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R L Nagel

Publications and source records attributed to R L Nagel.

At least 19 recordsLinked to original sources
PubMed1992

High expression of human beta S- and alpha-globins in transgenic mice: hemoglobin composition and hematological consequences.

A line of transgenic mice (alpha H beta S-11; where alpha H is human alpha-globin) was created in which the human beta S and human alpha 2 globin genes, each linked to the beta-globin locus control region, were cointegrated into the mouse genome. On a normal genetic background, the transgenic mice produced 36% human beta S-globin chains with an alpha H/beta S ratio of 1.3. Higher levels of beta S were achieved by breeding the transgenic mice with mutant mice carrying a mouse beta major-globin gene deletion. Mice heterozygous for the beta major deletion (alpha H beta S[beta MD]; MD, mouse deletion) had 54% beta S with an alpha H/beta S ratio of 1.0; mice homozygous for the beta major deletion (alpha H beta S[beta MDD]) had 72.5% beta S and an alpha H/beta S ratio of 0.73. Because mouse alpha chains inhibit hemoglobin (Hb) S polymerization, we bred the mice to heterozygosity for a mouse alpha-globin deletion. These mice (alpha H beta S[alpha MD beta MDD]) had an increased alpha H/beta S ratio of 0.89 but expressed 65% beta S. Expression of the human genes cured the thalassemic phenotype associated with the murine beta major deletion. Transgenic alpha H beta S[beta MDD] mice had normal hematocrit and Hb and somewhat elevated reticulocytes (6% vs. 3% for control), whereas the mice carrying the alpha-globin deletion (alpha H beta S[alpha MD beta MDD]) had a normal hematocrit and Hb and more elevated reticulocytes (10.3 +/- 7.6% vs. 3.4 +/- 1.0%). Expression of the transgene restored a normal distribution of erythrocyte densities when compared to thalassemic mice; however, the average mean corpuscular Hb concentration of alpha H beta S[beta MDD] mice increased to 35.7 g/dl (vs. control 33.7 g/dl) whereas that of alpha H beta S[alpha MD beta MDD] mice was further elevated to 36.3 g/dl. The intrinsic oxygen affinity was increased in transgenic mouse erythrocytes at 280 milliosmolal, and the PO2 at midsaturation of alpha H beta S[alpha MD beta MDD] erythrocytes was higher than that of alpha H beta S[beta MDD] cells (37.4 +/- 2 vs. 33.5 +/- 1 mmHg). The higher values of the mean corpuscular Hb concentration and intrinsic PO2 at midsaturation, which favor in vivo sickling, may explain the slightly more severe hematological picture in alpha H beta S[alpha MD beta MDD] mice. We conclude that the transgenic mouse with high Hb S expression does not exhibit adult anemia but does have abnormal hematological features: increased erythrocyte density, high oxygen affinity, and reticulocytosis with increased stress reticulocytes.

Anemia, Sickle Cell↗
PubMed1992

High expression of human beta S- and alpha-globins in transgenic mice: erythrocyte abnormalities, organ damage, and the effect of hypoxia.

A line of transgenic mice with two cointegrated transgenes, the human beta S- and alpha 2-globin genes, linked to the beta-globin locus control region was produced and bred with mice carrying a deletion of the mouse beta major-globin gene. In transgenic mice homozygous for the beta major deletion (alpha H beta S[beta MDD]; where alpha H is human alpha-globin and MD is mouse deletion), 72.5 +/- 2.4% (mean +/- SD) of the beta-chains are beta S and the ratio of alpha H- to beta S-globin was 0.73. Introduction of a heterozygous mouse alpha-globin deletion into mice homozygous for the beta major deletion (alpha H beta S[alpha MD beta MDD]) resulted in 65.1 +/- 8.5% beta S and a human alpha/beta ratio of 0.89 +/- 0.2. Sickling occurs in 95% of erythrocytes from alpha H beta S[beta MDD] mice after slow deoxygenation. Transmission electron microscopy revealed polymer fiber formation but not fascicles of fiber. Increased organ weight was noted in lung, spleen, and kidney of transgenic mice vs. controls that may be due to hypertrophy or increased blood volume in the lungs and/or increased tissue water content. The hemoglobin content of lung, spleen, and kidney was also elevated in transgenic animals due to trapped hemoglobin and/or increased blood volume. When transgenic and control mice were examined by magnetic resonance imaging at 9.4 tesla, some transgenic animals had enlarged kidneys with prolonged relaxation time, consistent with increased organ weight and water content. The glomerular filtration rate was elevated in transgenic animals, which is characteristic of young sickle cell patients. Furthermore, exposure to hypoxia resulted in significantly decreased hematocrit, increased erythrocyte density, and induced a urine-concentrating defect. We conclude that the transgenic mouse line reported here has chronic organ damage and further hematological and organ dysfunction can be induced by hypoxia.

