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Biomedical subjects

S N Wickramasinghe

Publications and source records attributed to S N Wickramasinghe.

At least 73 records · Page 4Linked to original sources
PubMed1994

Reaction of 14C-acetaldehyde with whole blood in vitro: further evidence for the formation of unstable complexes with plasma proteins and red cells.

When heparinised whole blood was incubated with 5, 10, 45 or 180 microM 14C-acetaldehyde for 1 hr, an average of 33%, 34%, 33% and 41%, respectively, of the radioactivity was associated with red cells and the remainder with plasma. Although 71-80% of the radioactivity in the plasma was TCA-precipitable, only 0.9-3.1% was non-dialysable after 48 hr of dialysis, indicating that much of the acetaldehyde was reversibly bound to protein. When blood was incubated with 10-180 microM 14C-acetaldehyde for 1 hr and the plasma subjected to Sephacryl S300 gel filtration, 0.3-1.9% of the added radioactivity was found in the albumin and IgG fractions; this radioactivity is presumed to reside in both unstable and stable acetaldehyde-protein adducts. Plasma derived from whole blood which was incubated with 5-180 microM acetaldehyde and dialysed for 24 hr displayed cytotoxic activity against A9 cells. These data indicate that when 14C-acetaldehyde is incubated with whole blood, even at concentrations as low as 5-10 microM, a substantial proportion of the radioactive molecules form unstable cytotoxic adducts with plasma proteins and a much smaller proportion form stable adducts. Blood cells (mainly red cells) that were incubated with 14C-acetaldehyde were able to transfer radioactivity to cocultured K562 cells, supporting the possibility that not only acetaldehyde-modified plasma proteins but also acetaldehyde-modified blood cells may transport acetaldehyde and be cytotoxic in vivo.

Acetaldehyde↗
PubMed1993

Beta-thalassemia unlinked to the beta-globin gene in an English family.

An inherited hypochromic microcytic anemia transmitted in an autosomal manner has been observed in three generations of an English family. Affected members had the hallmarks of heterozygous beta-thalassemia, ie, elevated levels of hemoglobin A2 and imbalanced globin chain synthesis. However, despite extensive sequence analysis, no mutations could be found in or around the beta-globin genes of either the propositus or two other affected members from two different generations. Linkage analysis using restriction fragment length polymorphisms in the beta-globin gene cluster clearly showed that the gene responsible for the beta-thalassemia phenotype segregates independently of the beta-gene complex. Therefore, this condition represents a novel form of the disease.

Adolescent↗
PubMed1993

Apoptotic bodies in a murine model of alcoholic liver disease: reversibility of ethanol-induced changes.

The aim of this study was to investigate the effects of ethanol exposure on the location and number of apoptotic bodies in murine liver. Livers from 21 control mice were compared with those of 41 animals exposed to ethanol vapour by inhalation for up to 23 days. In the control livers, apoptotic bodies were identified in association with 36 per cent of the terminal hepatic venules examined and 88 per cent of these bodies were located in the first two rows of perivenular hepatocytes. By comparison, in the ethanol-exposed group, apoptotic bodies were observed in association with 72 per cent of the terminal hepatic venules examined and more were found further from them. This increase in the number of apoptotic bodies was shown to depend on the duration of ethanol exposure and, furthermore, the effects of ethanol were shown to be completely reversed by a period of abstinence. These results indicate that ethanol exposure has time-dependent, reversible effects on both the location and the number of apoptotic bodies in murine liver. This extends knowledge gained from previous work on rat livers.

Animals↗
PubMed1993

Erythropoietin and the human kidney: evidence for an evolutionary link from studies of Salmo gairdneri.

1. Ultrastructural evidence is provided of renal erythropoiesis in Salmo gairdneri. 2. Supernatants prepared from homogenates of the kidney of this teleost contain more immunoreactive erythropoietin than autologous plasma or serum or supernatants prepared from homogenates of spleen or liver. Thus, the trout kidney is not only a major erythropoietic organ but also a major source of erythropoietin. 3. These data provide the first immunochemical demonstration of the production of erythropoietin in fish and indicate that locally generated erythropoietin may be important in the regulation of renal erythropoiesis in the trout. 4. The results also suggest that the role of the kidney in the regulation of bone marrow erythropoiesis in humans may have an evolutionary basis.

Animals↗
PubMed1993

Activation of monocytes for the immune clearance of red cells in beta zero-thalassaemia/HbE.

