Transgenic mice expressing human sickle hemoglobin are partially resistant to rodent malaria

The polymorphic frequency of the gene for beta s-globin involved in the generation of sickle trait and sickle cell anemia in the human population is caused by the enhanced resistance of sickle trait individuals to Plasmodium falciparum malaria, as supported by epidemiologic and in vitro studies. However, the mechanism for the protective effect of sickle hemoglobin in vivo has not been fully defined. The generation of transgenic mice expressing high levels of human beta s- and alpha-chains has allowed us to study this phenomenon in vivo in an experimental model. We infected the transgenic beta s mice with two species of rodent malaria and found a diminished and delayed increase in parasitemia as compared with controls. This is in contrast to our previous studies involving the introduction of a beta A transgene, which does not alter the infection. The use of this model allowed us to address the question of the mechanism of protection against malaria in mice expressing sickle hemoglobin. We find that splenectomy of transgenic mice completely reverses the protection against Plasmodium chabaudi adami infection. The results reported have shown a relationship between the presence of the beta s gene product and partial resistance to malaria in an experimental model in vivo and shows that the spleen plays an important role in this protection.     

Lymphocyte depletion during treatment with intensive chemotherapy for cancer

Recently we have observed an increased incidence of opportunistic infections in patients treated with intensive chemotherapy for cancer. Because T-cell depletion is associated with similar clinical events in human immunodeficiency virus infection and after bone marrow transplantation, we have analyzed peripheral blood lymphocyte populations in a series of patients during treatment with intensive chemotherapy for cancer. Although neutrophil, monocyte, and platelet numbers consistently recovered to greater than 50% of pretreatment values after each sequential cycle of therapy, lymphocyte numbers did not recover within the same time period. B cells decreased rapidly from a mean value of 149 +/- 46/mm3 before chemotherapy to 4 +/- 1/mm3 during chemotherapy (P = .01). CD4+ T cells decreased from a mean of 588 +/- 76/mm3 before chemotherapy to 105 +/- 28/mm3 during chemotherapy (P = .0002) and CD8+ T cells decreased from a mean of 382 +/- 41/mm3 before chemotherapy to 150 +/- 46/mm3 during chemotherapy (P = .0009). Natural killer cell numbers did not show significant declines (171 +/- 30/mm3 before, 114 +/- 24/mm3 during, P = .19). Based on the history of opportunistic complications in patients with other disorders who display similar degrees of CD4+ T-cell lymphopenia and preliminary observations in this population, immune incompetence could surface as a dose-limiting toxicity for highly dose-intensive chemotherapy regimens.     

Further evidence of the in vivo role of erythropoietin or companion molecules induced by hypoxia on proliferation and continuing differentiation of BFU-e in PCDC

Normal and plethoric bone marrow cells were grown in plasma clot diffusion chambers (PCDC) implanted into the peritoneum of normal mice or mice submitted to 7 her of hypoxia (23,000 ft) daily, on a single day or on 2 consecutive days at different times after implantation of the PCDC's. Daily discontinuous hypoxia (DDH) produced more 6-day bursts than other treatments. Hypoxia on days 1 and 2 after implantation was nearly as effective as DDH on day-6 bursts. Later bouts of hypoxia or a singly hypoxic exposure on day 1 or 2 was less effective. Erythropoietin (Ep) levels were measured by bioassay on both diffusion chamber (DC) contents and serum. Serum Ep levels peaked at 160 mU/ml after a 7-hr hypoxic exposure while the DC content Ep levels were in the nondetectable range (less than 50 mU/ml). The data implies that either higher than normal Ep levels or a companion molecules (s) produced by hypoxia are required for 1-2 days early in the culture period of force an increasing number of BFU-d-e down the erythrocytic pathway and thus increase red cell production at times of need in vivo.     

