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At least 19 recordsLinked to original sources

PubMed2006

Pitting of malaria parasites and spherocyte formation.

BACKGROUND: A high prevalence of spherocytes was detected in blood smears of children enrolled in a case control study conducted in the malaria holoendemic Lake Victoria basin. It was speculated that the spherocytes reflect intraerythrocytic removal of malarial parasites with a concurrent removal of RBC membrane through a process analogous to pitting of intraerythrocytic inclusion bodies. Pitting and re-circulation of RBCs devoid of malaria parasites could be a host mechanism for parasite clearance while minimizing the anaemia that would occur were the entire parasitized RBC removed. The prior demonstration of RBCs containing ring-infected erythrocyte surface antigen (pf 155 or RESA) but no intracellular parasites, support the idea of pitting. METHODS: An in vitro model was developed to examine the phenomenon of pitting and spherocyte formation in Plasmodium falciparum infected RBCs (iRBC) co-incubated with human macrophages. In vivo application of this model was evaluated using blood specimens from patients attending Kisumu Ditrict Hospital. RBCs were probed with anti-RESA monoclonal antibody and a DNA stain (propidium iodide). Flow cytometry and fluorescent microscopy was used to compare RBCs containing both the antigen and the parasites to those that were only RESA positive. RESULTS: Co-incubation of iRBC and tumor necrosis factor-alpha activated macrophages led to pitting (14% +/- 1.31% macrophages with engulfed trophozoites) as opposed to erythrophagocytosis (5.33% +/- 0.95%) (P < 0.01). Following the interaction, 26.9% +/- 8.1% of the RBCs were spherocytes as determined by flow cytometric reduction in eosin-5-maleimide binding which detects RBC membrane band 3. The median of patient RBCs with pitted parasites (RESA+, PI-) was more than 3 times (95,275/muL) that of RESA+, PI+ RBCs (28,365/muL) (P < 0.01). RBCs with pitted parasites showed other morphological abnormalities, including spherocyte formation. CONCLUSION: It is proposed that in malaria holoendemic areas where prevalence of asexual stage parasites approaches 100% in children, RBCs with pitted parasites are re-circulated and pitting may produce spherocytes.

Anemia↗

PubMed1995

Beta spectrin PRAGUE: a truncated beta spectrin producing spectrin deficiency, defective spectrin heterodimer self-association and a phenotype of spherocytic elliptocytosis.

Spherocytic elliptocytosis is a phenotypic hybrid between hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) characterized by the presence of spheroovalocytes and spherocytes which exhibit increased osmotic fragility, indicating a deficiency of surface area. Both the spherocytic red cell morphology and the increased osmotic fragility distinguish this clinical entity from common HE. In contrast to common HE, the molecular basis of spherocytic elliptocytosis is unknown. Here we describe two members of a family who both have the characteristic features of spherocytic HE. We show that the underlying defect involves a G to C transversion at the -1 position of the acceptor splice site upstream of exon X of beta spectrin leading to skipping of exon X from the mutant beta spectrin mRNA allele. The mutant mRNA is present in reticulocytes in similar amounts as the normal mRNA. Pulse-labelling of erythroblasts prepared from peripheral blood in a two-phase liquid-culture system reveals a decreased synthesis of the truncated beta spectrin, a finding which is likely to underlie the moderately severe spectrin deficiency in the two patients. In addition, this mutant spectrin, similar to the previously reported spectrins, is defective in spectrin heterodimer self-association. The spectrin deficiency, which represents a common finding in the majority of patients with HS, together with weakened spectrin heterodimer self-association, as found in the majority of patients with common HE, provides a molecular explanation for the phenotype of spherocytic elliptocytosis in this kindred and, most likely, in other patients carrying similar beta spectrin mutations.

Base Sequence↗

PubMed1997

Thrombosis and secondary hemochromatosis play major roles in the pathogenesis of jaundiced and spherocytic mice, murine models for hereditary spherocytosis.