Anemia, Sickle Cell↗
PubMed1992

An alanine-to-threonine substitution in protein 4.2 cDNA is associated with a Japanese form of hereditary hemolytic anemia (protein 4.2NIPPON).

Erythrocyte (RBC) protein 4.2 (P4.2)-deficiency observed in Japanese individuals results in a hemolytic anemia associated with abnormally shaped (spherocytic, ovalocytic, and elliptocytic), osmotically fragile RBCs, the clinical presentation of which resembles hereditary spherocytosis (HS). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, P4.2-deficient individuals contain less than 1% of the normal membrane content of P4.2 and immunologic analysis shows that the P4.2 present exists as an equimolar doublet of 74-Kd and 72-Kd bands, in contrast to normal RBC membranes where a discrete 74-Kd band is not observed. RBC membranes from both of the biologic parents of a P4.2-deficient individual contained both the 74-Kd and the 72-Kd bands, demonstrating their heterozygosity for the P4.2 defect. The molecular basis of Japanese P4.2-deficiency was investigated by reverse transcription of total reticulocyte RNA, followed by polymerase chain reaction (PCR) amplification, subcloning, and sequencing. The complete cDNA sequence of a P4.2-deficient patient showed a single point mutation that changes codon 142 from GCT (alanine) to ACT (threonine) (Protein 4.2NIPPON). The mutation also eliminated an HgaI restriction site, therefore allowing rapid screening for the presence of the mutation. Screening of PCR-amplified genomic DNA showed that the mutation was present in the homozygous state in four (eight chromosomes) unrelated Japanese P4.2-deficient individuals and absent in 35 (70 chromosomes) P4.2-normal controls (including 15 Japanese [30 chromosomes]). The presence of the mutation was confirmed by allele-specific hybridization. The mutation occurred in an alternatively spliced exon that is present in two of four P4.2 mRNA splicing isoforms. These results demonstrate that Japanese P4.2-deficiency is closely associated with the P4.2 gene and does not arise secondarily to a defect in another membrane protein, and further suggest that the P4.2-deficiency is related to the pathogenesis of the hemolytic anemia in this variant form of recessively inherited spherocytosis.

Alanine↗
PubMed1992

Demonstration of endothelial adhesion of sickle cells in vivo: a distinct role for deformable sickle cell discocytes.

Different morphologic and density classes of sickle cells (SS) may play distinct roles in the generation of vasoocclusion, explaining the complexity of this phenomena. The densest SS red blood cells (RBCs) (SS4) can induce vasoocculsion in ex vivo microcirculatory preparations as well as in an intact animal model. Previous studies of the interaction of SS deformable discocytes with endothelial monolayers or the rat ex vivo mesocecum preparation have shown adhesion that is desmopressin (dDAVP)-stimulated, von Willebrand factor (vWF)-mediated, and limited to the small venules. However, in vivo adhesion of SS RBCs to the endothelium has neither been demonstrated nor characterized; and, in particular, the relation of adhesion to vasoocclusion is unknown. Using an intact animal model that involves injecting saline-washed, density-defined SS RBCs into the femoral artery of a rat, we find that: (1) Quantitative studies of RBCs retained in the rat thigh using 99mTc-labeled RBCs and gamma camera imaging showed that dDAVP induces a threefold increase in retention of normal (AA) cells and deformable SS discocytes (SS2). (2) electron microscopy and Microfil injection show that the retention of SS2 cells is due to adhesion to the vascular endothelium with no evidence of obstruction. (3) H-1 magnetic resonance imaging showed that retention of SS4 cells induced a dose-dependent increase in tissue edema (presumable secondary to tissue hypoxia), while retention of AA or SS2 cells produced no change. We conclude that endothelial adhesion of deformable SS discocytes can be demonstrated in an in vivo animal model, that this adhesion is enhanced by dDAVP (presumably related to, but not necessarily limited to the release of vWF), and that this phenomenon per se does not lead to vasoocclusion. Nevertheless, adhesion of deformable SS discocytes may have consequences. We hypothesize that adhesion of SS discocytes could narrow the lumen of postcapillary venules and facilitate secondary trapping of SS4 cells and lead to subsequent vasoocclusion.

