Low red cell production may protect against severe anemia during a malaria infection—Insights from modeling

The malaria parasite causes lysis of red blood cells, resulting in anemia, a major cause of mortality and morbidity. Intuitively, one would expect the production of red blood cells to increase in order to compensate for this loss. However, it has been observed that this response is weaker than would be expected. Furthermore, iron supplementation for iron deficient children in malaria endemic regions can paradoxically adversely affect the clinical outcome of malaria infection. A possible explanation may lie in the preference that some malaria parasites show for infecting immature red blood cells (reticulocytes). In the presence of a parasite preference for immature red cells, a rise in red cell production can ‘fuel the fire’ of infection by increasing the availability of the parasite's preferred target cell.We present a mathematical model of red blood cell production and infection in order to explore this hypothesis. We assess the effect of varying the reticulocyte replacement rate and preference of the parasite for reticulocytes on four key outcome measures assessing anemia and parasitemia.For a given level of parasite preference for reticulocytes we uncover an optimal erythropoietic response which minimizes disease severity. Increasing red blood cell production much above this optimum confers no benefit to the patient, and in fact can increase the degree of anemia and parasitemia. These conclusions are consistent with epidemiological studies demonstrating that both iron deficiency and anemia are protective against severe malaria, whilst iron supplementation in malaria endemic regions is with an increased number of malaria related adverse effects. Thus, suppression of red blood cell production, rather than being an unfortunate side effect of inflammation, may be a host protective effect against severe malarial anemia.     

Plasmodium yoelii: effects of red blood cell modification and antibodies on the binding characteristics of the 235-kDa rhoptry protein

The 235-kDa rhoptry protein of the rodent malaria parasite Plasmodium yoelii yoelii was shown to bind to the surface of mouse red blood cells in a calcium-independent process, using a erythrocyte-binding assay. This binding is affected by modification of the surface of the red blood cells by enzymatic treatment. Chymotrypsin and trypsin but not neuraminidase treatment of the erythrocytes significantly reduced the binding of the 235-kDa proteins. The binding of an unrelated 135-kDa protein was abolished by treatment with chymotrypsin. Although the 235-kDa proteins bind to both reticulocytes and mature red blood cells, the binding to mature cells was more pronounced. In the presence of hyperimmune infection serum or specific polyclonal antibodies to the 235-kDa protein its binding to erythrocytes was reduced, further demonstrating the specificity of this ligand-receptor interaction.     

Plasmodium berghei: relative immunogenicity of infected reticulocytes and infected oxyphilic red blood cells

Hyperbleeding of mice 1 day before and 1 day after infection with Plasmodium berghei resulted in a more aggravated infection. Parasitemia rose significantly faster, but the mean survival time of these mice was not significantly different from control mice. At Day 5 of infection, parasites were almost exclusively in reticulocytes in contrast to control infections in which parasites were found in oxyphilic erythrocytes at Day 5 after infection. Purified parasitized reticulocytes taken from hyperbled mice at Day 5 after infection contained more young developmental parasite stages than purified parasitized oxyphilic erythrocytes taken from normal mice at Day 5 to 7 after infection. Parasitized reticulocytes were more readily opsonized by antibodies from immune serum when compared to parasitized oxyphilic red blood cells and when used to stimulate immune spleen cells the former were better stimulator cells than the latter. Results suggest either that parasitized reticulocytes are more immunogenic then parasitized oxyphilic red blood cells or that suspensions of parasitized reticulocytes contain more immunogenic parasite stages than suspensions of parasitized oxyphilic red blood cells.     

Oxidative stress and rheology in severe malaria

There is mounting evidence that the release of haemozoin (beta-haematin), which is produced in large amounts during malaria infection and is released into the circulation during schizont rupture, is associated with damage to cell membranes through an oxidative mechanism. The red blood cell membrane is thus oxidised, causing rigidity of the cell. This can contribute to the pathophysiology of severe malaria, since red blood cells will have to deform considerably in order to squeeze through the microcirculation, the patency of which is disturbed by sequestered red blood cells containing the mature forms of the parasite. Rigidity of red blood cells forms a new target for intervention. Since this seems to be caused by oxidative damage to the red blood cell membrane, the anti-oxidant N-acetylcysteine is a promising candidate for adjunctive treatment in severe malaria, which still has a mortality rate as high as 20%.     