Jaundiced mice, ja/ja, suffer from a severe hemolytic anemia caused by a complete deficiency of erythroid beta-spectrin. We used these mice as a model to investigate the pathophysiological consequences of the deficiency, including the effects in the nonerythroid tissues where this protein is expressed. Because the ja/ja mice rarely survive beyond the fourth postnatal day, methods were assessed for extending lifespan into adulthood. Neonatal transfusion increased lifespan to a mean of 3.7 months, allowing a more complete characterization of the pathophysiology. Blood parameters and histopathology of the jaundiced mouse were compared with that from spherocytic mice, which have a hemolytic anemia caused by deficiency of erythroid alpha-spectrin, yet can survive the postnatal period transfusion free. The adult jaundiced and spherocytic mice present with greatly decreased hematocrit and red blood cell counts, reticulocytosis, and bilirubinemia, leading secondarily to hepatosplenomegaly and cardiomegaly. Jaundiced and spherocytic mice were analyzed histopathologically between 1.0 and 9.5 months of age. Interestingly, the complete absence of erythroid beta-spectrin in jaundiced mice leads to no detectable structural defects in brain, cardiac, or skeletal muscles. However, fibrotic lesions and lymphocytic infiltration were observed in cardiac tissue from 4 of 13 jaundiced mice and 15 of 15 spherocytic mice, and thrombi were detected at either the atrioventricular valves or within the atria of 2 of 13 jaundiced mice and 15 of 15 spherocytic mice. In addition, all affected mice had a progressive renal hemosiderosis concurrent with hydronephrosis and glomerulonephritis. The severity of the renal disease and its presence in all moribund mice suggests kidney failure rather than the fibrotic heart lesions as the major cause of death in these mice.

Alternative Splicing↗

PubMed1992

Transbilayer asymmetry of pyrene mobility in human spherocytic red cell membranes.

The diffusion-dependent formation of pyrene excimers (excited dimers) was studied in normal and spherocytic red cell membranes. Pyrene emission was alternatively quenched in either bilayer half by non radiative energy transfer to haemoglobin. Pyrene excimer to monomer fluorescence intensity ratio, I'/I, was 0.35 +/- 0.03 (S.E.) in washed red blood cells obtained from normal donors (n = 8) and 0.45 + 0.03 (n = 13) in the corresponding isolated, haemoglobin-free resealed membranes (P less than 0.02). In the spherocytic condition the respective values were 0.28 +/- 0.01 (n = 9) and 0.53 +/- 0.03 (n = 9), P less than 0.001. In contrast to the decrease of I'/I in red cells as compared to isolated membranes, being 22% in normal cells and 47% in spherocytic ones, haemoglobin added to the exofacial side of isolated membranes, respectively, reduced I'/I by 18% and 5%. In normal red cell membranes, pyrene mobility appears to be higher in the inner monolayer than in the outer one. In spherocytic membranes our results indicate an enhanced transmembrane asymmetry in lipid monolayer fluidity, probably due to a defect of the membrane protein skeleton organization.

Cholesterol↗

PubMed1988

Ca2+ influx in normal and spherocytic red cells.

The influx of 45Ca2+ into normal red cells and various types of spherocytic red cells was studied after blocking active Ca2+ extrusion by vanadate. The measurements were performed with and without verapamil, a calcium antagonist. The influx of Ca2+ into red cells from unsplenectomized persons was 22 +/- 7 mumol/l packed red cells/h (mean +/- SD), and 17 +/- 7 mumol/l per h when incubated with verapamil. The influx of Ca2+ into red cells from four splenectomized normal controls was of the same magnitude as in the unsplenectomized controls but there was no effect of verapamil on the influx rate. The influx of Ca2+ into red cells from nine splenectomized patients with hereditary spherocytosis (HS) was 27 +/- 9 mumol/l per h without and 24 +/- 9 mumol/l per h with verapamil. In 9 normal red cell samples made spherocytic by thermal damage the corresponding values were 32 +/- 16 and 31 +/- 19 mumol/l per h, respectively. The uptake of Ca2+ in chlorpromazine-induced spherocytic red cells was 20 +/- 4 mumol/l per h without and 19 +/- 5 mumol/l per h with verapamil in 9 experiments. These results indicate that although in HS erythrocytes changes in the cell membrane lead to an increased Ca2+ influx, the slow calcium channels are not affected, whereas in spherocytes induced by thermal damage or by incubation with chlorpromazine the channels are blocked, at least partly.

