased, and the mean corpuscular hemoglobin concentration decreased in all groups with a concomitant increase in the intracellular cation content. A loss of the densest red cell population was observed in conjunction with a shift from a high proportion of sickled to well-hydrated discoid erythrocytes, with some echinocytes present at the highest dosage. These data indicate feasibility for the potential use of Cl-conductance blockers to treat human sickle cell disease.

Anemia, Sickle Cell↗

PubMed2001

Erythrocyte-active agents and treatment of sickle cell disease.

The sickle hemoglobin (HbS)-containing erythrocyte and its membrane represent a logical target for sickle cell disease therapy. Several antisickling agents which interfere with HbS polymerization have been studied over the last 30 years, but none has overcome the challenge of delivering high concentrations inside the sickle red blood cell without toxicity. The sickle erythrocyte membrane has also been targeted for therapeutic developments. Prevention of sickle cell dehydration by use of specific blockers of ion transport pathways mediating potassium loss from the sickle erythrocyte has been shown to be a feasible strategy in vitro, in vivo in transgenic sickle mice, and in patients. Other approaches have focused on improving the hemorheology of sickle erythrocytes and reducing their abnormal adhesion to endothelial cells. These potential treatments could be used alone or in combination with other approved therapies, such as hydroxyurea.

Anemia, Sickle Cell↗

PubMed2001

Flow-cytometric analysis of erythrocytes and reticulocytes in congenital dyserythropoietic anaemia type II (CDA II): value in differential diagnosis with hereditary spherocytosis.

Congenital dyserythropoietic anaemia type II (CDA II) is the most common congenital dyserythropoietic anaemia. CDA II is frequently misdiagnosed as Hereditary Spherocytosis (HS) due to the presence of mild chronic haemolytic anaemia with splenomegaly, increased osmotic fragility, and presence of microspherocytes. Accurate diagnosis of CDA II is important to prevent severe iron overload. Erythrocyte and reticulocyte indices were assessed in 10 patients from six families with CDA II, 18 patients from eight families with HS, and 50 normal controls. Characteristic increases in distribution width were present in CDA II for cell volume (RDW, anisocytosis) and in HS for cell haemoglobin concentration (HDW, anisochromia), resulting in an RDW/HDW ratio which was significantly greater in CDA than HS (P < 0.0002). A cut-off value for RDW/HDW of 5.34 resulted in 89% sensitivity and 70% specificity in distinguishing CDA II from HS. Distribution width for cell haemoglobin content of reticulocytes (CHDWr) was characteristically increased in CDA II, resulting in a CHDW/CHDWr ratio significantly lower in CDA II than HS (P < 0.0002). A cut-off value of 0.98 provided 89% sensitivity and 80% specificity in distinguishing CDA II from HS. These differences in distribution widths of flow-cytometric parameters of reticulocytes and mature erythrocytes reflect the different pathogeneses of the two diseases and are helpful for the differential diagnosis of these two conditions.

Adolescent↗

PubMed2001

K-Cl cotransport modulation by intracellular Mg in erythrocytes from mice bred for low and high Mg levels.

Mg is an important determinant of erythrocyte cation transport system(s) activity. We investigated cation transport in erythrocytes from mice bred for high (MGH) and low (MGL) Mg levels in erythrocytes and plasma. We found that K-Cl cotransport activity was higher in MGL than in MGH erythrocytes, and this could explain their higher mean corpuscular hemoglobin concentration, median density, and reduced cell K content. Although mouse KCC1 protein abundance was comparable in MGL and MGH erythrocytes, activities of Src family tyrosine kinases were higher in MGH than in MGL erythrocytes. In contrast, protein phosphatase (PP) isoform 1 alpha (PP1 alpha) enzymatic activity, which has been suggested to play a positive regulatory role in K-Cl cotransport, was lower in MGH than in MGL erythrocytes. Additionally, we found that the Src family kinase c-Fgr tyrosine phosphorylates PP1 alpha in vitro. These findings suggest that in vivo downregulation of K-Cl cotransport activity by Mg is mediated by enhanced Src family kinase activity, leading to inhibition of the K-Cl cotransport stimulator PP1.

Animals↗

PubMed2001

Novel therapies for prevention of erythrocyte dehydration in sickle cell anemia.

Sickle cell anemia is a genetic disorder characterized by mutant hemoglobin (Hb) polymerization and resultant cell deformation (sickling) under conditions of reduced oxygen tension. The disease is caused by mutation of wild-type Glu to Val in position 6 of the beta-chain of hemoglobin, yielding hemoglobin S (HbS). The sickling process is markedly accelerated when the intracellular concentration of HbS is increased. A variable fraction of dehydrated erythrocytes is seen in the majority of patients, and these cells are believed to play an important role in the pathophysiology of the vasoocclusive events of sickle cell disease. Therapy of sickle cell disease is extremely limited in range and efficacy. Many patients still receive treatment only for symptomatic relief of sickle crises, painful episodes due to vasoocclusion by sickled cells. The last 15 years, however, have seen the identification of the principal transport pathways that mediate sickle erythrocyte dehydration, and the last 6 years have witnessed promising clinical tests of specific inhibitors of these pathways, with the intent of reducing cell sickling via inhibition of red cell dehydration. This review discusses the pathophysiology of sickle cell dehydration and explores current and future treatment options for in vivo prevention of sickle cell dehydration.

Journal Article↗

PubMed2000

Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: role of intracellular Mg++.

An increase in the activity of membrane-associated protein phosphatase type 1 (mb-PP1) is associated with stimulation of erythrocyte K-Cl cotransport (KCC). We have recently proposed that membrane-associated protein phosphatase type 2A (mb-PP2A) is also involved in KCC regulation by cell swelling (Bize et al., 1999. Am. J. Physiol. 277:C899-C912). We used two protein phosphatase inhibitors, okadaic acid (OA) and calyculin A (CalA), and two KCC activating treatments, n-ethylmaleimide (NEM) and Mg(i)++-depletion, and determined KCC transport activity and mb-PP1 and mb-PP2A activities. OA, an inhibitor of erythrocyte mb-PP2A, partially prevents stimulation of KCC activity by NEM but not by Mg(i)++-depletion. CalA, an inhibitor of both mb-PP1 and mb-PP2A prevents stimulation of KCC activity by both treatments. NEM and Mg(i)++-depletion inhibit mb-PP1 activity, suggesting that activation of KCC can take place in the absence of mb-PP1 activation. Mb-PP2A activity is stimulated in NEM-treated cells but not in Mg(i)++-depleted cells. In NEM-treated cells, Mg(i)++-depletion inhibits both KCC and mb-PP2A. In Mg(i)++-depleted cells, NEM does not stimulate KCC or mb-PP2A. The strong correlation between KCC stimulation and mb-PP2A stimulation provides further support to the idea that mb-PP2A plays an important role in KCC regulation. Our results are consistent with the hypothesis that KCC regulation involves at least two distinguishable phosphorylation sites.

Carrier Proteins↗

PubMed2000

Erythropoietin, iron, and erythropoiesis.

Recent knowledge gained regarding the relationship between erythropoietin, iron, and erythropoiesis in patients with blood loss anemia, with or without recombinant human erythropoietin therapy, has implications for patient management. Under conditions of significant blood loss, erythropoietin therapy, or both, iron-restricted erythropoiesis is evident, even in the presence of storage iron and iron oral supplementation. Intravenous iron therapy in renal dialysis patients undergoing erythropoietin therapy can produce hematologic responses with serum ferritin levels up to 400 microg/L, indicating that traditional biochemical markers of storage iron in patients with anemia caused by chronic disease are unhelpful in the assessment of iron status. Newer measurements of erythrocyte and reticulocyte indices using automated counters show promise in the evaluation of iron-restricted erythropoiesis. Assays for serum erythropoietin and the transferrin receptor are valuable tools for clinical research, but their roles in routine clinical practice remain undefined. The availability of safer intravenous iron preparations allows for carefully controlled studies of their value in patients undergoing erythropoietin therapy or experiencing blood loss, or both.

Animals↗

PubMed2000

Structure and genetic polymorphism of the mouse KCC1 gene.

The KCC1 K-Cl cotransporter is a major regulator of erythroid and non-erythroid cell volume, and the KCC1 gene is a candidate modifier gene for sickle cell disease and other hemoglobinopathies. We have cloned and sequenced the mouse KCC1 (mKCC1) gene, defined its intron-exon junctions, and analyzed (AC)/(TG) intragenic polymorphisms. A highly polymorphic (AC) repeat of mKCC1 intron 1 was characterized in musculus strains, and used to prove lack of linkage between the mKCC1 gene and the rol (resistant to osmotic lysis) locus. The intron 1 (AC) repeat in CAST/Ei and SPRET/Ei was not only more divergent in length but also underwent additional sequence variation. A dimorphic (TG) repeat in intron 2 distinguished CAST/Ei from other strains, and an intron 17 B1 Alu-like SINE present in all musculus strains was found to be absent from intron 17 in SPRET/Ei. These and additional described strain-specific polymorphisms will be useful mapping and genetic tools in the study of mouse models of sickle cell disease.

Anemia, Sickle Cell↗

PubMed2000

Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter.

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMTi. A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.

Amino Acid Sequence↗

PubMed2000

Maximum urine concentrating ability in children with Hb SC disease: effects of hydroxyurea.

Studies in adults with Hb SC disease suggested that hydroxyurea reduced hemolysis and increased red cell hydration. Because increased hydration should diminish the polymerization tendency of Hb S we hypothesized that hydroxyurea might repair the urine concentration defect of HbSC disease. Eight Hb SC disease patients, aged 10 to 17 years, were given hydroxyurea daily. Maximal urine concentrating ability following overnight fasting and after subcutaneous arginine vasopressin (dDAVP), blood counts, and cell volumes were observed for 12-15 months. All patients had impaired urine concentrating ability prior to hydroxyurea treatment and failed to increase their ability to concentrate urine following treatment (maximum urine concentration after an overnight fast and dDAVP, 520-530 mOsm). Mean corpuscular volume (MCV) and reticulocyte MCV increased after administration of hydroxyurea, and the reticulocyte count and ratio of red cell hemoglobin to reticulocyte hemoglobin fell but there was little change in PCV. Hb F increased substantially in 2 patients but showed little change in the remaining patients. There was no evidence that hydroxyurea was associated with increased urine concentrating ability in children with Hb SC disease. These results may reflect irreversible renal medullary damage prior to beginning treatment or insufficient intensity or duration of treatment.

Adolescent↗

PubMed2000

E2F4 is essential for normal erythrocyte maturation and neonatal viability.

The retinoblastoma protein (pRB) plays a key role in the control of normal development and proliferation through the regulation of the E2F transcription factors. We generated a mutant mouse model to assess the in vivo role of the predominant E2F family member, E2F4. Remarkably, loss of E2F4 had no detectable effect on either cell cycle arrest or proliferation. However, E2F4 was essential for normal development. E2f4-/- mice died of an increased susceptibility to opportunistic infections that appeared to result from craniofacial defects. They also displayed a variety of erythroid abnormalities that arose from a cell autonomous defect in late stage maturation. This suggests that E2F4 makes a major contribution to the control of erythrocyte development by the pRB tumor suppressor.

Animals↗

PubMed2000

Oral magnesium pidolate: effects of long-term administration in patients with sickle cell disease.

Prevention of erythrocyte dehydration by specific blockade of the transport pathways promoting loss of potassium (K) is a potential therapeutic strategy for sickle cell (SS) disease. Dietary magnesium (Mg) pidolate supplementation over a 4-week period has been shown to inhibit K-Cl co-transport and reduce dehydration. We report here the results in 17 of 20 patients with SS disease treated in an open-label unblinded study of the effects of long-term (6 months) oral Mg pidolate administration (540 mg Mg/d). A significant decrease (P < 0.0025) was observed with Mg therapy in the distribution widths for red cell mean cell haemoglobin concentration (MCHC) (haemoglobin distribution width; HDW), reticulocyte mean cell volume (red cell distribution width of reticulocytes; RDWr) and MCHC (reticulocyte HDW; HDWr), activity of red cell K-Cl co-transport, Na/Mg exchanger and Ca2+-activated (Gardos) K+ channel, whereas red cell K and Mg contents were significantly increased. Hb levels and absolute reticulocyte counts did not change with Mg therapy. Two patients did not complete the trial because of diarrhoea and one did not complete the trial for unrelated reasons. Although the median number of painful days in a 6-month period decreased from 15 (range 0-60) in the year before the trial to 1 (range 0-18; P < 0.0005) during the period of Mg therapy, no firm conclusion on therapeutic efficacy could be drawn from this unblinded open-label trial.