Anemia, Sickle Cell↗
PubMed1992

Genetic epidemiology of beta-thalassemia in Sicily: do sequences 5' to the G gamma gene and 5' to the beta gene interact to enhance HbF expression in beta-thalassemia?

The present epidemiological study of the molecular characteristics of beta-thalassemia in Sicily was prompted by the disparate phenotypic expression (in clinical status and absolute HbF level) observed in two beta-thalassemic homozygotes who were also homozygous for the beta-like globin gene cluster haplotype III. We suspected that polymorphisms within haplotype III could be the cause for the discrepancy. Based on the association of particular conformations of the (AT)xT(y) motif (-540 5' to the beta gene) with milder forms of thalassemia and sickle cell anemia, 38 homozygous beta-thalassemia patients were studied to define their haplotypes, the -158 site 5' to the G gamma gene (linked to haplotype III) and the structure of the (AT)xT(y) motif. We found that the patient who was phenotypically mild and homozygous for beta-thalassemia, haplotype III, and the -158 C----T mutation was homozygous for the rare (AT)9T5 motif. In contrast, the patient homozygous for beta-thalassemia, haplotype III, and the -158 mutation, but exhibiting a severe clinical course, was homozygous for the (AT)7T7 configuration. Others have suggested that (AT)9T5 is a negative regulatory protein binding sequence, and it is a silent carrier state for beta-thalassemia. The usual configuration (AT)7T7, has considerably less affinity for regulatory protein binding, and it is the most common configuration in Sicilian beta-thalassemics (67 of the 78 chromosomes studied). Within the 38 patients studied, seven were informative because they had various combinations of the (AT)9T5 and (AT)7T7 motif, and the -158 C----T mutation. The results in these patients suggest that only the co-presence of the (AT)9T5 configuration and a C----T change at -158 5' to the G gamma gene is associated with high HbF expression and a mild clinical phenotype. We postulate that these two regions of the beta-like globin gene cluster interact, when endowed with the proper sequences, to enhance the expression of HbF secondary to anemia.

Adolescent↗
PubMed1992

Presence of an African beta-globin gene cluster haplotype in normal chromosomes in Sicily.

African admixture in Sicily has been long suspected because of the presence of the sickle gene. Nevertheless, the degree of African admixture cannot be derived from the study of HbS frequency, since this gene was most likely expanded by the selective pressure of malaria, for a long time endemic to the region. We have examined 142 individuals from the Sicilian town of Butera (12% sickle trait) to search for other markers of the globin gene cluster less likely to be selected for by malaria. The TaqI polymorphism in the intervening sequences between the two gamma genes is informative. We have found only two instances of this African marker (TaqI(-)) among 267 normal chromosomes, demonstrating that the admixture occurred at a much lower level than previously thought.

Adolescent↗
PubMed1992

Elliptocytosis associated with an abnormal alpha glycophorin.

