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C Brugnara

Publications and source records attributed to C Brugnara.

At least 73 records · Page 4Linked to original sources
PubMed1997

Anemia in hemodialysis patients: variables affecting this outcome predictor.

Despite the prevalent use of recombinant human erythropoietin (rhEPO), anemia is a frequent finding in hemodialysis patients. The goal of this study was to evaluate the impact of anemia on patient survival and characterize the determinants of hematopoiesis that may be amenable to therapeutic manipulation to enhance rhEPO responsiveness and reduce death risk. Patient characteristics and laboratory data were collected for 21,899 patients receiving hemodialysis three times per week in dialysis centers throughout the United States in 1993. Hemoglobin concentrations (Hb) < or =80 g/L were associated with a twofold increase in the odds of death (odds ratio = 2.01; P = 0.001) when compared with Hb 100 to 110 g/L. No improvement in the odds of death was afforded for Hb >110 g/L. Using multiple linear regression, variables of rhEPO administration (rhEPO dose and percentage of treatments that rhEPO was administered), variables of iron status (serum iron, transferrin saturation, and ferritin), variables of nutritional status (serum albumin and creatinine concentration), and the dose of dialysis (urea reduction ratio) were found to be significantly associated with hemoglobin concentration (P < 0.001). Age, race, and gender were also found to be significantly associated with hemoglobin concentrations (P < 0.001). From this report, the following conclusions may be made. (1) Anemia may be predictive of an increased risk of mortality in some hemodialysis patients. (2) Hemoglobin concentrations > 110 g/L are not associated with further improvements in the odds of death. (3) Laboratory surrogates of iron stores, nutritional status, and the delivered dose of dialysis are predictive of hemoglobin concentration. Whether manipulation of the factors that improve anemia will also enhance the survival of patients on hemodialysis is unknown and should be evaluated by prospective, interventional studies.

Adult↗
PubMed1996

A multiparameter analysis of sickle erythrocytes in patients undergoing hydroxyurea therapy.

During 24 weeks of hydroxyurea treatment, we monitored red blood cell (RBC) parameters in three patients with sickle cell disease, including F-cell and F-reticulocyte profiles, distributions of delay times for intracellular polymerization, sickle erythrocyte adherence to human umbilical vein endothelial cells in a laminar flow chamber, RBC phthalate density profiles, mean corpuscular hemoglobin concentration and cation content, reticulocyte mean corpuscular hemoglobin concentration, 1H-nuclear magnetic resonance transverse relaxation rates of packed RBCs, and plasma membrane lateral and rotational mobilities of band 3 and glycophorins. Hydroxyurea increases the fraction of cells with sufficiently long delay times to escape the microcirculation before polymerization begins. Furthermore, high pretreatment adherence to human umbilical vein endothelial cells of sickle RBCs decreased to normal after only 2 weeks of hydroxyurea treatment, preceding the increase in fetal hemoglobin levels. The lower adhesion of sickle RBCs to endothelium would facilitate escape from the microcirculation before polymerization begins. Hydroxyurea shifted several biochemical and biophysical parameters of sickle erythrocytes toward values observed with hemoglobin SC disease, suggesting that hydroxyurea moderates sickle cell disease toward the milder, but still clinically significant, hemoglobin SC disease. The 50% reduction in sickle crises documented in the Multicenter Study of Hydroxyurea in Sickle Cell Disease is consistent with this degree of erythrocyte improvement.

Adult↗
PubMed1996

The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells.

Clotrimazole (CLT) prevents dehydration of the human HbSS red cell through inhibition of Ca++-dependent (Gardos) K+ channels in vitro (1993. J. Clin Invest. 92:520-526.) and in patients (1996. J. Clin Invest. 97:1227-1234.). Basolateral membrane K+ channels of intestinal crypt epithelial cells also participate in secretagogue-stimulated Cl- secretion. We examined the ability of CLT to block intestinal Cl- secretion by inhibition of K+ transport. Cl- secretion was measured as short-circuit current (Isc) across monolayers of T84 cells. CLT reversibly inhibited Cl- secretory responses to both cAMP- and Ca2+-dependent agonists with IC50 values of approximately 5 microM. Onset of inhibition was more rapid when CLT was applied to the basolateral cell surface. Apical Cl- channel and basolateral NaK2Cl cotransporter activities were unaffected by CLT treatment as assessed by isotopic flux measurement. In contrast, CLT strongly inhibited basolateral 86Rb efflux. These data provide evidence that CLT reversibly inhibits Cl- secretion elicited by cAMP-, cGMP-, or Ca2+-dependent agonists in T84 cells. CLT acts distal to the generation of cAMP and Ca2+ signals, and appears to inhibit basolateral K+ channels directly. CLT and related drugs may serve as novel antidiarrheal agents in humans and animals.

Antifungal Agents↗
PubMed1996

Modulation of erythrocyte potassium chloride cotransport, potassium content, and density by dietary magnesium intake in transgenic SAD mouse.

Prevention of erythrocyte dehydration is a potential therapeutic strategy for sickle cell disease. Increasing erythrocyte magnesium (Mg) could inhibit sickle cell dehydration by increasing chloride (CI) and water content and by inhibiting potassium chloride (K-CI) cotransport. In transgenic SAD 1 and (control) C57BL/6 normal mice, we investigated the effect of 2 weeks of diet with either low Mg (6 +/- 2 mg/kg body weight/d) or high Mg (1,000 +/- 20 mg/kg body weight/ d), in comparison with a diet of standard Mg (400 +/- 20 mg/ kg body weight/d). The high-Mg diet increased SAD 1 erythrocyte Mg and K contents and reduced K-CI cotransport activity, mean corpuscular hemoglobin concentration (MCHC), cell density, and reticulocyte count. SAD 1 mice treated with low-Mg diet showed a significant reduction in erythrocyte Mg and K contents and increases in K-CI cotransport, MCHC, cell density, and reticulocyte counts. In SAD 1 mice, hematocrit (Hct) and hemoglobin (Hb) decreased significantly with low Mg diet and increased significantly with high-Mg diet. The C57BL/6 controls showed significant changes only in erythrocyte Mg and K content, and K-CI cotransport activities, similar to those observed in SAD 1 mice. Thus, in the SAD 1 mouse, changes in dietary Mg modulate K-CI cotransport, modify erythrocyte dehydration, and ultimately affect Hb levels.

Anemia, Sickle Cell↗
PubMed1996

Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton.

The red blood cell (RBC) membrane protein AE1 provides high affinity binding sites for the membrane skeleton, a structure critical to RBC integrity. AE1 biosynthesis is postulated to be required for terminal erythropoiesis and membrane skeleton assembly. We used targeted mutagenesis to assess AE1 function in vivo. RBCs lacking AE1 spontaneously shed membrane vesicles and tubules, leading to severe spherocytosis and hemolysis, but the levels of the major skeleton components, the synthesis of spectrin in mutant erythroblasts, and skeletal architecture are normal or nearly normal. The results indicate that AE1 does not regulate RBC membrane skeleton assembly in vivo but is essential for membrane stability. We postulate that stabilization is achieved through AE1-lipid interactions and that loss of these interactions is a key pathogenic event in hereditary spherocytosis.

Animals↗
PubMed1996

Therapy with oral clotrimazole induces inhibition of the Gardos channel and reduction of erythrocyte dehydration in patients with sickle cell disease.

Pathologic water loss from sickle erythrocytes concentrates the abnormal hemoglobin and promotes sickling. The Ca2+-activated K+ channel (Gardos channel) contributes to this deleterious dehydration in vitro, and blockade of K+ and water loss via this channel could be a potential therapy in vivo. We treated five subjects who have sickle cell anemia with oral clotrimazole, a specific Gardos channel inhibitor. Patients were started on a dose of 10 mg clotrimazole/kg/d for one week. Protocol design allowed the daily dose to be escalated by 10 mg/kg each week until significant changes in erythrocyte density and K+ transport were achieved. Blood was sampled three times a week for hematological and chemical assays, erythrocyte density, cation content, and K+ transport. At dosages of 20 mg clotrimazole/kg/d, all subjects showed Gardos channel inhibition, reduced erythrocyte dehydration, increased cell K+ content, and somewhat increased hemoglobin levels. Adverse effects were limited to mild/moderate dysuria in all subjects, and a reversible increase in plasma alanine transaminase and aspartic transaminase levels in two subjects treated with 30 mg clotrimazole/kg/d. This is the first in vivo evidence that the Gardos channel causes dehydration of sickle erythrocytes, and that its pharmacologic inhibition provides a realistic antisickling strategy.