Lipid antigens derived from erythrocytes infected with Plasmodium berghei

Lipids were extracted from red blood cells infected with Plasmodium berghei, from the membranes of infected red cells and from free parasites. A radioimmunoassay was used to detect antibodies to these lipids in sera from convalescent and immune rats. Most of the antigenic activity could be attributed to the parasite although some activity was found in lipids isolated from the membranes of infected red blood cells. Absorption studies showed that the binding was specific for malarial lipid antigens. Immune sera showed no cross-reactivity with lipids from red blood cells of non-infected rats. However, sera from non-infected control rats showed low levels of cross-reactivity with the parasitized red cell-derived lipids. Levels of anti-lipid antibodies were directly correlated with the progress of the infection. The highest antibody level occurred when the parasitaemia reached zero. The malarial lipids had no effect on lymphoblast transformation of immune splenocytes in vitro. However, liposomes prepared from either malarial or non-specific lipids caused an increased response to antigen by the blast cells.     

Oxidative stress and rheology in severe malaria

Abstract

There is mounting evidence that the release of haemozoin (β-haematin), which is produced in large amounts during malaria infection and is released into the circulation during schizont rupture, is associated with damage to cell membranes through an oxidative mechanism. The red blood cell membrane is thus oxidised, causing rigidity of the cell. This can contribute to the pathophysiology of severe malaria, since red blood cells will have to deform considerably in order to squeeze through the microcirculation, the patency of which is disturbed by sequestered red blood cells containing the mature forms of the parasite. Rigidity of red blood cells forms a new target for intervention. Since this seems to be caused by oxidative damage to the red blood cell membrane, the anti-oxidant N-acetylcysteine is a promising candidate for adjunctive treatment in severe malaria, which still has a mortality rate as high as 20%.     

Kinetics of antigen specific and non-specific polyclonal B-cell responses during lethal Plasmodium yoelii malaria.

In order to study the kinetics and composition of the polyclonal B-cell activation associated to malaria infection, antigen-specific and non-specific B-cell responses were evaluated in the spleens of mice infected with Plasmodium yoelii 17XL or injected with lysed erythrocytes or plasma from P. yoelii infected mice or with P. falciparum culture supernatants. Spleen/body weight ratio, numbers of nucleated spleen cells and Immunoglobulin-containing and Immunoglobulin-secreting cells increased progressively during the course of infection, in parallel to the parasitaemia. A different pattern of kinetics was observed when anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell plaque forming cells response were studied: maximum values were observed at early stages of infection, whereas the number of total Immunoglobulin-containing and Immunoglobulin-secreting cells were not yet altered. Conversely, at the end of infection, when these latter values reached their maximum, the anti-sheep red blood cell and anti-trinitrophenylated-sheep red blood cell specific responses were normal or even infranormal. In mice injected with Plasmodium-derived material, a higher increase in antigen-specific PFC was observed, as compared to the increase of Immunoglobulin-containing and Immunoglobulin-secreting cell numbers. This suggested a "preferential" (antigen-plus mitogen-induced) stimulation of antigen-specific cells rather than a generalized non-specific (mitogen-induced) triggering of B-lymphocytes. On the basis of these and previous results, it is suggested that the polyclonal B-cell activation that takes place during the course of infection appears as a result of successive waves of antigen-specific B-cell activation.     

The importance of the spleen in malaria

There are several malaria vaccine candidates at various stages of development. Many of these target blood stages of Plasmodium falciparum. The spleen is a key site for removal of parasitized red blood cells, generation of immunity and production of new red blood cells during malaria. This article describes how all of these processes are modified following infection, and suggests that until we fully understand how these processes function and are modulated by infection, appropriate malaria vaccine design and delivery will be extremely difficult to achieve.     