A case of elliptocytosis associated with an undescribed abnormal alpha glycophorin (alpha GP) is reported. Using immunoblotting techniques, a clear-cut minor band 6' was detected emerging just behind the monomer of delta GP (band 6) when probed with anti-alpha GP antiserum. It also reacted with anti-peptide C antiserum, suggesting that this new band with a molecular weight of 24 K is related to the structural alteration of alpha GP and not delta GP. The erythrocyte membrane proteins of the patient exhibited a quite normal pattern, with a normal alpha spectrin/beta spectrin ratio, but the reaction with anti-protein 4.1 serum confirmed the increase in proteolytic susceptibility of her protein 4.1. The results of DNA mapping implied that the abnormality may be due to a short deletion of the heterozygote. The significance of deviation involving the alpha GP and protein 4.1 to the elliptocytic change of erythrocyte shape is briefly discussed.

Aged↗
PubMed1992

Genetic epidemiology of the beta s gene.

The beta s gene arose at least four times in Africa, with three of these mutations expanding through diverse ethnic groups, but limited to definite geographical areas: Atlantic west Africa for the Senegal haplotype linked beta s; central west Africa for the Benin haplotype; and equatorial, eastern and southern Africa for the Bantu haplotype. The fourth mutation (linked to the Cameroon haplotype) is restricted to a single ethnic group, the Eton of central Cameroon. The Benin haplotype linked beta s gene was spread by gene flow to the Mediterranean (north, south and east) and to the western portions of Saudi Arabia. An independent mutation linked to a fifth haplotype, Arab-India, is found among the tribals of India (independent from their geographical origin) and in the eastern oases of Saudi Arabia. It is also suspected of being associated with the beta s gene found in Afghanistan, Iran, Transcaucasia and central Asia. The selective force involved in the expansion of the gene was most likely P. falciparum malaria, and the time of the gene frequency increase was likely to have been during the expansion of agriculture about 4000 or more years ago in India and about 3000 years ago in Africa. The partial protection against severe and life-threatening malaria is through the limitation of P. falciparum parasitaemia. This is a complex process which involves at least two mechanisms: early intraerythrocyte parasite forms are in a suicidal position through increasing the tendency of HbAS cell to sickle and then be destroyed by the spleen; intraerythrocyte growth is inhibited during deep vascular schizogony. Although there is evidence that P. falciparum (and P. malariae) parasitaemias are limited in HbSS red cells, malaria is a major trigger to haemolytic and infarctive crises in sickle-cell disease, and a common cause of morbidity and mortality.

Africa↗
PubMed1992

The gene for human erythrocyte protein 4.2 maps to chromosome 15q15.

Protein 4.2 (P4.2), one of the major components of the red-blood-cell membrane, is located on the interior surface, where it binds with high affinity to the cytoplasmic domain of band 3. Individuals whose red blood cells are deficient in P4.2 have osmotically fragile, abnormally shaped cells and moderate hemolytic anemia. cDNA clones from both the 5' and the 3' coding regions of the P4.2 gene were used to map its chromosomal location by fluorescence in situ hybridization. The probes, individually or in combination, gave specific hybridization signals on chromosome 15. The hybridization locus was identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by Alu-PCR (R-like) probe and by DAPI staining (G-like). Our results demonstrate that the locus of the P4.2 gene is located within 15q15.

Biotin↗
PubMed1991

The unique red cell heterogeneity of SC disease: crystal formation, dense reticulocytes, and unusual morphology.

Knowledge concerning SS (homozygous for the beta s gene) red blood cell (RBC) heterogeneity has been useful for understanding the pathophysiology of sickle cell anemia. No equivalent information exists for RBCs of the compound heterozygote for the beta s and beta c genes (SC) RBCs. These RBCs are known to be denser than most cells in normal blood and even most cells in SS blood (Fabry et al, J Clin Invest 70:1284, 1981). We have analyzed the characteristics of SC RBC heterogeneity and find that: (1) SC cells exhibit unusual morphologic features, particularly the tendency for membrane "folding" (multifolded, unifolded, and triangular shapes are all common); (2) SC RBCs containing crystals and some containing round hemoglobin (Hb) aggregates (billiard-ball cells) are detectable in circulating SC blood; (3) in contrast to normal reticulocytes, which are found mainly in a low-density RBC fraction, SC reticulocytes are found in the densest SC RBC fraction; and (4) both deoxygenation and replacement of extracellular Cl- by NO3- (both inhibitors of K:Cl cotransport) led to moderate depopulation of the dense fraction and a dramatic shift of the reticulocytes to lower density fractions. We conclude that the RBC heterogeneity of SC disease is very different from that of SS disease. The major contributions of properties introduced by HbC are "folded" RBCs, intracellular crystal formation in circulating SC cells, and apparently a very active K:Cl cotransporter that leads to unusually dense reticulocytes.