We have recently provided evidence that IgG antibodies play a role in the destruction of red cells in thalassaemia syndromes. In order further to delineate factors involved in the clearance of thalassaemic cells, monocytes of 30 Thai patients with beta zero-thal/HbE (17 non-splenectomized and 13 splenectomized) and 16 normal controls were examined for their ability to bind and phagocytose normal red cells coated with IgG anti-Rh(D). In beta zero-thal/HbE, the mean number of red cells attached to the monocytes was approximately 3-fold greater than in normal controls and the number ingested 30% higher. Among the non-splenectomized patients, the number of red cells attached to and ingested by the monocytes, correlated inversely with mean basal Hb levels, suggesting that activation of mononuclear phagocytes for the immune clearance of red cells is a factor in determining the severity of the anaemia. As Fc-gamma-RI is of primary importance in the recognition of IgG-coated red cells by monocytes, leucocytes from 10 beta zero-thal/HbE patients (four non-splenectomized and six splenectomized) and five normal controls were investigated for their expression of Fc-gamma-RI by flow cytometry. In beta zero-thal/HbE there was an approximately 3-fold increase in the percentage of leucocytes expressing this receptor; the receptor was up-regulated on monocytes and induced on granulocytes. The up-regulation of Fc-gamma-RI in beta zero-thal/HbE is likely to be an important component in the activation of monocytes and in mediating their enhanced effector function towards antibody-coated cells.

Erythrocytes↗
PubMed1993

Possible role of macrophages in the pathogenesis of ethanol-induced bone marrow damage.

Adherent-cell-depleted human marrow cells (MC) were cultured on their own or co-cultured with monolayers of blood-monocyte-derived macrophages or marrow-derived adherent cells with and without 2 mg ethanol/ml for 24 h. The incorporation of 3H-thymidine and 3H-leucine by MC cultured on their own was not significantly influenced by ethanol. By contrast, the incorporation of both radiolabelled compounds was significantly lower in MC from the co-cultures containing ethanol than from those not containing ethanol. This effect was mediated by a diffusible factor produced by macrophages in the ethanol-containing cultures and was independent of intercellular contact. Supernatants from ethanol-containing cultures of marrow-derived adherent cells displayed cytotoxic activity against A9 cells due to the presence of unstable acetaldehyde-albumin complexes. Ethanol inhibited rather than stimulated nitrite production in the MC/marrow-derived adherent cell co-culture system, suggesting that macrophage-derived nitric oxide did not play a role in causing the observed ethanol-related effects. The data indicate that, in the presence of ethanol, macrophages cause inhibition of the incorporation of 3H-thymidine and 3H-leucine into overlying MC, at least partly by oxidizing ethanol to acetaldehyde and releasing some of the potentially cytotoxic acetaldehyde thus formed extracellularly. Bone marrow macrophages may therefore play an important role in the pathogenesis of alcohol-related marrow damage in vivo.

Bone Marrow↗
PubMed1993

Misincorporation of uracil into the DNA of folate- and B12-deficient HL60 cells.

HL60 cells were cultured for 10 days under various experimental conditions. They were then incubated with 1 mumol/l [5-3H] uridine for 2 hours and their DNA extracted. The DNA was hydrolysed to deoxyribonucleosides with phosphodiesterase and alkaline phosphatase and the hydrolysate subjected to Aminex A6 chromatography. The elution profiles showed that, when compared with control cells, DNA from cells grown in medium deficient in folate, B12 or both folate and B12 contained increased amounts of deoxyuridine (dU) and increased radioactivity in the dU peak. The data demonstrate that misincorporation of uracil into DNA occurs in a myeloid cell line cultured in growth medium deficient in folate, B12 or both folate and B12.

Chromatography, Ion Exchange↗
PubMed1993

Observations on two members of the Swedish family with congenital dyserythropoietic anaemia, type III.

Two affected individuals of the Swedish family with CDA, type III, in which the disease is transmitted as an autosomal dominant character, were studied. Both cases displayed features hitherto undescribed in this family but described in patients with CDA, type III, in whom the inheritance may have been as an autosomal recessive character. Such features were: (a) haemosiderinuria, (b) grossly disorganised erythroblast nuclei, (c) differences in the ultrastructural appearances of individual nuclei within the same multinucleate erythroblast and (d) intraerythroblastic inclusions resembling precipitated globin chains. In both cases the giant mononucleate erythroblasts and the multinucleate erythroblasts had total DNA contents up to 28c (1c = haploid DNA content) and 48c respectively, and some DNA synthesising bi- and multinucleate erythroblasts contained one or more nuclei which were unlabelled with 3H-thymidine. These findings are similar to those in patients with the autosomal recessive type of disease. Thus no major phenotypic differences are yet apparent between cases of CDA, type III, with different patterns of inheritance. Analysis of the surface erythrocyte proteins of the 2 Swedish CDA, type III, patients with monoclonal antibodies recognising Band 3, glycophorins A, B, C and D, Rh, CD44, CD47, CD55, CD58, CD59, Lutheran, Kell, LW and acetylcholinesterase did not reveal any gross abnormality of expression of these proteins. A slightly altered expression of blood group antigens A and H was revealed by the lectins Dolichos biflorus and Ulex europaeus and the Mr of Band 3 as judged by SDS polyacrylamide gel electrophoresis was also slightly reduced, suggesting that there may be minor alterations in the degree of N-glycosylation of some red cell membrane constituents.