Administration, Oral↗
PubMed1996

Combination therapy of erythropoietin, hydroxyurea, and clotrimazole in a beta thalassemic mouse: a model for human therapy.

beta thalassemia (beta thal) in DBA/2J mice is a consequence of the spontaneous and complete deletion of the beta major globin gene. Homozygous beta thal mice have clinical and biological features similar to those observed in human beta thal intermedia. Erythrocytes in human beta thal are characterized by a relative cell dehydration and reduced K+ content. The role of this erythrocyte dehydration in the reduced erythrocyte survival, which typifies the disease, has not previously been evaluated. We examined for 1 month the effects on the anemia and the erythrocyte characteristics of beta thal mice of daily treatment with either clotrimazole (CLT), an inhibitor of red blood cell (RBC) dehydration via the Gardos channel, or human recombinant erythropoietin (r-HuEPO), or hydroxyurea (HU). The use of either r-HuEPO or HU induced a significant increase in hemoglobin (Hb), hematocrit (Hct), erythrocyte K+ and a decrease in percent reticulocytes, suggesting improved erythrocyte survival. CLT alone decreased only mean corpuscular hemoglobin concentration (MCHC) and cell density and increased cell K+. Thus, though the Gardos channel plays a major role in cell dehydration of murine beta thal erythrocyte survival. Combination therapy with r-HuEPO plus HU produced no incremental benefit beyond those of single drug therapy. However, addition of CLT to r-HuEPO, to HU, or to combined r-HuEPO plus HU led to statistically significant increase in Hb, Hct, and erythrocyte K+ compared with any of the regimens without CLT. These results suggest that CLT not only inhibits erythrocyte dehydration, but also potentiates the erythropoietic and cellular survival responses to r-HuEPO and HU.

Animals↗
PubMed1996

Clotrimazole and efaroxan inhibit red cell Gardos channel independently of imidazoline I1 and I2 binding sites.

In the present report, we investigated the potential involvement of imidazoline I1 and I2 binding sites in the inhibition of the Ca(2+)-activated K+ channel (Gardos channel) by clotrimazole in human red cells. Ca(2+)-activated 86Rb influx was inhibited by clotrimazole and efaroxan but not by the imidazoline binding site ligands clonidine, moxonidine, cirazoline and idazoxan (100 microM). Binding studies with [3H]idazoxan and [3H]p-aminoclonidine did not reveal the expression of I1 and I2 binding sites in erythrocytes. These data indicate that the effects of clotrimazole and efaroxan on the erythrocyte Ca(2+)-activated K+ channel may be mediated by a 'non-I1/non-I2' binding site.

Adrenergic alpha-Antagonists↗
PubMed1996

Resistance to osmotic lysis in BXD-31 mouse erythrocytes: association with upregulated K-Cl cotransport.

The decreased osmotic fragility and reduced K+ content of BXD-31 mouse erythrocytes arise from variation at a single genetic locus. We compared ion transport in erythrocytes from BXD-31 mice and the parental strain, DBA/2J. The strains had similar rates for Na-K pump, Na/H exchange, Na-K-2Cl cotransport, Ca2+ activated K+ channel, or AE1-mediated SO4 transport. In contrast, K-Cl cotransport was twice as active in BXD-31 as in DBA/2J cells. Cl- dependent K+ efflux from BXD-31 cells displayed steep activation by acid pH (with maximal transport occurring at pH 6.75), whereas DBA/2J erythrocytes displayed a far less dramatic response to pH. Both strains displayed regulatory volume decrease in response to cell swelling. However, a 62% greater loss of cell K+ via K-Cl cotransport was observed in the BXD-31 strain. Furthermore the decreased osmotic fragility of BXD-31 red blood cells was normalized by treatment with nystatin to achieve normal cell K+ and water content. Thus upregulated K-Cl cotransport induces cell dehydration and K+ deficit in BXD-31 erythrocytes and causes their characteristic resistance to osmotic lysis.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Mutations of conserved arginines in the membrane domain of erythroid band 3 lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis.

To elucidate the molecular basis of band 3 deficiency in a recently defined subset of patients with autosomal dominant hereditary spherocytosis (HS), we screened band 3 cDNA for single-strand conformation polymorphism (SSCP). In 5 of 17 (29%) unrelated HS subjects with band 3 deficiency, we detected substitutions R760W, R760Q, R808C, and R870W that were all coinherited with the HS phenotype. The involved arginines are highly conserved throughout evolution. To examine whether or not the product of the mutant allele is inserted into the membrane, we studied one HS subject who was doubly heterozygous for the R760Q mutation and the K56E (band 3sMEMPHIS) polymorphism that results in altered electrophoretic mobility of the band 3 Memphis proteolytic fragments. We detected only the band 3MEMPHIS in the erythrocyte membrane indicating that the protein product of the mutant, R760Q, band 3 allele is absent from the red blood cell membrane. These findings suggest that the R760Q substitution, and probably the other arginine subsitutions, produce band 3 deficiency either by precluding incorporation of the mutant protein into the red blood cell membrane or by leading to loss of mutant protein from differentiating erythroid precursors.

Alleles↗
PubMed1995

Ca(2+)-activated K+ channels of human and rabbit erythrocytes display distinctive patterns of inhibition by venom peptide toxins.

Despite recent progress in the molecular characterization of high-conductance Ca(2+)-activated K+ (maxi-K) channels, the molecular identities of intermediate conductance Ca(2+)-activated K+ channels, including that of mature erythrocytes, remains unknown. We have used various peptide toxins to characterize the intermediate conductance Ca(2+)-activated K+ channels (Gardos pathway) of human and rabbit red cells. With studies on K+ transport and on binding of 125I-charybdotoxin (ChTX) and 125I-kaliotoxin (KTX) binding in red cells, we provide evidence for the distinct nature of the red cell Gardos channel among described Ca(2+)-activated K+ channels based on (i) the characteristic inhibition and binding patterns produced by ChTX analogues, iberiotoxin (IbTX) and IbTX-like ChTX mutants, and KTX (1-37 and 1-38 variants); (ii) the presence of some properties heretofore attributed only to voltage-gated channels, including inhibition of K transport by margatoxin (MgTX) and by stichodactyla toxin (StK); (iii) and the ability of scyllatoxin (ScyTX) and apamin to displace bound 125I-charybdotoxin, a novel property for K+ channels. These unusual pharmacological characteristics suggest a unique structure for the red cell Gardos channel.

Animals↗
PubMed1995

Clotrimazole inhibits cell proliferation in vitro and in vivo.

Cell proliferation is critically dependent on the regulated movement of ions across various cellular compartments. The antimycotic drug clotrimazole (CLT) has been shown to inhibit movement of Ca2+ and K+ across the plasma membrane. Our results show that CLT inhibits the rate of cell proliferation of normal and cancer cell lines in a reversible and dose-dependent manner in vitro. Moreover, CLT depletes the intracellular Ca2+ stores and prevents the rise in cytosolic Ca2+ that normally follows mitogenic stimulation. In mice with severe combined immunodeficiency disease (SCID) and inoculated intravenously with MM-RU human melanoma cells, daily subcutaneous injections of CLT induced a significant reduction in the number of lung metastases. Modulation of early ionic mitogenic signals and potent inhibition of cell proliferation both in vitro and in vivo are new and potentially useful clinical effects of CLT.

3T3 Cells↗
PubMed1995

A high-performance liquid chromatographic assay for the determination of itraconazole concentration using solid-phase extraction and small sample volume.

Itraconazole has been shown to be highly effective against a broad spectrum of fungi, particularly aspergillus. In order to achieve the desired pharmacological effect, the itraconazole plasma concentration should be > 250 ng/ml. We developed a high-performance liquid chromatography assay for the rapid determination of plasma itraconazole concentration using solid-phase extraction and small sample volume. The assay possessed linearity up to 5,000 ng/ml, sensitivity of 50 ng/ml, average recovery of 100.8%, and run-to-run precision (n = 10) for concentrations of 100, 200, and 500 ng/ml of 12, 6.6, and 5.8%, respectively. Furthermore, the assay proved to be free of interference from 48 commonly prescribed and some over-the-counter medications. We conclude that the method described here is ideally suited for the therapeutic monitoring of itraconazole.

Acetonitriles↗
PubMed1995

Erythrocyte dehydration in pathophysiology and treatment of sickle cell disease.

A prominent feature of sickle cell disease is the presence of cells with markedly increased sickle cell hemoglobin concentration, as a consequence of the loss of potassium, chloride, and water from the erythrocyte. Because of the extreme dependency of the kinetic of polymerization on sickle cell hemoglobin concentration, these dehydrated erythrocytes have an increased tendency to polymerize and sickle. Thus blockade of the loss of potassium from the erythrocyte should prevent the increase in sickle cell hemoglobin concentration and reduce sickling. The availability of this potential therapeutic option is based on a detailed knowledge of the mechanisms leading to cell dehydration. Two ion transport pathways, the K-Cl cotransport and the Ca(2+)-activated K+ channel, play a prominent role in the dehydration of sickle erythrocytes. Possible therapeutic strategies include inhibition of K-Cl cotransport by increasing erythrocyte Mg2+ content and inhibition of the Ca(2+)-activated K channel by oral administration of clotrimazole.

Anemia, Sickle Cell↗
PubMed1995

Cation transport in mouse erythrocytes: role of K(+)-Cl- cotransport in regulatory volume decrease.

We investigated cation transport and cell volume regulation in erythrocytes of CD1 and C57/B6 mice. Swelling of cells from either strain stimulated K+ efflux that was insensitive to ouabain, bumetanide, and clotrimazole. Seventy-five percent of swelling-induced K+ efflux was Cl- dependent (inhibited by sulfamate or methanesulfonate, partially by NO3-, but not by SCN-) and was inhibited by okadaic acid (OA; 50% inhibitory concentration = 18 +/- 6 nM in CD1 and 10 +/- 4 nM in C57/B6). In both strains, K+ efflux into isotonic medium was stimulated by staurosporine or by N-ethylmaleimide, and the latter was partially blocked by pretreatment of cells with OA. When cells of either strain were incubated in hypotonic medium or preswollen isosmotically with nystatin, OA-sensitive regulatory volume decrease (RVD) and K+ loss were observed. RVD produced by hypotonic swelling was prevented by Cl- replacement with sulfamate or methanesulfonate. These properties suggest the presence in outbred and inbred mouse erythrocytes of RVD mediated by K(+)-Cl- cotransport.

Animals↗
PubMed1995

Sulfhydryl oxidation and activation of red cell K(+)-Cl- cotransport in the transgenic SAD mouse.

The SAD mouse is characterized by the expression of human SAD hemoglobin (Hb), a super S Hb with a higher tendency to polymerize than HbS due to the presence of two additional mutations, Antilles beta 23Ile and D Punjab beta 121Glu. Monovalent cation transport was studied in erythrocytes from SAD-1 (Hb SAD = 19%) and beta-thal/SAD-1 (Hb SAD = 26%) mice. Erythrocytes containing Hb SAD exhibited dehydration, increased maximal rate of Na(+)-K+ pump, unchanged Rb+ flux via the Gardos channel, and increased K(+)-Cl- cotransport. K(+)-Cl- cotransport was defined as Cl(-)-dependent (substitution with sulfamate or methanesulfonate) okadaic acid-sensitive K+ efflux. Volume regulatory decrease via K(+)-Cl- cotransport was also increased in swollen SAD erythrocytes compared with controls. K(+)-Cl- cotransport was stimulated by staurosporine in all mouse strains, but the extent of stimulation was reduced in beta-thal/SAD-1 mice. Treatment with dithiothreitol reduced K(+)-Cl- cotransport activity in SAD-1 and beta-thal/SAD-1 mice to levels similar to that of control strains, indicating that reversible sulfhydryl oxidation contributes to the activated state of K(+)-Cl- cotransport in mouse erythrocytes that express transgenic human Hb SAD.

Animals↗