Adult↗

PubMed2005

Effects of bilirubin and phototherapy on osmotic fragility and haematoporphyrin-induced photohaemolysis of normal erythrocytes and spherocytes.

AIM: To study the effects of phototherapy on erythrocyte haemolysis in vitro and to determine possible differences in sensitivity to phototherapy between normal erythrocytes and spherocytes. METHODS: Erythrocytes from four normal healthy donors and two donors with hereditary spherocytosis were treated with bilirubin (160 microM) in the presence of human serum albumin in the molecular ratio bilirubin/albumin 0.8. Treated cells were maintained either in the dark or in blue light (450 nm, 8 mW/cm2, 30 min). The experimental light dose was comparable to 2 h of clinical phototherapy. The osmotic fragility of the treated cells was measured by scoring haemolysis in hypo-osmolar solutions (0.10-0.90% NaCl). The sensitivity to photohaemolysis of cells pre-treated with bilirubin (BR) and/or phototherapy was tested by exposing the cell suspensions to haematoporphyrin and UVA radiation. The delayed (18 h) photohaemolysis was measured by spectrophotometry. RESULTS: Osmotic fragility, expressed as percentage haemolysis, of normal erythrocytes was more than doubled in the presence of BR combined with phototherapy (n = 6, p < 0.05). In contrast, osmotic fragility of spherocytes was unaffected by either treatment (n = 8, p < 0.05). Increased photohaemolysis was seen in spherocytes treated with BR (n = 13, p < 0.05), phototherapy (n = 13, p < 0.05) and a combination of the two agents (n = 13, p < 0.05) compared with spherocytes without BR in the dark (n = 6). CONCLUSION: Bilirubin may make the plasma membrane of normal erythrocytes more fragile. Newborns with hereditary spherocytosis may be sensitive to phototherapy.

Bilirubin↗

PubMed1979

Reconstitution of spectrin-deficient, spherocytic mouse erythrocyte membranes.

To study directly the role of spectrin in erythrocyte membrane function, we have designed a reconstituted membrane system using erythrocyte membranes from spectrin-deficient mice and purified spectrin from normal mice. The normal spectrin is inserted into the spectrin-deficient spherocytes by exchange hemolysis. Thereafter, raising the ionic strength and temperature reseals the cells and, with time, facilitates binding of the spectrin to the spectrin-deficient membranes. The binding is apparently specific as shown by its dependence upon the concentration of undenatured spectrin and the concentration of salt used, as well as by the immunofluorescent appearance of the reconstituted cells after treatment with specific antispectrin antibody. In terms of in vitro cellular behavior, the reconstituted preparations show marked changes in comparison to the untreated spherocytes. In particular, membrane stability, as measured by the reduction of myelin figure formation and lipid loss, is considerably enhanced. In addition, membrane fusion, which occurs readily with the untreated spherocytes, is virtually eliminated. Finally, the osmotic behavior of the native spherocytes is appreciably altered, such that the early phase of osmotically induced swelling, as measured in a high-speed stop-flow apparatus, is delayed and modified. Taken together, these findings indicate specific roles for spectrin in the stabilization of the erythrocyte membrane, in the limitation of membrane fusion, and in the modulation of the membrane's response to osmotic stress.

Animals↗

PubMed1976

Red cell velocity and plasma transit time in the cerebral microcirculation of spherocytic deer mice.