ABO Blood-Group System↗
PubMed1993

Correlations between holo-transcobalamin II, holo-haptocorrin, and total B12 in serum samples from healthy subjects and patients.

AIMS: To study the correlations between total vitamin B12(B12), holo-haptocorrin, and holo-transcobalamin II (holo-TCII) concentrations in human sera; the association between reduced holo-TCII concentrations and macrocytosis attributable to B12 deficiency. METHODS: Serum samples from 38 healthy volunteers, 113 patients with normal total serum B12 concentrations and 93 patients with low total serum B12 were studied. Holo-TCII was removed from whole serum by adsorption with amorphous precipitated silica, and both whole serum and adsorbed serum were assayed for B12 using the Becton Dickinson vitamin B12 [57Co] radioassay kit. RESULTS: In all three groups of subjects studied there were strong correlations between the logarithms of the total serum B12 and the holo-haptocorrin concentrations with regression coefficients between 0.884 and 0.967. By contrast, the correlations between the logarithms of the total serum B12 and holo-TCII concentrations were weaker, especially in the patients with normal or low total serum B12, for whom the regression coefficients were 0.491 and 0.391, respectively. Analysis of the clinical records of a proportion of the patients studied indicated that there were many more patients with low holo-TCII concentrations than with haematological disturbances related to B12 deficiency. CONCLUSIONS: The total serum B12 concentration is a relatively poor indicator of holo-TCII concentrations and, therefore, of the ability of serum to deliver B12 to tissues. Additional information regarding B12 values can therefore be gleaned from measuring holo-TCII concentrations in the serum. Low holo-TCII concentrations, however, are an early sign of negative B12 balance and are frequently unassociated with haematological abnormalities caused by B12 deficiency.

Adult↗
PubMed1992

In vitro effects of vitamin C, thioctic acid and dihydrolipoic acid on the cytotoxicity of post-ethanol serum.

The serum of subjects consuming ethanol contains a non-dialysable cytotoxic activity, which is thought to reside in unstable acetaldehyde-protein adducts: the cytotoxic effects have been attributed to the transfer of acetaldehyde molecules from such adducts to target cells. When post-alcohol sera are incubated for 3 hr with ascorbic acid, thioctic acid or dihydrolipoic acid at a concentration of 10-500 micrograms/mL, their cytotoxicity against A9 cells is reduced. Post-alcohol sera incubated with these concentrations of thioctic acid or dihydrolipoic acid also had reduced cytotoxic activity against phytohaemagglutinin-transformed normal human lymphocytes. Studies with artificially produced [14C]acetaldehyde-125I-albumin complexes showed that treatment with thioctic acid or dihydrolipoic acid resulted in a reduced transfer of [14C]acetaldehyde to K562 cells. If these in vitro data also apply in vivo and if circulating acetaldehyde-protein adducts play a role in alcohol-mediated tissue damage, vitamin C and, to a greater extent, thioctic acid may have a beneficial effect in patients with acute and chronic alcohol toxicity.

Acetaldehyde↗
PubMed1992

Ultrastructure of the bone marrow in HIV infection: evidence of dyshaemopoiesis and stromal cell damage.