Anemia, Sickle Cell↗
PubMed1991

Rosetting of Plasmodium falciparum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions.

The occurrence of rosetting of Plasmodium falciparum-infected human red blood cells (IRBC) with uninfected red blood cells (RBC) and its potential pathophysiologic consequences were investigated under flow conditions using the perfused rat mesocecum vasculature. Perfusion experiments were performed using two knobby (K+) lines of P falciparum, ie, rosetting positive (K+R+) and rosetting negative (K+R-). The infusion of K+R+ IRBC resulted in higher peripheral resistance (PRU) than K+R- IRBC (P less than .0012). Video microscopy showed that under conditions of flow, in addition to cytoadherence of K+R+ IRBC to the venular endothelium, rosette formation was also restricted to venules, especially in the areas of slow flow. Rosettes were absent in arterioles and were presumably dissociated by higher wall shear rates. The presence of rosettes in the venules must therefore reflect their rapid reformation after disruption. Cytoadherence of K+R+ IRBC was characterized by formation of focal clusters along the venular wall. In addition, large aggregates of RBC were frequently observed at venular junctions, probably as a result of interaction between flowing rosettes, free IRBC, and uninfected RBC. In contrast, the infusion of K+R+ IRBC resulted in diffuse cytoadherence of these cells exclusively to the venular endothelium but not in rosetting or large aggregate formation. The cytoadherence of K+R+ IRBC showed strong inverse correlation with the venular diameter (r = -.856, P less than .00001). Incubation of K+R+ IRBC with heparin and with monoclonal antibodies to glycoprotein IV/CD36 abolished the rosette formation and resulted in decreased PRU and microvascular blockage. These findings demonstrate that rosetting of K+R+ IRBC with uninfected RBC enhances vasocclusion, suggesting an important in vivo role for rosetting in the microvascular sequestration of P falciparum-infected RBC.

Animals↗
PubMed1991

Subunit composition of plasma von Willebrand factor multimers: evidence for a non-proteolytic mechanism resulting in apparent increase in proteolytic fragments.

Plasma von Willebrand factor (vWf) consists of a series of multimers of different molecular sizes. When analyzed for subunit composition, plasma vWf contained a major polypeptide of approximately 225 kD, and at least three smaller proteolytic fragments. In the present study, the subunit composition of each of these multimers was analyzed using a two dimensional electrophoresis technique. Although nearly all the multimers, as separated by SDS 1.5% agarose gel electrophoresis, contained both the major polypeptide and the smaller fragments, the relative amount of the fragments varied among the multimers. The smaller multimers contained relatively more proteolytic fragments. After the larger multimers were depleted by incubation of plasma with formaldehyde-fixed platelets and ristocetin, the intact subunit decreased, while the amount of the proteolytic fragments remained relatively unchanged. The findings of this study are consistent with the scheme that plasma multimers undergo proteolytic cleavage after multimer assembly. Furthermore, an apparent increase in the amount of these proteolytic fragments may result from mechanisms unrelated to proteolysis, such as selective adsorption of the larger multimers onto platelets.

Humans↗
PubMed1991

Rapid increase in red blood cell density driven by K:Cl cotransport in a subset of sickle cell anemia reticulocytes and discocytes.

