Invasion of erythroblasts by Pasmodium vivax: A new mechanism contributing to malarial anemia.

Severe malarial anemia causes considerable mortality and morbidity in endemic areas. Possible mechanisms underlying the anemia include lysis of parasitized and nonparasitized red cells as well as parasite product-mediated effects on erythropoiesis. The latter include suppression of erythropoiesis, dyserythropoiesis, and ineffective erythropoiesis. Present transmission electron microscope data in two cases of Pasmodium vivax malaria show a hitherto undescribed mechanism contributing to malarial anemia, namely, infection of erythroblasts by parasites and their subsequent degradation. No parasites were detected in the peripheral blood but parasites were found in the bone marrow. These findings emphasise the value of bone marrow examination in the diagnosis and eradication of malaria.

Congenital dyserythropoietic anaemia, type I, in a Caucasian patient with retinal angioid streaks (homozygous Arg1042Trp mutation in codanin-1).

A congenital dyserythropoietic anaemia (CDA) was recognised in a French Caucasian male patient. Blood smears showed a pronounced aniso-poikilocytosis. Bone marrow light microscopy showed signs of dyserythropoesis, but no internuclear chromatin bridges. Electron microscopy disclosed erythroblast nuclei with the Swiss cheese aspect and the presence of cytoplasmic organelles, assessing the diagnosis of CDA I. The presence of internuclear chromatin bridges may thus be missing in CDA I. The patient proved to be homozygous for the Arg1042Trp mutation in codanin-1 (the 'Bedouin mutation'). By the age of 25, the patient's vision started to deteriorate as a result of retinal angioid streaks and macular abnormalities. Evolution was controlled and the patient, being nearly 50 yr old now, still has a partial use of his eyes. This second case of retinal angioid streaks reported in CDA I adds to the non-haematological features likely to be associated with this condition.

Diagnosis of megaloblastic anaemias.

There are a large number of causes of megaloblastic anaemia. The most frequent are disorders resulting in vitamin B(12) or folate deficiency. The diagnostic process often consists first of establishing the presence of B(12) or folate deficiency and then of determining the cause of deficiency. The blood count, blood film, serum B(12) assay, and red cell and serum folate assays are the primary investigations. Other useful investigations include serum/plasma methylmalonic acid (MMA), plasma total homocysteine (tHCYS) and serum holo-transcobalamin II assays. All currently used tests have limitations regarding specificity or sensitivity or both and the metabolite assays are not widely available. An understanding of these limitations is essential in formulating any diagnostic strategy. The wide use of serum B(12) and metabolite assays has resulted in the increasingly early diagnosis of B(12) deficiency, often in patients without B(12)-related symptoms (subclinical deficiency). Food cobalamin malabsorption is the most frequent cause of a low serum B(12). At least 25% of low serum B(12) levels are not associated with elevated metabolite levels and may not indicate B(12) deficiency. Some of these are caused by partial deficiency of transcobalamine I.

Congenital dyserthropoietic anaemia other than type I to III with a peculiar erythroblastic morphology.

Here, we report the case of a child who, since birth, showed persistent macrocytosis and elevated mean corpuscular volume of the erythrocytes. Bone marrow biopsy revealed gross disorganisation of the erythroblastic series both at the light and electron microscopic examination, with complete absence of dysplastic features in the granulocytic and megakaryocytic series. Common causes of macrocytosis were excluded. The spectrum of morphological findings were not consistent with any of the classical types of congenital dyserythropoietic anaemias (CDAs) and serological findings of CDA type II were absent. The most outstanding feature was a marked irregularity of the nuclear outline of the late erythroblasts that presented thick-ending finger-like projections. The combination of macrocytosis without anaemia and these morphologic erythroblastic changes have not been previously reported in the setting of classical and variant forms of CDAs.

Natural history of congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemia type II (CDA-II) is an autosomal recessive disease characterized by anemia, jaundice, splenomegaly, and erythroblast multinuclearity. The natural history of the disease is unknown. The frequency, the relevance of complications, and the use of splenectomy are poorly defined. This study examined 98 patients from unrelated families enrolled in the International Registry of CDA-II. Retrospective data were obtained using an appropriate questionnaire. The mean age at presentation was 5.2 +/- 6.1 years. Anemia was present in 66% and jaundice in 53.4% of cases. The mean age at correct diagnosis was 15.9 +/- 11.8 years. Twenty-three percent of patients for whom data were available developed anemia during the neonatal period, and 10 of these individuals required transfusions. Splenectomy produced an increased hemoglobin (P <.001) and a reduced bilirubin level (P =.007) in comparison with values before splenectomy. Preliminary data indicate that iron overload occurs irrespective of the hemochromatosis genotype. (Blood. 2001;98:1258-1260)

Changes in murine bone marrow macrophages and erythroid burst-forming cells following the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP).

In order to explore the effect on bone marrow macrophages of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP), mice were injected intravenously with a preparation of such liposomes at a dose known to deplete spleen and liver macrophages. Two days later, the macrophages in the marrow of the femoral bones were quantified by flow cytometry using a macrophage-specific monoclonal antibody (F4/80), and their ultrastructure and phagocytic activity towards zymosan particles was assessed. To determine the effect on erythropoiesis of liposome-encapsulated Cl2MDP-induced changes in bone marrow macrophages, red blood cell parameters and the formation of erythroid burst-forming unit (BFU-E)-derived colonies in vitro were evaluated. In mice injected with liposome-encapsulated Cl2MDP, there was a 54% and 67% decrease in the total number of bone marrow macrophages as compared to uninjected controls and mice treated with empty liposomes, respectively. Moreover, residual macrophages showed an abnormal ultrastructure, with reduced numbers of crystalloid inclusions and increased numbers of large myelin figures. However, the phagocytic activity of these cells was unimpaired or slightly enhanced. In mice injected with liposome-encapsulated Cl2MDP there was an approximately 60% decrease in the percentage and total number of circulating reticulocytes and a 54% reduction in the BFU-E number, demonstrating deregulation of erythropoiesis under conditions of macrophage loss and impairment. The results suggest that mice treated with liposome-encapsulated Cl2MDP are a model for studying the role of macrophages in erythropoiesis.

Severe congenital dyserythropoietic anaemia type I: prenatal management, transfusion support and alpha-interferon therapy.

We report a case of congenital dyserythropoietic anaemia, type I, with severe pre- and postnatal manifestations. Exchange transfusions were required for fetal anaemia (3.5 g/dl) at 28 and 30 weeks of gestation. Transfusions were administered at birth (Caesarean section at week 35) and at regular intervals thereafter. At 14 months, alpha-interferon therapy was initiated (106 units three times a week). This resulted in stabilization of the haemoglobin at or above 11 g/dl and a reduction in the percentage of erythroblasts with ultrastructurally abnormal heterochromatin. After 9 months, the dose of alpha-interferon was decreased to 106 units twice a week. No relapse of anaemia was noted during an additional 4 months of follow-up.

Transfusion-dependent congenital dyserythropoietic anaemia with intraerythroblastic inclusions of a non-globin protein.

The congenital forms of dyserythropoiesis comprise a group of hereditary disorders characterized by ineffective erythropoiesis as the predominant mechanism of anaemia and morphologically abnormal erythroblasts. Up to now three major forms and four variants have been described. Group VII is characterized by dyserythropoiesis with intraerythroblastic precipitation of a non-globin protein. Here we described a case of dyserythropoietic anaemia presenting neonatally and requiring regular blood transfusions. Optical and electronic microscopy studies confirmed that this case was very similar to those in two previously reported transfusion-dependent patients with an unusual type of congenital dyserythropoietic anaemia.

Blood and bone marrow changes in malaria.

A variety of abnormalities in the number, morphology and function of blood and bone marrow cells may be found in Plasmodium falciparum and P. vivax malaria. In a non-immune individual, the nature of such abnormalities depends on the time after infection. In others it is determined by the pattern and intensity of malaria transmission in the area and the extent of host immunity. Severe anaemia may occur in children with chronic falciparum malaria and low parasitaemia as well as in patients with complicated acute falciparum malaria with high parasitaemia. However, the mechanisms underlying the anaemia in these two situations appear to be different. The possible roles of parasite products, T-cell-derived cytokines produced in response to the infection, macrophage activation and hyperplasia, macrophage-derived factors such as tumour necrosis factor-alpha, and macrophage dysfunction in the pathogenesis of the haematological abnormalities are discussed.

Geographic distribution of CDA-II: did a founder effect operate in Southern Italy?

BACKGROUND AND OBJECTIVE: Congenital dyserythropoietic anemia type II (CDA-II) is an autosomal recessive condition, whose manifestations range from mild to moderate. Its exact prevalence is unknown. Based on a recently established International Registry of CDA-II (64 unrelated kindreds), a high frequency of CDA II families living in South Italy became evident. DESIGN AND METHODS: The aim of this study was to define the haplotypes of the CDA II kindreds living in Southern Italy based on markers D20S884, D20S863, RPN, D20S841 and D20S908. These markers map to 20q11.2, within the interval of the CDAN2 gene that is responsible for CDA II. Next, we looked at these markers in kindreds from other regions of Italy and from other countries, with special attention to families having ancestors in Southern Italy. RESULTS: Evaluation of the geographic distribution of the ancestry of Italian CDA-II patients clearly demonstrated the unusually high incidence of this condition in Southern Italy. Our statistical calculations and linkage disequilibrium data also clearly demonstrate a strong association of the markers of chromosome 20 with the disease locus in our sample. Almost all the regions defined by the markers here used is in disequilibrium with the disease. Combining the data from the Italian sample together with those obtained from the non-Italian ones, we can restrict the area of highest disequilibrium to that defined by markers D20S863-D20S908. INTERPRETATION AND CONCLUSIONS: Despite the presence of this linkage disequilibrium the search for a common haplotype failed. This could suggest that the mutation was very old or that it occurred more than once on different genetic backgrounds.

Congenital dyserythropoietic anemias.

The congenital dyserythropoietic anemias (CDAs) are an uncommon and heterogeneous group of disorders characterized by markedly ineffective erythropoiesis and, usually, striking dysplastic changes in erythroblasts. Each of the three originally described forms, designated CDA types I to III, is defined by the presence of distinctive morphologic (including ultrastructural) abnormalities in erythroblasts. CDA type II is also characterized by a marked reduction in polylactosamine structures associated with the erythrocyte membrane glycoprotein, band 3 (detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis), and, usually, a positive result on the acidified serum lysis test. The course of CDA is often complicated by cholelithiasis. Even patients who have not had transfusions sometimes develop substantial iron overload. Recent studies have extended our knowledge on the clinical manifestations of CDA types I and III and have revealed the existence of forms of CDA distinct from types I to III. Information is now available on the chromosomal localization of the genes involved in CDA types I and II and in the Swedish cases of CDA type III. A few patients with CDA type I have been treated with interferon-alpha2, with a good response.

Demonstration of mRNA for five species of cytochrome P450 in human bone marrow, bone marrow-derived macrophages and human haemopoietic cell lines.

The expression of mRNA for five cytochrome P450s (CYP1A1, 2A6/7, 2D6, 2E1 and 3A4) was studied in human bone marrow, bone-marrow-derived macrophages and blood monocyte-derived macrophages. Reverse transcriptase polymerase chain reaction (RT-PCR) detected expression of all five CYPs in each of these cell populations. All five CYPs were also expressed in the haemopoietic cell lines HL-60 and HEL and in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines. The data suggest that bone marrow macrophages and probably other bone marrow cell types are capable of metabolizing xenobiotics. This metabolic potential may play a role in the bone marrow damage induced by some drugs and chemicals.

Determinants of iron status and bilirubin levels in congenital dyserythropoietic anaemia type I.

Seven untransfused patients with congenital dyserythropoietic anaemia type I were investigated to assess the determinants of both iron overload and serum bilirubin levels. The serum ferritin concentration was increased in all patients and non-transferrin-bound iron (NTBI) was increased in all but one patient. None of the patients showed the C282Y mutation in the hereditary haemochromatosis gene, HFE. One patient was homozygous for the H63D mutation in this gene. The data indicated that differences in the extent of iron overload were not mediated by co-inheritance of the C282Y mutation in the HFE gene but could largely be explained by differences in the severity of anaemia and ineffective erythropoiesis, and in the age of the patient. In one patient an unusually high plasma bilirubin level was associated with the variant A[TA]7TAA configuration in the TATA box of the uridine diphosphate glucuronosyltransferase (UGT-1A) gene promoter, the mutation found in most patients with mild Gilbert's syndrome.

Role of FcgammaRI (CD64) in erythrocyte elimination and its up-regulation in thalassaemia.

To examine any role of the high affinity Fcgamma class I receptor (FcgammaRI) (CD64) in erythrocyte elimination by mononuclear phagocytes (MP) in thalassaemia (thal), we investigated the in vitro interaction of beta-thalassaemic erythrocytes with monocytes (Mo) whose FcgammaR expression had been modulated by cytokines. Treatment of Mo with interferon (IFN)-gamma or interleukin (IL)-10 which up-regulate FcgammaRI, caused a dose-dependent increase in binding of beta-thalassaemic erythrocytes, whereas stimulation with IL-4 which down-regulates the receptor, reduced this interaction, in a dose-dependent manner, to that of normal erythrocytes. Binding of thalassaemic erythrocytes by IFN-gamma or IL-10-treated Mo was inhibited by FcgammaRI-specific reagents. In addition, Mo expression of FcgammaRI and HLA class II DR was determined by flow cytometry in Thai patients with HbH disease (alpha1/alpha2 or alpha1/Hb Constant Spring) (n = 15) or beta degrees -thal/HbE (n = 16). In both groups of patients FcgammaRI expression was increased as compared to normal controls (n = 14): mean fluorescence intensity (+/-SD) 124.79 +/- 38. 77 in HbH disease and 121.86 +/- 18.23 in beta degrees -thal/HbE versus 91.94 +/- 17.36 in normal controls (P < 0.01 and P < 0.001, respectively). In contrast, HLA class II DR expression was similar in patients and controls. The results suggest that, in thalassaemia, up-regulated FcgammaRI on mononuclear phagocytes plays a role in their interaction with erythrocytes and that this process can be modified by cytokines.

Suppression of CDA II expression in a homozygote.

The CDAN2 gene, responsible for congenital dyserythropoietic anaemia, type II (CDA II), was recently mapped to 20q11.2. We report data on an additional member of a previously studied CDA II family. This member had always been regarded as haematologically normal. Unexpectedly, she had the same microsatellite assortments around the CDAN2 alleles as her three sisters with CDA II. In particular, she was a homozygote for microsatellites D20S863 and D20S841. This prompted an analysis of all facets of her phenotype. The Ham test was negative. The bone marrow smears contained a normal proportion of binucleate erythroblasts. Electron microscopy revealed the absence of extensive stretches of cisternae beneath and parallel to the inner surface of the erythroblast plasma membrane. Proteins of the endoplasmic reticulum, which contaminate the reticulocyte plasma membrane in CDA II patients, were missing. Only the shape of the band 3 peak appeared slightly altered. This case exemplifies that homozygosity (or compound heterozygosity) for a deleterious gene may be silenced, or almost completely silenced. In recessively inherited diseases, suppressed phenotypes tend to be overlooked in siblings where both patients and unaffected individuals are expected.

Expression of CYP2E1 by human monocyte-derived macrophages.

