Emperipolesis Observed in Immature Cells in Blast Crisis of Chronic Myelogenous Leukaemia

In a 62 year-old male with chronic myelogenous leukaemia, an acute phase developed with appearance of large immature cells in the bone marrow. A significant proportion of these cells was found to have engulfed autologous immature and polymorphonuclear neutrophils, erythroblasts, red cells and platelets. These abnormal cells were Philadelphia chromosome positive, and considered to be derived from the leukaemic cell line. Electron microscopic examinations revealed, in the cytoplasm of engulfed cells, no lysosomes or phagosomes which are typically seen in phagocytic cells, nor any evidence of degenerative changes in either engulfing or engulfed cells. These findings suggest that this phenomenon be considered as emperipolesis rather than phagocytosis. The pathophysiological mechanism of this phenomenon is uncertain.     

The Anaemia of P. falciparum Malaria

The haematological changes in a group of young Gambian children with P. falciparum malaria have been analysed. In children with acute infection anaemia was most marked during the period after treatment. Although many of these patients developed a positive direct Coombs test during this period of the illness it is not clear whether the anaemia which occurs after treatment has an immune basis. A second group of children showed quite different haematological findings. They appear to have a more chronic form of P. falciparum malaria infection, were profoundly anaemic at presentation, showed gross dyserythropoietic changes in their bone marrows, and had a full reticulocyte response and rise in haemoglobin after treatment. A third group of children were encountered whose haematological abnormalities were intermediate to those of the acute and chronic groups. These findings indicate that the patho-physiological mechanisms responsible for the anaemia of P. falciparum malaria are different at different stages of the illness.     

Nuclear bridging of erythroblasts in acquired dyserythropoiesis: an early and transient preleukemic marker

The clinical, hematologic, and histological characteristics of two patients who progressed from refractory anemia to acute leukemia are described. When first studied, nuclear bridging of erythroblasts, similar to that seen in congenital dyserythropoietic anemia type I and megakaryocytic dysplasia, were the only abnormalities. Within 6 years, both patients died, the first of acute nonlymphocytic leukemia, the second of erythroleukemia. Nuclear bridging of erythroblasts in the marrow of these patients was an early and transient phenomenon and was not observed during the terminal phase of leukemia.     

Ultrastructural evaluation of periodate-reactive glycoconjugates in human leukaemia cells

Periodate-reactive glycoconjugates in human leukaemic cells were examined electron microscopically by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method. Granules in ALL cells were classified into 4 types based on PA-TCH-SP staining features. Abnormal granules containing glycogen were observed only in children with treatment-resistant ALL. Cytoplasmic granules in leukaemic cells of patients with AML and acute monocytic leukaemia exhibited moderate reactivity. The distribution pattern of glycogen in the cytoplasm of leukaemic cells was classified into 3 types, one lacking glycogen, one containing small glycogen particles scattered throughout cytoplasm, and one showing clusters of glycogen particles. Cells with glycogen clusters were observed in ALL cells and in erythroblasts from patients with erythroleukaemia. PA-TCH-SP reactivity was detected in the rough endoplasmic reticulum in acute promyelocytic leukaemia but not in ALL or other types of AML. Megakaryoblasts in megakaryocytic crisis of chronic myelogenous leukaemia exhibited characteristic PA-TCH-SP reactivity similar to that of normal megakaryocytes.     

CTLA-4 positive T cells in contrast to procalcitonin plasma levels discriminate between severe and uncomplicated Plasmodium falciparum malaria in Ghanaian children

Procalcitonin (PCT) plasma levels and the fraction of CTLA-4-positive T cells are both elevated in acute Plasmodium falciparum malaria in human adults and the degree of elevation is positively correlated with other markers of disease severity, for example with parasitaemia. However, the clinical manifestations of malaria are strongly age-dependent and children from endemic areas carry the main disease burden. Therefore, we measured PCT plasma levels and CTLA-4 expression by T cells in four groups of children from the Ashanti Region in Ghana: asymptomatic children with or without parasitaemia, children with uncomplicated P. falciparum malaria and children with severe disease. PCT levels were highly elevated in both groups with acute malaria but they did not discriminate between uncomplicated and severe disease. In contrast, CTLA-4-expression by T cells was increased only in severe malaria. The fraction of CTLA-4 positive T cells in the blood of children with severe disease differed significantly from that in uncomplicated malaria, which was not elevated in spite of the high parasite loads observed in these children. This was unexpected, as in adults uncomplicated malaria is associated with a dramatic sixfold increase of the fraction of CTLA-4-positive T cells. The data from this study support the hypothesis that strong T cell activation as measured here by CTLA-4 expression is not just the by-product of a high parasite burden, but that it contributes to the pathogenesis of P. falciparum malaria.     

Nitric oxide, malaria, and anemia: inverse relationship between nitric oxide production and hemoglobin concentration in asymptomatic, malaria-exposed children.

The cause of the anemia associated with chronic, intermittent, asymptomatic, low-level parasitemia in children in malaria-endemic endemic areas is not well understood. Nitric oxide (NO) decreases erythropoiesis, and it is likely an important mediator of anemia of chronic disease. Production of NO is decreased in acute uncomplicated and cerebral malaria, but it is increased in asymptomatic Tanzanian children (with or without parasitemia). We hypothesized that chronic overproduction of NO in these asymptomatic children contributes to the anemia associated with subclinical/subpatent malaria. In 44 fasting, asymptomatic, malaria-exposed, Tanzanian children, NO production (measured using fasting urine NOx excretion) was inversely associated with hemoglobin concentration (P = 0.03, controlling for age and gender). Using multiple linear regression, hemoglobin concentration was negatively associated with parasitemia (P = 0.005). After controlling for age and parasitemia, NO was no longer an independent predictor of anemia. One of the mechanisms of parasite-related anemia in such children may be through the adverse hematologic effects of parasite-induced NO production.     

Mechanisms of malarial anaemia: Potential involvement of the Plasmodium falciparum low molecular weight rhoptry-associated proteins

Plasmodium falciparum malaria is a major cause of morbidity and mortality throughout the tropics. Anaemia is a constant feature of the disease. Pregnant women mostly primigravidae and children below the age of 5 years are the most afflicted. Its pathogenesis is multifactorial and incompletely understood. Among several factors, the destruction of erythrocytes (RBCs) is the most frequently observed cause of severe malarial anaemia and the removal of non-parasitized RBCs (nEs) is thought to be the most important, accounting for approximately 90% of the reduction in haematocrit in acute malaria. Previous studies demonstrated that the tagging of nEs with the parasite antigen RAP-2 (rhoptry-associated protein-2; also designated RSP-2) due to either failed or aborted invasion by merozoites resulted in the destruction of these cells.In this study we further investigated the mechanisms mediating the destruction of nEs in the development of severe malarial anaemia and the possible involvement of RAP-2/RSP-2 and other members of the low molecular weight rhoptry complex (RAP-1: rhoptry-associated protein-1 and RAP-3: rhoptry-associated protein-3). Antibodies to the rhoptry-associated proteins were found to recognise the surface of nEs in a parasitaemia-dependent manner after merozoite release in P. falciparum in vitro cultures. These cells, as well as erythroblasts co-cultured with infected RBCs (IEs), could then be destroyed by either phagocytosis or lysis after complement activation. The ability of anti-rhoptry antibodies to mediate the destruction of RAP-2/RSP-2-tagged erythroblasts in the presence of effector cells was also investigated. Data obtained suggest that mouse monoclonal antibodies to the low molecular weight RAP proteins mediate the death of RAP-2/RSP-2-tagged erythroblasts on interaction with adherent monocytes. The mechanism of cell death is not yet fully known, but seems to involve primarily apoptosis. The above observations suggest that the antibody response against RAP-2/RSP-2 and other members of the complex could trigger the destruction of RAP-2/RSP-2-tagged host cells.Taken together it appears that during severe anaemia a defective bone marrow or dyserythropoiesis possibly due to erythroblast cell death, may overlap with the accelerated destruction of normal erythroid cells, either by opsonisation or complement activation further aggravating the anaemia which may become fatal. These observations could therefore have implications in the design, development and deployment of future therapeutic interventions against malaria.     

Phenotypic heterogeneity of erythroblasts in erythroblastic leukemia revealed by monoclonal antibodies