The ultrastructure of bone marrow cells was studied in nine patients infected with the human immunodeficiency virus (HIV). Two of these (cases 1 and 3) were thrombocytopenic, had never suffered from opportunistic infections and had not received any drugs prior to the time of study. A number of ultrastructural abnormalities were found in a variable proportion of the affected cell types in all nine patients. These were: (a) an increased prevalence of multivesicular bodies within several cell types and of abnormalities of the nuclear membrane in neutrophil granulocytes, (b) an increase in the size of the Golgi apparatus and in the quantity of endoplasmic reticulum in neutrophil granulocytes, (c) dysplastic features, including multiple long intranuclear clefts and large cytoplasmic vacuoles in some erythroblasts and (d) vacuolation of the plasma cells. Other abnormalities seen in a proportion of the patients were: (a) cylindrical confronting cisternae (CCC) in some of the lymphocytes, macrophages (phagocytic reticular cells), non-phagocytic reticular cells (including adventitial cells) and endothelial cells of marrow sinusoids, (b) tubuloreticular structures (TRS) in some lymphocytes, plasma cells, monocytes and endothelial cells and (c) precipitates of protein within occasional erythroblasts and marrow reticulocytes. There was also a striking and hitherto undescribed abnormality of the structure of the nucleus in intersinusoidal and perisinusoidal non-phagocytic reticular cells. This was seen in six patients, including case 3, and was characterized by the extensive detachment of masses of abnormally electron-dense heterochromatin from the nuclear membrane, the presence of a uniformly thin layer of electron-dense material at the inner surface of the areas of nuclear membrane denuded of heterochromatin masses and an abnormal electron lucency of areas containing euchromatin. The CCC and TRS were found in the six patients with the lowest number of circulating CD4-positive T cells. The precipitation of protein within erythroid cells may have been caused by the oxidant effect of dapsone or high doses of co-trimoxazole. The abnormalities in the stromal cells and in particular the nuclear changes seen in the non-phagocytic reticular cells support the possibility that one of the mechanisms underlying the cytopenia in patients infected with HIV may be a disturbance of the microenvironmental regulation of haemopoiesis.

Adult↗
PubMed1992

In vitro effects of ethanol on the phagocytic and microbial killing activities of normal human monocytes and monocyte-derived macrophages.

Human blood monocytes were cultured within the wells of chamber slides in growth medium to which 0, 1, 2, or 3 mg ethanol per ml was added at the start of the culture. After incubation for 1 h, 1 day and 7 days, their ability to phagocytoze IgG-sensitized red cells and to phagocytoze and kill non-opsonized Candida albicans was assessed. In all alcohol-containing wells, the concentration of alcohol in the growth medium fell progressively, reaching negligible values after 3 days. When compared with control cells, monocytes incubated with 1, 2 or 3 mg ethanol/mg for 1 h showed impaired phagocytosis of IgG-sensitized RBC and non-opsonized C. albicans and those incubated with 1 or 2 mg ethanol/ml for 1 h showed impaired killing of Candida. After incubation for 7 days, the monocyte-derived macrophages in wells initially containing 1, 2 or 3 mg ethanol/ml showed increased phagocytic activity towards C. albicans but not towards sensitized RBC. In addition, in 4 of 5 experiments, the percentage of phagocytozed organisms killed was increased in wells initially containing 1 mg ethanol/ml. The results support the view that the susceptibility of chronic alcoholics to certain infections may be partly dependent on an ethanol-induced depression of the phagocytic and killing functions of macrophages. They also suggest that a few days after a brief period of exposure to ethanol there may be stimulation of certain but not all effector functions of macrophages.

Candida albicans↗
PubMed1991

Stromal cell-dependent terminal maturation of K562 erythroleukaemia cells.

K562 cells were cultured over monolayers of various cell types for four days and the characteristics of those cells which became firmly adherent to such monolayers studied. The monolayers used were composed of stromal cells derived from human bone marrow, two human fibroblast lines, human umbilical cord-derived endothelial cells, bone marrow macrophages, blood-monocyte-derived macrophages, a neuroglial cell line (U-251 MG) or mixtures of some of the above. The data showed that K562 cells adhered much more frequently to fibroblasts than to other cell types. In the case of some marrow-derived stromal cell layers grown in human group AB serum, the firmly adherent K562 cells showed morphological evidence of nuclear and cytoplasmic maturation and, especially in the presence of haemin, sometimes developed into erythrocyte-like cells. The maturing cells were weakly or moderately benzidine-positive. By contrast, K562 cells which adhered to monolayers of fibroblasts, endothelial cells or macrophages showed little or no maturation and were either benzidine-negative or only very weakly positive. In experiments employing monolayers made up by mixing skin fibroblasts and endothelial cells or skin fibroblasts, endothelial cells and macrophages, some adherent K562 cells developed into cells resembling early and late polychromatic normoblasts and erythrocytes. The maturing cells were moderately or strongly benzidine-positive. Maturation was most marked in K562 cells adherent to regions of the monolayer containing all three of the stromal cell types. The results indicate that the optimal microenvironmental niche for the development of K562 cells into erythroblasts and erythrocytes may consist of a cluster of closely associated fibroblasts, macrophages and endothelial cells.

Benzidines↗
PubMed1991

Comparison of the generation of albumin-associated cytotoxic activity in supernatants from ethanol-containing cultures of human blood monocyte-derived macrophages and of two human hepatoma cell lines.

The rates of oxidation of ethanol to acetate by human blood monocyte-derived macrophages and the two human hepatoma cell lines PLC/PRF/5 and Hep G2 were studied. The average rates obtained were, respectively, 621, 447 and 596 nmol/h/mg cellular protein. