Reticulocyte-rich Blood Research Articles

What triggers the gametocyte pathway in Plasmodium falciparum?

Among the many important questions about Plasmodium falciparum that remain to be answered, none is more fascinating to the biologist than what determines the gametocyte pathway of differentiation? We know [ 1 Janse C.J. Waters A.P. Sexual development of malaria parasites. in: Waters A.P. Janse C.J. Malaria Parasites: Genomes and Molecular Biology. Caister Academic Press, 2004: 445-474 Google Scholar , 2 Lobo C.A. Kumar N. Sexual differentiation and development in the malaria parasite. Parasitol. Today. 1998; 14: 146-150 Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar , 3 Smith T.G. et al. Sexual differentiation and sex determination in the Apicomplexa. Trends Parasitol. 2002; 18: 315-323 Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar ] that a single haploid merozoite produces among its offspring not only many merozoites like itself but also male and female gametocytes. It obviously contains the genetic information for two very different pathways of development. In the first, the merozoite multiplies by schizogony to form up to 30 daughter merozoites within 48 h. In the second, the merozoite differentiates into a single male or female gametocyte. Ordinarily, only a small proportion develops into gametocytes. Under culture conditions, this proportion can be influenced by a variety of additions to the medium but none of these is likely to be a determining factor in nature. There is evidence from in vitro experiments [ 4 Smalley M.E. Brown J. Plasmodium falciparum gametocytogenesis stimulated by lymphphocytes and serum from infected Gambian children. Trans. R. Soc. Trop. Med. Hyg. 1981; 75: 316-317 Abstract Full Text PDF PubMed Scopus (67) Google Scholar ], in addition to field observations [ 5 Coatney G.R. et al. The primate malarias. US Govt Printing Office, 1971 Google Scholar , 6 Carter R. Gwadz R. Infectious and gamete immunization in malaria. in: Kreier J. Malaria. Vol. 3. Academic Press, 1980: 263-297 Google Scholar ], that a developing immunity of the host might tend to increase the relative number of gametocytes. It is, however, perfectly clear that this is not a necessary condition [ 6 Carter R. Gwadz R. Infectious and gamete immunization in malaria. in: Kreier J. Malaria. Vol. 3. Academic Press, 1980: 263-297 Google Scholar ]. The first gametocytes appear in the circulation of a newly infected case of falciparum malaria approximately one week after the first parasitemia. Because the gametocytes of P. falciparum require eight to ten days to mature, the differentiation of these first gametocytes is unlikely to be dependent on host immunity. Perhaps the triggering of the gametocyte pathway is just a matter of chance. However, there is strong evidence that it is strikingly affected by a feature of reticulocytes or very young erythrocytes [ 7 Trager W. Gill G.S. Enhanced gametocyte formation in young erythrocytes by Plasmodium falciparum in vitro. J. Protozool. 1992; 39: 429-432 Crossref PubMed Scopus (65) Google Scholar , 8 Trager W. et al. Plasmodium falciparum: enhanced gametocyte formation in vitro in reticulocyte-rich blood. Exp. Parasitol. 1999; 91: 115-118 Crossref PubMed Scopus (59) Google Scholar ].

Plasmodium falciparum: Enhanced Gametocyte Formationin Vitroin Reticulocyte-Rich Blood

Trager, W., Gill, G. S., Lawrence, C., and Nagel, R. L. 1999.Plasmodium falciparum: Enhanced gametocyte formationin vitroin-reticulocyte-rich blood.Experimental Parasitology91,115–118. Concentrates of late stage parasites of the gametocyte-forming clone HB-3 were mixed with blood rich in reticulocytes from anemic patients, or with normal control blood, and kept under culture conditions for 4 days. Significantly more gametocytes were always formed in the reticulocyte-rich blood than in the control. This was true whether the anemic blood supported a larger asexual parasitemia than the control, or a lower one, or the same and without regard to the cause of the anemia. Gametocytes as a percentage of asexual forms were up to 10 times higher in reticulocyte-rich blood than in normal blood.

Ouabain-binding sites of reticulocytes from guinea pigs treated with triiodothyronine

To understand the mechanism of reduced number of sodium pump units (ouabain-binding sites, OBS) of erythrocytes in hyperthyroidism, OBS of reticulocytes from guinea pigs injected with triiodothyronine (T 3) were measured. Reticulocytosis was induced in 16 guinea pigs by withdrawing 6 to 10 mL of blood for 5 days followed by 2 days rest. T 3 ( 50 μ g 100 g body weight) was injected into seven animals on days 4 and 6, and the remainder were injected with solvent for T 3. Reticulocyte-rich blood was withdrawn on day 8. Oxygen consumption determined 24 hours after the first injection of T 3 was 30% higher in the T 3-treated animals ( P < .001). OBS of reticulocytes from control animals were sixfold higher than that of mature erythrocytes. Reticulocytes from T 3-treated guinea pigs had significantly lower OBS ( P < .01) compared with controls. We conclude that the loss of OBS induced by thyroid hormones probably occurs during the formation of reticulocytes.

Induction of the ATP-dependent proteolytic system in guinea pig reticulocyte lysates by triiodothyronine

The mechanism involved in the decreased numbers of several trans-membrane proteins such as sodium pump sites, sodium-lithium countertransport, sodium potassium cotransport proteins, proteins mediating the passive efflux of sodium and insulin receptors in erythrocytes from patients with hyperthyroidism is not known. The ATP-dependent proteolytic system which is involved in the loss of trans-membrane proteins during the maturation of the reticulocyte may be involved in the accelerated loss of these membrane proteins. Therefore, the effect of thyroid hormones on the ATP-dependent proteolytic activity of reticulocyte lysates was examined in this study. Reticulocytosis was induced in 14 guinea pigs by phenylhydrazine hydrochloride injections for 5 consecutive days followed by 2 days of rest. T 3 (10μg/100g body weight) was injected into 7 animals on day 4 and day 6. Reticulocyte-rich blood was withdrawn on day 8. Oxygen consumption determined 24 hours after injection of T 3 was 25% higher (p<0.01) and T 3 treated animals had a 2.5 fold higher (p<0.01) weight loss than control animals. The ATP-dependent proteolytic activity measured in reticulocyte lysates using 125I labelled lysozyme was 3.6 fold higher in the T 3 than in the control group of guinea pigs (p<0.01). We conclude that thyroid hormones induce the ATP-dependent proteolytic activity of reticulocyte lysates which may be responsible for the reduced number of several trans-membrane proteins found in erythrocytes from patients with hyperthyroidism.

Hemorheologic effects of reticulocytosis

Erythrocyte deformability indexes and packed cell viscosities of reticulocyte-rich blood samples of guinea pigs were compared with the values obtained from the same animals at the beginning of the experiments. Both of these parameters were increased in reticulocyte rich samples. Erythrocyte deformability indexes correlated with the reticulocyte ratio of the blood samples, but there was no significant correlation between the packed cell viscosities and reticulocyte ratios.

A Soluble Adenosine Triphosphate-Dependent Proteolytic System in Human Peripheral Red Blood Cells

A soluble adenosine triphosphate (ATP)-dependent proteolytic system has been detected in human peripheral blood erythroid cells. Hemolysates prepared from reticulocyte-rich blood of subjects with autoimmune hemolytic anemia, treated pernicious anemia, and iron deficiency anemia or from pools of red blood cells enriched for reticulocytes by density gradient centrifugation were tested against a radioactive casein standard. Up to 57% of the casein was rendered trichloroacetic acid (TCA) soluble after incubation with such hemolysates for 60 minutes in the presence of 1.0 mmol/L ATP. In the absence of ATP or in hemolysates prepared from reticulocyte-poor blood as little as 6% to 10% of the casein was hydrolyzed. The proteolytic activity was found in the 100,000-g supernatant of active hemolysates and was blocked by hemin, N-ethylmaleimide, and sodium vanadate and thus resembles a previously described activity in rabbit reticulocytes. In the presence of ATP, similar lysates prepared from rabbit reticulocytes preferentially hydrolyzed the abnormal human hemoglobins Leiden and Gun Hill compared with hemoglobin A. These results suggest that there is an active ATP-dependent proteolytic system in young human erythroid cells that can degrade certain abnormal globin chains; the enzymatic activity is lost in the transition from reticulocyte to erythrocyte.

A soluble adenosine triphosphate-dependent proteolytic system in human peripheral red blood cells

A soluble adenosine triphosphate (ATP)-dependent proteolytic system has been detected in human peripheral blood erythroid cells. Hemolysates prepared from reticulocyte-rich blood of subjects with autoimmune hemolytic anemia, treated pernicious anemia, and iron deficiency anemia or from pools of red blood cells enriched for reticulocytes by density gradient centrifugation were tested against a radioactive casein standard. Up to 57% of the casein was rendered trichloroacetic acid (TCA) soluble after incubation with such hemolysates for 60 minutes in the presence of 1.0 mmol/L ATP. In the absence of ATP or in hemolysates prepared from reticulocyte-poor blood as little as 6% to 10% of the casein was hydrolyzed. The proteolytic activity was found in the 100,000-g supernatant of active hemolysates and was blocked by hemin, N- ethylmaleimide, and sodium vanadate and thus resembles a previously described activity in rabbit reticulocytes. In the presence of ATP, similar lysates prepared from rabbit reticulocytes preferentially hydrolyzed the abnormal human hemoglobins Leiden and Gun Hill compared with hemoglobin A. These results suggest that there is an active ATP- dependent proteolytic system in young human erythroid cells that can degrade certain abnormal globin chains; the enzymatic activity is lost in the transition from reticulocyte to erythrocyte.