The exported Plasmodium berghei protein IBIS1 delineates membranous structures in infected red blood cells

The importance of pathogen-induced host cell remodelling has been well established for red blood cell infection by the human malaria parasite Plasmodium falciparum. Exported parasite-encoded proteins, which often possess a signature motif, termed Plasmodium export element (PEXEL) or host-targeting (HT) signal, are critical for the extensive red blood cell modifications. To what extent remodelling of erythrocyte membranes also occurs in non-primate hosts and whether it is in fact a hallmark of all mammalian Plasmodium parasites remains elusive. Here we characterize a novel Plasmodium berghei PEXEL/HT-containing protein, which we term IBIS1. Temporal expression and spatial localization determined by fluorescent tagging revealed the presence of IBIS1 at the parasite/host interface during both liver and blood stages of infection. Targeted deletion of the IBIS1 protein revealed a mild impairment of intra-erythrocytic growth indicating a role for these structures in the rapid expansion of the parasite population in the blood in vivo. In red blood cells, the protein localizes to dynamic, punctate structures external to the parasite. Biochemical and microscopic data revealed that these intra-erythrocytic P. berghei-induced structures (IBIS) are membranous indicating that P. berghei, like P. falciparum, creates an intracellular membranous network in infected red blood cells.     

Red Blood Cells Preconditioned with Hemin Are Less Permissive to Plasmodium Invasion In Vivo and In Vitro

Malaria is a parasitic disease that causes severe hemolytic anemia in Plasmodium-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of Plasmodium immunity in vitro and in vivo, mice preconditioned with hemin develop lower parasitemia when challenged with Plasmodium chabaudi adami blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and P. c. adami infection. This strategy allowed discriminating hemin-treated from de novo generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased P. c. adami red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in in vitro P. falciparum cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during in vivo infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host.     

Possible mechanisms responsible for the increased ascorbic acid content of Plasmodium vinckei-infected mouse erythrocytes

The possible mechanisms underlying the acquisition of an increased ascorbic acid content by mouse erythrocytes containing the malarial parasite Plasmodium vinckei were investigated. Ascorbic acid was taken up readily by parasitized red blood cells but not by controls, whilst its partly oxidized form, dehydroascorbic acid, entered both. The uptake of both ascorbic acid and dehydroascorbic acid into erythrocytes was increased as a result of malarial infection. Lysates prepared from parasitized red blood cells reduced exogenous dehydroascorbic acid to ascorbic acid at a higher rate than control red blood cell lysates; this difference was abolished following dialysis of the lysates, a process which removes endogenous reduced glutathione (GSH). The rates of chemical and enzymatic reduction of dehydroascorbic acid to ascorbic acid by GSH were of similar magnitude, thus calling into question the existence of a specific dehydroascorbate reductase in erythrocytes and parasites. These observations suggest that the increased uptake of dehydroascorbic acid into parasitized red blood cells may be a result of enhanced dehydroascorbate-reducing capacity, whilst the presence of the parasite induces a selective increase in the permeability of the erythrocyte plasma membrane to ascorbic acid. The endogenous ascorbic acid content of livers obtained from infected mice was 55% below the normal concentration and its relative rate of destruction during incubation in vitro was enhanced in comparison with that of control livers. Furthermore, the capacity of liver homogenates to synthesize ascorbic acid from glucuronic acid was greatly reduced in infected mice. Therefore it is unlikely that the increase in ascorbic acid content of parasitized red blood cells is a consequence of increased biosynthesis and release of ascorbic acid by the host liver. We have not been able to exclude the possibility that the malarial parasite itself may be capable of de novo synthesis of ascorbic acid.     