Phospholipase abolishes the effect of stimulated platelets on the thrombin activation of factor VIII

Factor VIII functions as a cofactor in the intrinsic coagulation pathway and must first be activated to function optimally in this capacity. Low concentrations of thrombin activate factor VIII, and the presence of stimulated platelets is known to enhance the activation of factor VIII complexed to von Willebrand factor. The current studies show that platelets stimulated by thrombin, collagen, or calcium ionophore will increase the activation of isolated factor VIII by thrombin. Ongoing platelet release is not necessary for the enhanced factor VIII activation, nor is platelet von Willebrand factor or platelet membrane glycoproteins Ib or IIb/IIIa. Platelet membrane phospholipids, on the other hand, are important for the enhanced activation of factor VIII by thrombin because the effect of stimulated platelets is abolished by incubation of the stimulated platelets with phospholipases. These results suggest that the enhanced activation of factor VIII by thrombin in the presence of stimulated platelets may be mediated by factor VIII binding to platelet phospholipid or to a receptor whose functional integrity is dependent on surrounding membrane phospholipid.     

Association between morphologic distortion of sickle cells and deoxygenation-induced cation permeability increase

We hypothesized that the deoxygenation-induced increase in cation permeability of sickle cells was related to mechanical distention of the membrane by growing HbS polymer within the cell. To test this hypothesis, we determined the effect of deoxygenation on cation fluxes in sickle cells under conditions that restricted or permitted extensive growth of polymer, producing different degrees of membrane distention. Manipulation of suspending medium osmolality for density-isolated high and low mean cell hemoglobin concentration (MCHC) cells was used to regulate the extensional growth of polymer bundles and hence membrane distortion. For initially low MCHC cells, the deoxygenation-induced increase in both Na and K fluxes was markedly suppressed when the MCHC was increased by increasing the osmolality. This suppression corresponded to the inhibition of extensive morphologic cellular distortion. For initially high MCHC, ISC-rich cells, deoxygenation had minimal effect on K permeability. However, reduction of MCHC by a decrease in osmolality produced a concomitant increase in cation permeability and cellular distortion. These observations support the idea that the sickling-associated increase in membrane permeability is related to mechanical stress imposed on the membrane by bundles of HbS polymer.     

Paroxysmal nocturnal hemoglobinuria erythrocytes are of two distinct types: positive or negative for acetylcholinesterase

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder. Erythrocytes isolated from PNH patients show increased sensitivity to complement and decreased acetylcholinesterase (AChE) activity. In this study, indirect immunofluorescence analysis of a monoclonal antibody specific for a surface epitope of human erythrocyte AChE is used to quantitate the content of this enzyme at the single-cell level. Flow- cytofluorimetric analysis of erythrocytes from normal donors indicates that all erythrocytes contain detectable levels of the surface epitope with a strong correlation between cell size and enzyme content. In contrast, erythrocytes from PNH patients show two distinct populations of erythrocytes; namely, those containing a normal content of AChE and a second population containing no detectable AChE. The AChE-negative population of cells is quantitatively complement-sensitive. These data support suggestions that PNH is a clonal disorder resulting in two distinct types of circulating erythrocytes. The abnormal clone produces cells that are both surface-AChE-negative and complement-sensitive. In addition, the method described provides an attractive alternative for the diagnosis and quantitative evaluation of abnormal erythrocytes in PNH patients.     

Treatment of hypoplastic anemia in mice with placental transplants

A genetic mutation in mice (W/Wv) causes an autosomal recessive disease characterized by hypoplastic anemia which lasts throughout life. Double- dominant W/Wv anemic mice were sublethally irradiated to facilitate repopulation of marrow with transplanted cells and were injected intravenously with suspensions of 5-10 million placental cells of 15 days gestation derived from normal, isogeneic donors. Red cell counts fell promptly after irradiation and then rose progressively over a period of weeks, reaching normal levels of the nonmutant. Mean corpuscular volume and hemoglobin electrophoresis patterns of red cells in recipient W/Wv mice resembled those of normal donor animals. The therapeutic effect lasted for the duration of the observation period, in some instances over 9 mo. W/Wv mice that were administered Hanks' solution or fetal blood, instead of placental transplants, remained anemic. Late gestation placentas (18 days) were also ineffective.