Crude spectrin extraction from reticulocyte-rich blood samples

Crude spectrin was extracted from the isolated red cell ghosts with low ionic strength buffer at 37°C for 30 min. No significant alterations in crude spectrin extractability in wide range of patients with various hematologie diseases were observed. However, blood samples characterized by elevated reticulocytosis provided crude extracts with increased amount of non-heme membrane skeletal proteins. The presence of ribose in the crude spectrin extracts obtained from reticulocyte-rich blood samples indicates also the presence of nucleic acids which causes the shift of protein peak in the extract from 280 nm towards lower wavelengths. A model experiment with a normal crude spectrin extract mixed with various amounts of RNA allowed us to obtain the correction curve which served for determination of non-heme protein (crude spectrin) extractability.

Abnormality of phospholipid transverse diffusion in sickle erythrocytes.

We have used spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to compare the transverse diffusion rates of lipids in normal and sickle erythrocytes. The beta-chain of the spin-labeled lipids was a short chain (five carbons) providing the spin labels with a relative water solubility, and hence permitting their rapid incorporation into cell membranes. The orientation of the labeled lipids in the membranes was assayed by selective chemical reduction of the nitroxide labels embedded in the outer leaflet. We have found that all three spin-labeled phospholipids are initially incorporated in the outer leaflet. Upon incubation at 4 degrees C the aminophospholipids, not the phosphatidylcholine, diffuse toward the inner leaflet within 3 h. The transverse diffusion rate of aminophospholipids is reduced by 41% (phosphatidylserine) and 14% (phosphatidylethanolamine) in homozygote sickle cells (SS) when compared with normal cells (AA) or heterozygote cells (AS or SC). At equilibrium the asymmetric distribution of spin-labeled phospholipids resulting from this selective diffusion is also reduced in SS cells when compared with AA, SC, or AS cells. This reduced asymmetry was not found in a reticulocyte-rich blood sample (hemoglobin A), indicating that the age of the cell cannot be responsible for this phenomenon. Moreover, because at low temperatures the sickling process does not occur, the observed perturbations in phospholipid organization reflect preexisting membrane abnormalities in sickle cells. Ghosts loaded with ATP give the same results. Varying the concentration of intracellular calcium had no effect on lipid diffusion, except at very high free calcium concentrations (3 microM) when diffusion was practically abolished. We suggest that membrane protein alterations may be part of the explanation of the observed abnormalities.

Effect of transfused reticulocytes on iron exchange

A animal model was developed whereby reticulocyte-rich blood was introduced into normal rats by exchange transfusion. Measurements of plasma iron turnover was made at similar plasma iron concentrations before and after exchange transfusions. High reticulocyte blood obtained from animals rendered iron deficient by diet or by treatment with phenylhydrazine resulted in a mean increase of 86% in internal iron exchange, while the plasma iron turnover was unaffected by exchange with normal red cells. Since iron input from reticuloendothelial cells could have increased due to breakdown of transfused cells, iron absorption was also measured. Within 1 hr and for a least 6 hr after exchange with high reticulocyte blood, mean absorption in six groups of animals was increased over control animals by 50%-130%. The increased plasma iron turnover and absorption was not mediated by a decrease in plasma iron or an increase in unsaturated iron-binding capacity. Indeed, a higher plasma iron and transferrin saturation augmented the movement of iron into the plasma from iron-donating tissues. It is proposed that the donation of iron by transferrin in some way immediately facilitates the procurement of more iron by transferrin.