Changes in the immunotropic activity of neutrophilokins in the course of experimental staphylococcal infection

The influence of the products secreted by activated neutrophils (neutrophilokins) of mice, both intact and infected with staphylococci, on the activity of mouse spleen cells in the graft-versus-host reaction, immune response to sheep red blood cells and the antigen-presenting function of peritoneal macrophages was studied. Neutrophilokins of intact mice stimulated the activity of immunocompetent cells. Neutrophilokins obtained from infected mice on day 3 after infection produced an immunosuppressing effect. On day 7 after infection the immunostimulating activity of neutrophils was restored and showed practically no difference from the normal level.     

Phagocytic capacity of Kupffer cells during hepatic amoebiasis in guinea pigs.

Very few studies have been carried out on the role of liver macrophages (Kupffer cells) during the course of hepatic amoebiasis. The kinetics of phagocytic activity of Kupffer cells and blood monocytes was studied in guinea pigs intra-mesenterically infested with Entamoeba histolytica. The phagocytic capacity of blood monocytes of normal animals was comparatively lower than Kupffer cells for both latex and haemolysin coated sheep red blood cells. Significant decline in phagocytic response of Kupffer cells and blood monocytes of infected animals was observed right from 2nd post infection day and it kept on decreasing with the progress of infection. Depression in phagocytic response of Kupffer cells and blood monocytes was more marked in those animals who had higher grades of pathological lesions. Hence, an inverse correlation was obtained between the phagocytic capacity and severity of amoebic lesions (P less than 0.01). The significance of depression in phagocytic response of Kupffer cells and blood monocytes may be responsible for the development of hepatic lesions.     

Adenine nucleotide content and energy charge of Azotobacter vinelandii during batch growth in dialysed-soil medium

Abstract In human peripheral blood, a factor which induces gonococcal resistance to complement-mediated killing by fresh human serum is more concentrated in the white blood cells of buffy coat than in red blood cells. Futhermore, the resistance-inducing factor is present in both polymorphonuclear phagocytes and mononuclear cells (monocytes and lymphocytes) separated from the buffy coat by centrifugation on Ficoll-Hypaque gradients. These results imply that inflammatory cells mobilised from the blood to sites of infection carry a host factor which, if it is available to the gonococci, would materially increase their ability to resist a major defence mechanism and hence enhance their capacity to maintain and increase infection.     

Cost of allogeneic and autologous blood transfusion in Canada. Canadian Cost of Transfusion Study Group.

OBJECTIVE: To determine the cost, from a societal perspective, of blood transfusion in Canada. STUDY DESIGN: Cost-structure analysis. SETTING: Data were collected from eight hospitals and from six blood centres operated by the Canadian Red Cross Society in four provinces. OUTCOME MEASURES: Costs associated with four stages of transfusion-- collection, production, distribution and delivery--in 1933 were assessed. Costs were divided into the following categories; personnel, purchases, external services, overhead, donors'' time, patients'' time (for autologous transfusion), wastage and infection. RESULTS: The mean overall cost of a transfusion performed on an inpatient basis was $210 per unit of red blood cells for an allogeneic transfusion and $338 per unit of blood for an autologous transfusion. The mean cost of an allogeneic transfusion performed on an outpatient basis was $280 per unit of red blood cells. CONCLUSION: The costs determined in this study can be used in future studies comparing the cost-effectiveness of allogeneic transfusion with that of alternative methods.     

A Pirhemocyton-like parasite of the Blenny, Blennius pholis L. (Teleostei; Blenniidae) and its relationship to Immanoplasma Neumann, 1909

A light and electron microscopic study was made of a Pirhemocyton-like infection of the red blood cells of B. pholis, an intertidal fish. Electron micrographs show that polygonal particles occur in the cytoplasm of infected cells; these particles resemble the supposed icosahedral virus of Pirhemocyton. Inclusion bodies associated with the infection also resemble those seen in Pirhemocyton.Immanoplasma Neumann, 1909, was re-examined from blood films taken from three infected Scyliorhinus canicula collected in 1970. A marked similarity was noted between the inclusion bodies of the Blenny infection, Pirhemocyton and the Immanoplasma body. The nature of the numerous particles in the infected red blood cells of S. canicula is not known, therefore, Immanoplasma is only tentatively included in the group of known and suspected icosahedral cytoplasmic deoxyriboviruses.     

Autoantibodies to the thrombocytes and erythrocytes in HIV-infected patients

In 48 patients with HIV infection were tested for the presence of autologous and allogenic antibodies to red blood cells with the use of Coombs' direct and indirect tests. 18 HIV-infected patients had IgG antibodies to thrombocytes, circulating in the blood (detected by the method of EIA) and bound to thrombocytes (detected by the method of RIA). In 5 out of 48 patients Coombs' direct test yielded positive results with red blood cells. 6 out of 18 examined patients had an elevated content of thrombocyte-bound antibodies. The presence of cross reactions between gp120 of human immunodeficiency virus and gp3a thrombocytes led to the formation of antithrombocyte antibodies and, consequently, to a decrease in the number of thrombocytes.     

Does activation of the blood coagulation cascade have a role in malaria pathogenesis?

Plasmodium falciparum infection is often associated with a procoagulant state. Recent identification of tissue factor in the brain endothelium of patients who have died from cerebral malaria casts new light on our understanding of the coagulation disorder found in P. falciparum infection. It has also been revealed that parasitized red blood cells support the assembly of multimolecular coagulation complexes. Tissue factor expression by the endothelium and amplification of the coagulation cascade by parasitized red blood cells and/or activated platelets (particularly at sequestration sites) have crucial roles in mounting and sustaining a coagulation-inflammation cycle which contributes to organ dysfunction and coma in falciparum malaria.     

The effect of bursectomy and thymectomy on the course of avian influenza virus infection

The effect of hormonal bursectomy and neonatal surgical thymectomy on the course of an avian influenza virus infection in chickens was studied. Analysis of the immunologic status of the chickens prior to infection included assay of natural agglutinins to rabbit red blood cells and induced agglutinins to sheep red blood cells, serum immunoelectrophoresis patterns, and in vitro effects of phytohemagglutinin on lymphocyte transformation. At 6 wk of age the chickens received the influenza virus intratracheally. Daily temperatures and mortality were recorded and HAI antibody titers were measured 7 and 14 days later. Completely thymectomized chickens were characterized by a failure of lymphocyte transformation to take place in two successive studies and absence of thymic remnants at autopsy. Bursectomy was associated with a significantly higher occurrence of temperature elevation (P < 0.05) and mortality (P < 0.001). Thymectomy had no significant effect on the course of the virus infection. Preliminary findings with passive administration of serum from immune animals also suggested a role for antibody in host resistance. These studies suggest cell-mediated immunity is less important than humoral immunity in recovery from avian influenza virus infection.     

Therapeutic effect of chloroquine(CQ)-containing immunoliposomes in rats infected with Plasmodium berghei parasitized mouse red blood cells: comparison with combinations of antibodies and CQ or liposomal CQ

The potential therapeutic application of chloroquine-containing immunoliposomes (Fab'-lipCQ) in a Plasmodium berghei malaria model was studied. Extending a previously described in vivo model (Peeters et al. (1988) Biochim. Biophys. Acta 943, 137-147) it was demonstrated that injection of antimouse red blood cell (anti-mRBC) Fab'-lipCQ was significantly more effective than liposome-encapsulated chloroquine (lipCQ) or free chloroquine in delaying or preventing a patent infection after intravenous injection of parasitized mouse red blood cells (p-mRBC) in rats. The results could be improved by injecting synchronized infected cells instead of non-synchronous p-mRBC in order to minimize the presence of free parasites which could easily infect rat RBC. It was further demonstrated that sequential injection of anti-mRBC IgG and lipCQ or chloroquine resulted in complete inactivation of the injected parasitized cells while Fab'-lipCQ administration resulted in a maximum score of 50% at an equal chloroquine, protein and phospholipid dose. In this report the potential of the concept of drug targeting for the effective treatment of a disease, which manifests in blood cells, was demonstrated.