Administration, Oral↗

PubMed2000

Hemolytic anemia induced by ribavirin therapy in patients with chronic hepatitis C virus infection: role of membrane oxidative damage.

The antiviral drug ribavirin (RBV) is widely used in combination with interferon (IFN) in the treatment of chronic hepatitis C virus (HCV) infection. A major side effect of RBV is a reversible hemolytic anemia. We have evaluated the in vitro effects of RBV on erythrocyte adenosine triphosphate (ATP) content and on hexosemonophosphate shunt (HMS). The ATP levels were significantly decreased in the presence of RBV and the HMS was increased, suggesting the presence of red cell susceptibility to oxidation. In vivo, we have studied the hematologic effects of treatment with RBV alone or in combination with IFN in 11 patients with chronic hepatitis C: 6 were treated with RBV (1,000-1,200 mg/d) and 5 were treated with a combination of RBV and IFN (5 million U thrice weekly). Patients were studied at semi-monthly intervals from 0 to day 60 of therapy. Both treatments were associated with a significant reduction in hemoglobin levels (steady state level at day 45) and a marked increase in absolute reticulocyte counts. Erythrocyte Na-K pump activity was significantly diminished, whereas K-Cl cotransport and its dithiotreitol-sensitive fraction, malondialdehyde and methemoglobin levels were significantly increased. RBV-treated patients showed an increase in aggregated band 3, which was associated with a significantly increased binding of autologous antibodies and complement C3 fragments indicating an erithrophagocytic removal by reticuloendothelial system.

Adenosine Triphosphate↗

PubMed2000

Reticulocyte parameters as potential discriminators of recombinant human erythropoietin abuse in elite athletes.

This study investigated using reticulocyte (retic) parameters as indirect markers of human recombinant erythropoietin (r-HuEPO) abuse in elite athletes. Absolute reticulocyte count (# retic), the per cell haemoglobin content of reticulocytes (CHr), reticulocyte haemoglobin mass per litre of blood (RetHb) and red blood cell:reticulocyte haemoglobin (RBCHb:RetHb) ratio were assessed using flow cytometry. Venous blood was drawn from 155 elite athletes from six sports during regular training to establish reference ranges (95% confidence interval) for these parameters. The reference ranges were compared with those of a non-athletic population (n = 23), four groups of athletes (n = 24) before and after exposure to simulated altitudes (2,500-3,000 m for 11-23 nights), two groups of elite cyclists (n = 13) before and after four weeks of training at natural altitude (1,780 and 2,690 m), and with those of non-athletic subjects from a separate study (n =24) before and 1-2 days after they were injected with 1,200 U x kg(-1) r-HuEPO over a 9-10 day period. Generally the changes induced by r-HuEPO injection exceeded by approximately 100% the magnitude of the changes associated with natural altitude exposure. Simulated altitude exposure did not significantly alter the reticulocyte parameters. From the sample of 155 non-users and 24 r-HuEPO users, the population mean and variance, as well as the 95% confidence limits for the population mean and population variance, were estimated. Relative to arbitrarily chosen cut-off levels, the confidence limits for the rate of true positives and rate of true negatives were also calculated. Based on the lowest rate of false positives and highest rate of true positives, the best discriminator between r-HuEPO users and non-users was # retic, marginally superior to RBCHb: RetHb ratio and RetHb. At a cut-off for # retic of 221 x 10(9)x L(-1) we could be 95% sure that we would find no more than 7 false positives in every 100,000 tests. We would expect to pick up 51.8% of users, and could be 95% sure of picking up at least 38% of current or recent users. This result highlights the potential power of retic parameters for detecting r-HuEPO abuse among athletes. However, the efficacy of these cut-offs for detecting r-HuEPO abuse is unknown if an athlete is a chronic user or stops using r-HuEPO several weeks before being tested.

Bicycling↗

PubMed2000

Plasminogen activator inhibitor-1: defining characteristics in the cerebrospinal fluid of newborns.

We measured plasminogen activator inhibitor-1 levels in the cerebrospinal fluid and plasma of newborns with and without posthemorrhagic hydrocephalus. We found that plasminogen activator inhibitor-1 levels in the cerebrospinal fluid of healthy newborns are <10 mg/mL but are greatly elevated in patients who have posthemorrhagic hydrocephalus and correlate directly with cerebrospinal fluid D-dimer and protein levels.

Enzyme-Linked Immunosorbent Assay↗