Effect of transfused reticulocytes on iron exchange

A animal model was developed whereby reticulocyte-rich blood was introduced into normal rats by exchange transfusion. Measurements of plasma iron turnover was made at similar plasma iron concentrations before and after exchange transfusions. High reticulocyte blood obtained from animals rendered iron deficient by diet or by treatment with phenylhydrazine resulted in a mean increase of 86% in internal iron exchange, while the plasma iron turnover was unaffected by exchange with normal red cells. Since iron input from reticuloendothelial cells could have increased due to breakdown of transfused cells, iron absorption was also measured. Within 1 hr and for a least 6 hr after exchange with high reticulocyte blood, mean absorption in six groups of animals was increased over control animals by 50%-130%. The increased plasma iron turnover and absorption was not mediated by a decrease in plasma iron or an increase in unsaturated iron-binding capacity. Indeed, a higher plasma iron and transferrin saturation augmented the movement of iron into the plasma from iron- donating tissues. It is proposed that the donation of iron by transferrin in some way immediately facilitates the procurement of more iron by transferrin.

Plasmodium berghei: Glycolytic intermediate concentrations of the infected mouse erythrocyte

The concentrations of glycolytic intermediates and related metabolites were measured in blood from three groups of mice: (1) mice infected with the malarial parasite Plasmodium berghei; (2) mice with an induced reticulocytosis; and (3) uninfected control mice. The glycolytic intermediate concentrations of parasitized versus normal blood samples were not grossly different, yet the differences appeared to correlate well with previous in vitro observations of enzyme changes in infected red blood cells. The changes in intermediate pool sizes in P. berghei-infected blood as compared to normal blood indicate the presence of unique glycolytic control mechanisms. Finally, the intermediate pattern of infected blood was related to the presence of the parasite and did not correlate with the intermediate pattern of normal or reticulocyte-rich blood.

Plasmodium berghei: Glycolytic enzymes of the infected mouse erythrocyte

This paper documents the maximal activities of the glycolytic enzymes in the red blood cells of normal mice and mice infected with Plasmodium berghei. There appears to be sufficient parasite-related activity of each glycolytic enzyme to support the increased glycolytic rate, i.e., increased glucose consumption, of the parasite-infected red blood cell. The relative proportions of glycolytic enzyme activities in parasite-infected red cells are different from the proportions in either normal or reticulocyte-rich blood, indicating that the increased enzyme activities associated with infected cells are not due to contaminating host red cells or reticulocytes. A comparison of maximal enzyme activities to the rate of whole cell glucose consumption indicates that different glycolytic control mechanisms are operating in the infected RBC from those in the uninfected cells.

Unstable triosephosphate isomerase (TPI)

This study was undertaken in the investigation of a child with chronic non-spherocytic haemolytic anaemia. B.C. was born in 1977 of unrelated parents. His father has β-thalassaemia minor. He required an exchange transfusion for neonatal jaundice and subsequently has had persistent anaemia, splenomegaly and intermittent jaundice. Delayed gross motor development was noted at 6 mth of age, and increasing spasticity from 14 mth. Haemoglobin electrophoresis and globin chain synthesis studies established that B.C. also had thalassaemia minor. Microassays of 23 enzymes of the glycolytic and pentose phosphate pathways, and some enzymes and metabolites involved in glutathione metabolism were undertaken. Since his red cell population was invariably young, it was anticipated that increased activity of many enzymes known to be increased in reticulocyte-rich blood would be detected. This was found to be so, with the exception of TPI which was present at normal to low levels. This enzyme was shown to be unstable when haemolysate was stored on ice for 1 h (activity fell from 1973 I.U./g Hb to zero, control 3695 to 3096 I.U./g Hb) and when whole red cells were stored at –20°C (B.C. 1937 to 64 I.U/g Hb, control 3695 to 3068 I.U./g Hb). However, these phenomena did not clearly distinguish between the homozygous individual and presumed heterozygotes in a family study. Heat inactivation studies showed a clearer distinction, allowing for the possibility of antenatal diagnosis of this defect. Methods have been established for measurement of TPI in cultured foetal fibroblasts, and it is now planned to examine cultured fibroblasts from B.C. and his parents. The problems of identification of a TPI deficiency variant and the association with thalassaemia minor are discussed.

A role of red cell pyrimidine 5?-nucleotidase in experimental lead poisoning

Intravenous administration of lead acetate to rabbits for 10 weeks at 2 week intervals resulted in significantly elevated blood lead levels, slight anemia with marked microspherocytosis and moderate basophilic stippling, and marked depression of red cell delta-aminolevulinic acid (ALA) dehydratase activity. However the decrease in red cell pyrimidine 5'-nucleotidase (P5N) activity was slight when compared to the red cell P5N activity of comparable reticulocyte-rich blood, and intracellular accumulation of pyrimidine nucleotides could not be demonstrated. In the in vitro inhibition test the same degree of inhibition of red cell P5N activity seen in hereditary red cell P5N deficiency was obtained by using a lead concentration 200--400 times higher than the lead levels detected in human plumbism. Most importantly, there were no differences in the lead-induced inhibition of human and rabbit red cell P5N. From the results of the in vitro inhibition test, lead-induced red cell P5N deficiency appears to be one of several pathogenic mechanisms in chronic lead exposure associated with the accumulation of lead in bone marrow. A decrease in rec cell P5N activity could not be demonstrated despite the marked depression in red cell ALA dehydratase activity, and slight anemia with marked microspherocytosis and moderate basophilic stippling in this experiment. These results suggest that lead affects red cells at multiple metabolic loci.