Eight cases each of erythroleukemia (AML-M6) and erythroblastic crisis of chronic granulocytic leukemia (CGLBC-E) were immunophenotyped with the help of a panel of lineage-associated monoclonal antibodies (McAbs). The latter included those reactive with erythroid progenitor (BFU-E and CFU-E) and erythroid precursors at different stages of maturation. In six of eight cases of AML-M6, erythroblasts revealed an immature phenotype, as evident from reactivity of the blast cells with McAbs directed against the earlier stages of erythroid maturation. One case had the phenotype of CFU-E, and in the remaining case of AML-M6 the erythroblasts showed a "mature" surface antigenic profile. This immunophenotypic spectrum was unrelated to the morphologic maturity of the erythroblasts. In two cases of CGLBC-E, an early erythroblastic phenotype was observed, while in as many cases a "mature" phenotype was present. Four of eight cases, however, revealed a mixed, erythroid plus myeloid phenotype. In one of the four cases, two separate blast populations, which represented erythroblasts and myeloblasts, could be identified. In the remaining three cases the blasts were morphologically homogeneous and undifferentiated. High incidence of HLA-DR positivity in the latter three cases suggests the primitive nature of blasts cells and their closeness to the putative "bipotent" myeloid stem cell. Our study has shown phenotypic heterogeneity of blast cells in AML-M6 and CGLBC-E.     

Expression of sialosyl-Tn in colony-forming unit-erythroid, erythroblasts, B cells, and a subset of CD4+ cells

The epitopes Tn and sialosyl-Tn are expressed on erythrocytes of individuals with a very rare blood group, who often suffer from "Tn syndrome." We surveyed expression of Tn and sialosyl-Tn in normal blood cells, malignant transformed cells, and progenitor stem cells from bone marrow (BM). An anti-Tn antibody, IE3, and an anti-sialosyl-Tn antibody, TKH2, were used in this study. TKH2 reacted with erythroblasts, B cells, and a subset of CD4+ cells; but not with erythrocytes. Erythroblastic cell lines (K562, HEL, and UT7/EPO) and B- cell lines (Daudi, Raji, and B-cell lines transformed by Epstein-Barr virus) showed reactivity to TKH2. Similar results from the reactivity of TKH2 with transformed cells from leukemia patients and lymphoma patients were obtained; TKH2 reacted with blasts from erythroleukemia (M6; for 4 of 4 cases) and with lymphocytes from B-cell chronic lymphocytic leukemia (3 of 3), B-cell lymphoma (5 of 5), and CD4+ adult T-cell leukemia (4 of 4), but did not react with blasts from acute myeloid leukemia (M0 to M5; 0 of 22) or acute lymphoid leukemia (B- lymphoid leukemia, 0 of 11; T-lymphoid leukemia, 0 of 2; undifferentiated leukemia, 0 of 1). IE3 did not react with all of the tested cells. CD2-CD19-TKH2+ normal BM cells (BMC) contained blasts and various maturation stages of erythroblasts. The TKH2+ cells produced a large number of colony-forming unit-erythroid (CFU-E) colonies, whereas they produced a small number of burst-forming unit-erythroid colonies and CFU-granulocyte-macrophage colonies. CD34+ normal BMC did not express Tn and sialosyl-Tn. These findings suggest that sialosyl-Tn expresses in CFU-E to erythroblasts.     

Peripheral blood dendritic cells in children with acute Plasmodium falciparum malaria.

The importance of dendritic cells (DCs) for the initiation and regulation of immune responses not only to foreign organisms but also to the self has raised considerable interest in the qualitative and quantitative analysis of these cells in various human diseases. Plasmodium falciparum malaria is characterized by the poor induction of long-lasting protective immune responses. This study, therefore, investigated the percentage of peripheral blood DCs as lineage marker-negative and HLA-DR(+) or CD83(+) cells in healthy children and in children suffering from acute malaria in Kilifi, Kenya. Comparable percentages of CD83(+) DCs were found in peripheral blood of healthy children and children with malaria. However, the percentage of HLA-DR(+) peripheral blood DCs was significantly reduced in children with malaria. The results suggest that a proportion of peripheral blood DCs may be functionally impaired due to the low expression of HLA-DR on their surface.     

The plasma concentration of the B cell activating factor is increased in children with acute malaria

Malaria-specific antibody responses in children often appear to be short-lived but the mechanisms underlying this phenomenon are not well understood. In this study, we investigated the relationship between the B-cell activating factor (BAFF) and its receptors expressed on B cells with antibody responses during and after acute malaria in children. Our results demonstrate that BAFF plasma levels increased during acute malarial disease and reflected disease severity. The expression profiles for BAFF receptors on B cells agreed with rapid activation and differentiation of a proportion of B cells to plasma cells. However, BAFF receptor (BAFF-R) expression was reduced on all peripheral blood B cells during acute infection, but those children with the highest level of BAFF-R expression on B cells maintained schizont-specific immunoglobin G (IgG) over a period of 4 months, indicating that dysregulation of BAFF-R expression on B cells may contribute to short-lived antibody responses to malarial antigens in children. In summary, this study suggests a potential role for BAFF during malaria disease, both as a marker for disease severity and in shaping the differentiation pattern of antigen-specific B cells.     