Spherocytic deer mice provide a model of human spherocytosis. Their erythrocytes are abnormal in shape and are more rigid than normal red blood cells (RBC). Like their human counterparts, spherocytic mice are anemic. Measurements of RBC velocity in microvessels on the cerebral surface failed to reveal a difference between the velocity of cells in spherocytic as compared to normal deer mice. However, plasma transit, as measured by fluorescein microangiography, was faster than normal. Both decreased plasma transit time and increased RBC velocity are expected in nonspherocytic, anemic mice. Since the former, but not the latter, was found in the spherocytic, anemic mice, it appears that increased RBC rigidity has a greater effect on RBC movement than on plasma movement within the cerebral microcirculation. Thus iit would seem that this increased RBC rigidity prevents the increased RBC velocity that otherwise would be observed in anemia but does not prevent the decrease in plasma transit time.

Animals↗

PubMed1995

Hereditary spherocytic anemia with deletion of the short arm of chromosome 8.

We describe a 30-month-old boy with multiple anomalies and mental retardation with hereditary spherocytic anemia. His karyotype was 46,XY,del(8)(p11.23p21.1). Genes for ankyrin and glutathione reductase (GSR) were localized to chromosome areas 8p11.2 and 8p21.1, respectively. Six patients with spherocytic anemia and interstitial deletion of 8p- have been reported. In these patients, severe mental retardation and multiple anomalies are common findings. This is a new contiguous gene syndrome. Lux et al. [1990: Nature 345:736-739] established that ankyrin deficiency and associated deficiencies of spectrin and protein 4.2 were responsible for spherocytosis in this syndrome. We reviewed the manifestations of this syndrome. Patients with spherocytic anemia and multiple congenital anomalies should be investigated by high-resolution chromosomal means to differentiate this syndrome.

Adolescent↗

PubMedDate not supplied

Osmotic swelling and hole formation in membranes of thalassemic and spherocytic erythrocytes.

Osmotic swelling and kinetics of the pore formation in the membranes of spherocytic, thalassemic, and normal erythrocytes were studied by measuring the time-dependent capacitance and conductance at a frequency of 0.2 MHz. No significant difference between the swelling rate of control and spherocytic cells was observed, whereas slower kinetics of swelling were found for thalassemic cells. Time records of the conductance indicate that the probability of the pore formation in the stretched membrances varies in the following order: thalassemia < control < spherocytosis. Based on these findings it was concluded that the erythrocyte swelling is controlled by the initial cell shape, volume, intracellular hemoglobin concentration, and elastic membrane properties, whereas the kinetics of the pore formation depend solely on the resistivity of the stretched membrane of the swollen RBC to the osmotic shock. Therefore, it was assumed that investigations of the pore formation may be used not only for examinations of spherocytic and thalassemic cells, but also for normocytic, normochromic, biconcave-shaped RBCs with altered membrane elasticity.

Cell Membrane Permeability↗

PubMed1992

Band 3 Tuscaloosa: Pro327----Arg327 substitution in the cytoplasmic domain of erythrocyte band 3 protein associated with spherocytic hemolytic anemia and partial deficiency of protein 4.2.

Protein 4.2 is a major red blood cell (RBC) protein that interacts with the band 3 protein and with ankyrin. Inherited deficiencies of this protein are associated with spherocytic hemolytic anemia, but the molecular basis of this defect is unknown. We have studied the underlying defect in a patient with spherocytic hemolytic anemia whose RBCs had a partial (29% +/- 5%) deficiency of protein 4.2. We have first studied the binding of normal ankyrin and protein 4.2 to patient inside-out vesicles (IOVs) stripped of peripheral proteins. While the binding of ankyrin was normal, the predicted maximal binding capacity of patient IOVs for band 4.2 was 20% to 33% lower than that of control IOVs, suggesting a defect in the cytoplasmic domain of band 3 (cdb3). An additional line of evidence pointing to a possible abnormality of band 3 was an abnormal proteolytic digest of cdb3. To elucidate the underlying molecular defect, we have cloned and sequenced the cDNA coding for cdb3 from the patient. One band 3 allele was found to be normal, while clones corresponding to the other allele contained two mutations: substitution A----G in nucleotide 166, changing codon 56 from AAG to GAG (Lys----Glu), and substitution C----G in nucleotide 980, changing codon 327 from CCC to CGC (Pro----Arg). Since the Lys56----Glu56 substitution is found in a common asymptomatic variant of the band 3 protein designated band 3 Memphis, we conclude that either the Pro327----Arg327 substitution itself, or in combination with the band 3 Memphis polymorphism, underlies the abnormal binding of protein 4.2 to cdb3 and results in the spherocytic phenotype.

Amino Acid Sequence↗

PubMed1978

31P-NMR study on nucleotides and intracellular pH of hereditary spherocytes.

As determined by 31p-NMR spectroscopy, intracellular pH of hereditary spherocytes was lower (pH 6.7-6.9) than that of normal red cells. The level of adenosine diphosphate in hereditary spherocytes was found to be persistently high. The metabolism of nucleotides and other phosphoryl compounds in human red blood cells have been studied in detail by 31p-MNR spectroscopy. However, to our knowledge, there seems to be no report describing the result of 31p-NMR spectroscopy on red blood cells from hereditary spherocytosis.

Adenosine Diphosphate↗

PubMed1984

Lateral mobility of phospholipids in the external and internal leaflets of normal and hereditary spherocytic human erythrocytes.

The lateral diffusion coefficients (D) and the mobile fractions of the fluorescent phospholipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) and of membrane proteins labelled with fluorescein isothiocyanate, were measured by fluorescence photobleaching recovery on erythrocytes from healthy persons and from a hereditary spherocytosis patient. Measurements of lipid probe mobility were performed on ghosts labelled by NBD-PE exclusively at the external monolayer, or at both sides of the membrane. Our results indicate the following: (1) The mean values and the temperature dependence of D are different at the external and internal membrane leaflets. (2) In both normal and HS ghosts the mobile fraction of NBD-PE in the external monolayer does not depend significantly on temperature. On the other hand, the mobile fraction in the internal monolayer is reduced as the temperature is decreased. (3) At low temperatures, the mobile fraction of NBD-PE in the internal monolayer of spherocytic ghosts is significantly lower than the mobile fraction in the internal monolayer of normal ghosts. (4) No differences were observed between the mobilities of membrane proteins in normal and in spherocytic ghosts. However, differences were observed between the two cell populations in the temperature-dependence of the intrinsic fluorescence of unlabelled membrane proteins. The implications of these results for membrane phospholipid asymmetry and for cytoskeletal interactions with the internal lipid monolayer are discussed.

Erythrocyte Membrane↗

PubMed1980

Lateral mobility of integral membrane proteins is increased in spherocytic erythrocytes.

Alterations of glycoprotein distribution and lateral mobility in cell membranes can provide transmembrane signals for several membrane-related phenomena. Control of the transmembranous events has been ascribed to interaction between submembranous protein matrices (or 'cytoskeletons') and membrane glycoproteins. A consequence of such interaction would be differential inhibition of protein lateral diffusion in biological membranes. Measurements of the lateral diffusion coefficients of membrane proteins, in fact, have generally yielded values much less than were predicted for unhindered diffusion in a fluid bilayer. The mouse spherocytic erythrocyte, which lacks the major components of the normal erythrocyte membrane matrix (composed of spectrin, actin, bands 4.1 and 4.9 (ref. 16), in the nomenclature of Fairbanks et al.), provides a unique system for a direct evaluation of the effect of the matrix on protein lateral mobility. After using a modification of the technique of fluorescence redistribution after photobleaching (FRAP), we report here that membrane proteins diffuse about 50 times faster in spherocytic than in normal mouse erythrocytes.

Animals↗

PubMed1985

Transformation of erythrocytes to spherocytes following incubation with malignant cells.

Incubation of erythrocytes from controls and patients with colorectal carcinoma with tumor cells, or their metabolites, caused transformation of the normally-shaped cells to spherocytes. It is assumed that abnormal metabolites produced by malignant cells affect the cell membrane inducing the subsequent appearance of spherocytes. The increased destruction of these cells by the reticuloendothelial system may serve as an additional factor for the explanation of anemia accompanying malignant diseases.

Colonic Neoplasms↗

PubMed2000

Chronic non-spherocytic haemolytic anaemia due to congenital pyrimidine 5' nucleotidase deficiency: 25 years later.

In 1972, Valentine et al described, under the name of 'non-spherocytic haemolytic anaemia, high red cell ATP and ribose phosphate pyrophosphokinase (RPK; EC 2.7.6.1) deficiency', an obscure congenital haemolytic anaemia with the characteristic feature of red blood cell basophilic stippling. The activity of Embden-Meyerhof pathway and hexose monophosphate shunt were normal, and the concentrations of reduced glutathione and of ATP were raised 2 SD above the normal mean. The low values of RPK also encountered were considered to be an epiphenomenon rather than a causative defect. One year later, further studies performed in two new kindreds with the same haemolytic disorder associated with persistent basophilic stippling were described under the name of 'haemolytic disorders associated with increased ATP'. In 1974, two new and important observations contributed to the final identification of the disease: the patients' red blood cells (RBCs) contained large amounts of nucleotides (pyrimidine nucleotides), and in all cases they were deficient in a hitherto unrecognized enzyme called pyrimidine 5' nucleotidase (P5N). In conclusion, all these cases were formerly referred to as 'high ATP syndromes' because of the erroneous assumption that the large number of nucleotides within deficient RBCs were adenine phosphate rather than pyrimidine phosphate. Twenty-five years after its description, P5N deficiency has been reported in about 35 unrelated families from different parts of the world, and it has become one commonly identified cause of hereditary non-spherocytic haemolytic anaemia due to RBC enzymopathy. Genetic transmission is via the autosomic recessive mode, and only homozygous or compound heterozygous are clinically affected. Family members who are biochemically heterozygous are haematologically normal and difficult to detect. Unfortunately, the precise gene mutation or mutations causing the disease remain unknown.

5'-Nucleotidase↗

PubMed1975

Diphenylhydantoin and fragility of erythrocytes in normal subjects and in patients with hereditary spherocytic anemia.

To study the effects of diphenylhydantoin (Dilantin, DPH) on membrane function, DPH was incubated with erythrocytes from normal individuals and from patients with congenital spherocytic hemolytic anemia and osmotic fragility measured. The resulting curves show that DPH reduces the osmotic fragility of normal and spherocytic erythrocytes. In the presence of ouabain, DPH still had a protective effect on erythrocytes even though ouabain alone increases hemolysis of erythrocytes at certain saline concentrations. This study shows that erythrocyte fragility can be manipulated in vitro and provides a basis for studies in vivo.

Adolescent↗

PubMed2000

Mild spherocytic hereditary elliptocytosis and altered levels of alpha- and gamma-adducins in beta-adducin-deficient mice.

The membrane skeleton, a dynamic network of proteins associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Deficiencies or defects in membrane skeletal proteins are associated with inherited disorders of erythrocyte morphology and function. Adducin is one of the proteins localized at the spectrin-actin junction of the membrane skeleton. In this work we show that deficiency of beta-adducin produces an 80% decrease of alpha-adducin and a fourfold up-regulation of gamma-adducin in erythrocytes. beta-Adducin or any other isoform generated by translation of abnormally spliced messenger RNAs could not be detected by our antibodies either in ghosts or in cytoplasm of -/- erythrocytes. Actin levels were diminished in mutant mice, suggesting alterations in the actin-spectrin junctional complexes due to the absence of adducin. Elliptocytes, ovalocytes, and occasionally spherocytes were found in the blood film of -/- mice. Hematological values showed an increase in reticulocyte counts and mean corpuscular hemoglobin concentration, decreased mean corpuscular volume and hematocrit, and normal erythrocyte counts that, associated to splenomegaly, indicate that the mice suffer from mild anemia with compensated hemolysis. These modifications are due to a loss of membrane surface and dehydration that result in an increase in the osmotic fragility of red blood cells. The marked alteration in osmotic fragility together with the predominant presence of elliptocytes is reminiscent of the human disorder called spherocytic hereditary elliptocytosis. Our results suggest that the amount of adducin remaining in the mutant animals (presumably alphagamma adducin) could be functional and might account for the mild phenotype. (Blood. 2000;95:3978-3985)

Animals↗