Study of a case with severe red-cell pyrimidine 5'-nucleotidase deficiency

A deficiency of erythrocyte pyrimidine 5'-nucleotidase was found in a 23-year-old male suffering from severe congenital hemolytic disease. Results from exhaustive metabolic exploration are given and compared with reticulocyterich blood from subjects with auto-immune hemolytic disease. Evidence is given that the 20% apparent P5N residual activity corresponds to a non-specific acid phosphatase.

Maturation dependent decline of adenylate cyclase in rabbit red blood cell membranes

Adenylate cyclase (EC 4.6.1.1) was studied in membrane preparations of reticulocyte-rich blood obtained from phenylhydrazine-treated rabbits and compared to that of untreated animals. Basal and fluoride-stimulated activities were decreased 2- and 4-fold, respectively, during the process of maturation. Catalytic parameters such as time course, protein, ATP, Mg 2+ concentration curves and K m have been determined and were found to be similar in the reticulocyte and the erythrocyte. Adenylate cyclase was sensitive to GTP, 5′-guanylyl imidodiphosphate, prostaglandin E 1 and prostaglandin E 2. Activation by prostaglandin E 1 was higher than that produced by prostaglandin E 2. Only additive effect was found when 5′-guanylyl imidodiphosphate or GTP was added to hormone-stimulated activity. The sensitivity of the enzyme to these effectors was decreased over the transition reticulocyte-erythrocyte. In either cell the enzyme was not activated by catecholamines (epinephrine, norepinephrine, isoproterenol).

Changes in membrane fluidity of erythrocytes during cell maturation

The measurements of the fluorescence polarization of perylene embedded in erythrocyte membranes were carried out with normal and reticulocyte-rich blood, and the microviscosity of erythrocyte membranes was calculated from the polarization degree. In intact cells, reticulocyte membranes had a significantly lower microviscosity than normal erythrocyte membranes, while in ghosts no significant difference in membrane microviscosity was observed between reticulocytes and mature erythrocytes.

Hereditary hemolytic anemia with increased red cell adenosine deaminase (45- to 70-fold) and decreased adenosine triphosphate.

Hereditary hemolytic anemia, a dominantly transmitted disorder, has affected 12 family members spanning three generations. The concentration of adenosine triphosphate in the red cells was about half that of comparably reticulocyte-rich blood. Since adenosine deaminase and adenosine kinase compete for a common substrate, the greatly increased activity of the former may interfere with nucleotide salvage via the latter.

Effect of lead on globin synthesis in vitro.

A defect in heme synthesis is well documented to occur in lead intoxication. Globin synthesis and the alpha/beta globin chain synthesis ratio have been shown to be disturbed in lead poisoning. To elucidate further the nature of the inhibitory effect of lead on hemoglobin synthesis, reticulocyte-rich peripheral blood samples have been incubated with lead at concentrations of 10(-6), 10(-5), and 10(-4) M, equivalent to 20, 200, and 2,000 mug/dl (.96, 9.6 and 96.0 mumol/l), respectively. The incorporation of tritiated leucine into globin was determined. Globin chain synthesis decreased to 85, 49 and 15% of the control value with increasing lead concentrations. The effect of heme on inhibition of globin synthesis by lead was studied by incubating reticulocyte-enriched blood with lead 10(-5) M (200 mug/dl; 9.6 mumol/l) in the presence or absence of heme at a concentration of 10(-3) M. Lead alone depressed the incorporation of tritiated leucine to 45% of control, while globin synthesis with heme alone was 109% of control. Although in the presence of both heme and lead, the mean incorporation of leucine into globin was 69.2% of control, the increase was not significantly different from that obtained with lead alone. It is concluded that (1) lead has an inhibitory effect on globin synthesis at a concentration considered to be within the acceptable range encountered in environmental lead pollution, and the extent of inhibition is dose-dependent; (2) the effect of lead on globin synthesis is not significantly prevented by heme and, therefore, does not appear to be mediated through the well-documented inhibitory effect of lead on heme synthesis.