The expression of the ethanol-metabolizing cytochrome P450 (CYP2E1) in human monocyte-derived macrophages was studied at the mRNA and protein levels. The presence of mRNA was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and protein by immunocytochemistry. The data show that CYP2E1 is expressed in human monocyte-derived macrophages at a level similar to that demonstrated in other extrahepatic tissues. Although there is circumstantial evidence for the presence of CYP2E1 in human macrophages, it has not previously been demonstrated directly. Its presence in macrophages underlines the potential importance of these cells in initiating alcohol-induced cytotoxicity.

A normal beta-globin allele as a modifier gene ameliorating the severity of alpha-thalassemia in mice.

Thalassemia is a heritable human anemia caused by a variety of mutations that affect expression of the alpha- or the beta-chain of hemoglobin. The expressivity of the phenotype is likely to be influenced by unlinked modifying genes. Indeed, by using a mouse model of alpha-thalassemia, we find that its phenotype is strongly influenced by the genetic background in which the alpha-thalassemia mutation resides [129(sv/ev)/129(sv/ev) (severe) or 129(sv/ev)/C57BL/6 (mild)]. Linkage mapping indicates that the modifying gene is very tightly linked to the beta-globin locus (Lod score = 13.3). Furthermore, the severity of the phenotype correlates with the size of beta-chain-containing inclusion bodies that accumulate in red blood cells and likely accelerate their destruction. The beta-major globin chains encoded by the two strains differ by three amino acids, one of which is a glycine-to-cysteine substitution at position 13. The Cys-13 should be available for interchain disulfide bridging and consequent aggregation between excess beta-chains. This normal polymorphic variation between murine beta-globin chains could account for the modifying action of the unlinked beta-globin locus. Here, the variation in severity of the phenotype would not depend on a change in the ratio between alpha- and beta-chains but on the chemical nature of the normal beta-chain, which is in excess. This work also indicates that modifying genes can be normal variants that-absent an apparent physiologic rationale-may be difficult to identify on the basis of structure alone.

Serum levels of tumor necrosis factor-alpha, interleukin-1, and interferon-gamma in beta(o)-thalassemia/HbE and their clinical significance.

Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and interferon-gamma (IFN-gamma) were estimated by conventional ELISA kits in 60, 42, and 58 Thai patients, respectively, with beta(o)-thalassemia HbE and found to be above the normal range in 13%, 21%, and 33% of the patients, respectively. Using high-sensitivity ELISA systems, an additional 10 beta(o)-thal/HbE patients were compared with 9 controls for concentrations of circulating TNF-alpha and IL-1beta, and 9 and 5 patients, respectively, but only 1 and none of the controls, respectively, showed values above the normal ranges. In patients with abnormally high IFN-gamma levels, basal hemoglobin values were significantly lower than in those with normal levels of the cytokine (mean +/- SEM: 6.03+/-0.24 vs. 7.08+/-0.18, p < 0.05), although circulating concentrations of soluble transferrin receptors (sTrF) and absolute reticulocyte counts were similar in the two groups. Patients with raised or normal levels of TNF-alpha, IL-1alpha, or IL-1beta had similar basal hemoglobin values. In a phagocytosis assay, monocytes of patients with raised serum levels of IFN-gamma showed significantly more attached or ingested IgG-coated red cells than those of patients with normal concentrations of the cytokine (mean +/- SEM: 192+/-22 vs. 140+/-14 per 100 monocytes, p < 0.05). Moreover, in 3 of 4 of the former patients, the number of attached or ingested IgG-coated red cells per 100 monocytes was above the 95% reference limit for the latter patients. The results suggest that IFN-gamma aggravates the anemia of beta(o)-thal/HbE by activating mononuclear phagocytes for destruction of red cells but not by inhibiting erythropoiesis. The elevated serum levels of TNF-alpha and IL-1 could contribute to complications of the disease, such as cachexia and thromboembolic phenomena.