We have previously demonstrated that young normal (AA) and sickle cell anemia (SS) red blood cells are capable of a volume regulatory decrease response (VRD) driven by a K:Cl cotransporter that is activated by low pH or hypotonic conditions. We now report on the characteristics of young SS cells (SS2, discocytes) capable of rapid increase in density in response to swelling. We have isolated cells with high VRD response (H-VRD) and low VRD response (L-VRD) cells by incubation and density-gradient centrifugation under hypotonic conditions. Comparison of these cells in patients homozygous for hemoglobin (Hb)S indicated that H-VRD cells have 91% more reticulocytes (P less than 9 x 10(-9) than L-VRD cells, 25% less HbF (P less than 5.5 x 10(-5), 106% more NEM (N-methylmaleimide)-stimulated K:Cl cotransport activity (P less than 2 x 10(-4), and 86% more volume-stimulated K:Cl cotransport activity (P less than 1.8 x 10(-3). H-VRD and L-VRD cells have similar G-6-PD and Na+/H+ antiport activity. In agreement with the reduced percent HbF in H-VRD cells, F cells (red blood cells that contain fetal Hb) are depleted from the H-VRD population; however, F reticulocytes are enriched in the H-VRD population to the same extent as non-F reticulocytes, which suggests that both F and non-F reticulocytes have a similar initial distribution of volume-sensitive K:Cl cotransport activity but that it may be more rapidly inactivated in F than in S reticulocytes. We find that H-VRD cells consist of 20% reticulocytes (or 79% of all reticulocytes in SS2) and 80% more mature cells. This study demonstrates the role of K:Cl cotransport in determining red blood cell density, the heterogeneity of K:Cl cotransport activity in reticulocytes, and the capacity for rapid change in the density of reticulocytes with high K:Cl cotransport activity. We speculate that the H-VRD population may be more susceptible to generation of dense and irreversibly sickled cells.

Anemia, Sickle Cell↗
PubMed1991

The Senegal DNA haplotype is associated with the amelioration of anemia in African-American sickle cell anemia patients.

We have previously determined that in African sickle cell anemia (SS) patients three different beta-like globin gene cluster haplotypes are associated with different percent G gamma (one of the two types of non-alpha chains comprising hemoglobin F [HbF]), mean percent HbF, and percent dense cells. We report now that in adult New York SS patients, the presence of at least one chromosome with the Senegal haplotype is associated with higher Hb levels (1.2 g/dL higher) than is found for any other non-Senegal haplotype (P less than .004). The percent reticulocytes and the serum bilirubin levels were lower in these patients. When the effect of alpha-gene number was analyzed by examining a sample of SS patients with concomitant alpha-thalassemia, the same results were obtained. Because the HbF level is significantly higher among the Senegal haplotype carriers in this sample, the inhibitory effect on sickling of this Hb variant may be one of the reasons for the haplotype effect. We conclude that the Senegal beta-like globin gene cluster haplotype is associated with an amelioration of the hemolytic anemia that characterizes sickle cell disease.

Adult↗
PubMed1991

Blood zinc protoporphyrin is elevated only in sickle cell patients with low fetal hemoglobin.

Increased levels of various porphyrin species have been reported in sickle cell anemia (SS) patients in the absence of lead poisoning and iron deficiency anemia, but conflicting data remain. Suspecting that SS patients may be heterogenous for this abnormality, we have studied zinc protoporphyrin (ZPP) and protoporphyrin IX (PPIX) blood levels and find abnormally elevated levels of ZPP in those with low peripheral fetal hemoglobin (%HbF) levels. Two groups exist: one with less than 9% HbF and elevated ZPP, and one with greater than or equal to 9% HbF and normal ZPP levels (P less than 8.1 x 10(-4). There is a strong negative correlation of ZPP levels with %Hb F (r = -0.83, P less than 8.0 x 10(-5], and a moderate one with total hemoglobin levels (r = -0.55, P less than 0.05). These results suggest that ZPP may indeed contribute to the pathophysiology of the disease and/or serve as a marker of the severity of the disease.

Anemia, Sickle Cell↗
PubMed1991

Preliminary report on the use of desferrioxamine in the treatment of Plasmodium falciparum malaria.

The administration of 0.5 g (IM) of desferrioxamine every 12 hr for 3 days with 25 mg/kg (total dose for three days) of chloroquine abated the parasitemia of Plasmodium falciparum infected individuals more rapidly than chloroquine alone. Two patients with in vitro evidence of total or partial resistance to chloroquine also were successfully treated with the combination of drugs, and were free of parasitized red cells by day 7. Further clinical trials and development of desferrioxamine in the treatment of P. falciparum are warranted.

Animals↗