Hepcidin demonstrates a biphasic association with anemia in acute Plasmodium falciparum malaria

Hepcidin levels are high and iron absorption is limited in acute malaria. The mechanism(s) that regulate hepcidin secretion remain undefined. We have measured hepcidin concentration and cytokines in 100 Kenyan children with acute falciparum malaria and different degrees of anemia. Hepcidin was increased on admission and fell significantly one week and one month after treatment. The association of hepcidin with hemoglobin was not linear and hepcidin was very low in severe malarial anemia. Parasite density, IL-10 and IL-6 were significantly associated with hepcidin concentration. Hepcidin response to acute malaria supports the notion of iron sequestration during acute malaria infection and suggests that iron administration during acute malaria is futile. These data suggest iron supplementation policies should take into account the high hepcidin levels and probable poor utilization of iron for up to one week after treatment for the majority of patients with acute malaria.Key words: hepcidin, Plasmodium falciparum malaria, anemia, iron supplementation     

Haptoglobin levels are associated with haptoglobin genotype and alpha+ -Thalassemia in a malaria-endemic area

Haptoglobin (Hp) is an acute phase protein that removes free hemoglobin (Hb) released during hemolysis. Hp has also been shown to be toxic for malaria parasites. alpha(+)-Thalassemia is a hemoglobinopathy that results in subclinical hemolytic anemia. alpha(+)-Thassemia homozygosity confers protection against severe malarial disease by an as yet unidentified mechanism. Hp levels were measured in a serial cross-sectional survey of children in Madang Province, Papua New Guinea (PNG). Hp levels were related to age, Hp genotype, Hb levels, parasitemia, splenomegaly, and alpha(+)-thalassemia genotype. Surprisingly, children who were homozygous for alpha(+) -thalassemia had significantly higher levels of Hp than did heterozygotes, after controlling for relevant confounders. We suggest that this is the result of either reduced mean cell Hb associated with alpha(+) -thalassemia homozygosity or an elevated IL-6-dependent acute phase response.     

Acute P. falciparum malaria induces a loss of CD28- T IFN-gamma producing cells

P. falciparum malaria is associated with increased activation among peripheral lymphocytes. In the present study, we investigated markers of susceptibility to apoptosis and expression of IFN-gamma and IL-4 by CD28-and CD28+T cells in West African children with acute P. falciparum malaria. The study showed increased susceptibility to apoptosis and cytokine production among T lymphocytes during acute malaria but also that T cells, in particular IFN-gamma producing CD28-T cells, were substantially reduced. These results are in line with previous studies suggesting that certain T cell subsets are sequestered away from the peripheral blood during P. falciparum malaria.     

Red Cell Aplasia and Chronic Granulocytic Leukaemia

S ummary. Two patients with chronic granulocytic leukaemia developed red cell aplasia during the course of their disease. In one of them, cell culture studies demonstrated the presence in the patient's serum of an IgG inhibitor of haemoglobin synthesis by his own mature erythroblasts and erythroblasts grown in vitro from his erythroid colony forming cells. The IgG fraction was also found to be cytotoxic for the patients' marrow erythroblasts that were present after disappearance of the red cell aplasia. Treatment with corticosteroids resulted in reappearance of the erythroblasts in the marrow and decrease in the transfusion requirement. Red cell aplasia can occur before, at the same time or after the onset of chronic granulocytic leukaemia and may have the same immune pathogenesis as chronic idiopathic pure red cell aplasia. It occurs without busulphan treatment and seems to have no direct relation to the terminal metamorphosis. Treatment of the red cell aplasia with corticosteroids would appear worthwhile as it may reduce the transfusion requirement without affecting the course of the underlying leukaemia.     

Cellular and humoral inhibition of Plasmodium falciparum growth in vitro and recovery from acute malaria

Both mononuclear cell cytotoxicity and serum inhibition of Plasmodium falciparum growth in vitro were found to vary according to the stage of infection in Gambian children with clinical malaria. Cytotoxicity was displayed by mononuclear cells and serum from children with acute malaria but this form of parasite killing was more effective in children with low grade P. falciparum infections of at least 10 days duration. Parasite inhibitory antibody was not evident in sera from acutely infected children but was found in sera from children recovering from malaria and reached a peak in convalescent children when P. falciparum growth was inhibited by at least 50%. The humoral response in convalescent children was strain related, being more effective against the most recent infecting parasite strain than against other 'wild' P. falciparum isolates. In contrast, mononuclear cell cytotoxicity was not strain related; when effective, multiplication of all parasite isolates tested was retarded to the same degree. The discussion considers the role of mononuclear cell cytotoxicity in the development of protective immunity and suggests that it may be a 'front line' defense mechanism during each malaria attack.     

Pulmonary manifestations associated with malaria

Pulmonary manifestations are frequently observed in children, pregnant women and travellers with malaria. The pathophysiology of these pulmonary manifestations is poorly understood but would appear to be secondary to an interaction between the parasitized red cells and the pulmonary capillary endothelium. Bronchitis and pneumonia do not directly compromise outcome but, left unrecognized, the delay in diagnosis and treatment may be fatal. Acute respiratory distress in children is the first cause of overmortality, coming before neurological involvement. The acute respiratory distress caused by severe malaria has no specific characteristics. Iatrogenic complications and pulmonary superinfections must be differentiated. The prevention of pulmonary manifestations associated with malaria can easily be accomplished by limiting water intake and carefully monitoring urinary output and weight. Treatment is the same as for acute flare-ups in combination with symptomatic respiratory treatment when required.     

Establishment of an interleukin-3-dependent leukemic cell line from a patient with chronic lymphocytic leukemia in the acute phase

A novel human leukemic cell line, TMD2, which proliferates dependently on interleukin-3 (IL-3), was established from the peripheral blood (PB) of a patient with chronic lymphocytic leukemia (CLL) in the acute phase. After 8 years of the chronic phase of CLL, lymphoblastoid cells appeared and became dominant in the PB. After repeated subcultures of the patient's PB cells in the acute phase, lymphoblastoid cells have proliferated actively in the presence of recombinant human IL-3, and the TMD2 cell line has been established. The lymphoblastoid cells in acute phase and TMD2 cells proliferated dependently on IL-3, whereas growth of the small lymphocytes in chronic phase was not supported by IL-3. Other ILs (IL-1, 2, and 4 through 6) or CSF did not support the growth or survival of TMD2 cells. The existence of high-affinity receptors for IL-3 was shown on TMD2 cells (binding sites 88 per cell, Kd = 76.9 pmol/L). DNA extracted from the small lymphocytes in the chronic phase, the lymphoblastoid cells in the acute phase, and TMD2 cells showed the same rearrangement pattern of the immunoglobulin heavy-chain gene. Therefore, these cells were considered to have originated from the same clone. These results imply that a genetic event that caused the responsiveness to IL-3 in the cell of the chronic phase caused the acute transformation of CLL in this patient. We consider that TMD2 cell line is valuable as a model of cells of CLL in the acute phase and as a tool for studying the signal transduction system of IL-3.     

A study on malaria infection during the acute stage of measles infection

The recognized high mortality from measles in Africa is considered to be partly due to the flare-up of concomitant malaria infection. In 1987 there was a measles epidemic in the Rufiji Delta, Tanzania, in spite of recent vaccination campaigns. A comparative study was therefore conducted on the densities of malaria parasites in children during the acute stage of measles (67 consecutive cases, aged 5 months-19 years). The period of study was March-June, the peak season for malaria transmission. For each measles patient, a blood film was concomitantly taken from an asymptomatic age-matched child from the same village. Of 67 children with measles, 17 (25%) had parasitaemia ranging from 8 to 2480 parasites microliter-1 blood. Out of 67 asymptomatic control children 59 (88%) had parasitaemia ranging from 8 to 3400 parasites microliter-1 blood. This study indicates that malaria densities were lower during the acute stage of measles than in healthy children. The contribution of malaria to mortality in children with acute measles may be questioned.     

Radioautographic Investigations on DNA and Protein Metabolism in 2 Cases of Di Guglielmo's Disease

H³-thymidine and H³-DL-leucine incorporation in the erythroblasts of thebone marrow from a case of acute and one of chronic Di Guglielmo’s diseasewas studied. A similar investigation was also carried out with bone marrowfrom 4 normal subjects. The high resolution of the radioautographic technicemployed allowed a comparative study at the cellular and subcellular level.

A definitely lowered thymidine uptake was observed in the cells of acuteerythremic myelosis, while increased incorporation of DL-leucine was detectedin both the acute and the chronic form of this disease.

Submitted on August 4, 1959 Accepted on February 18, 1960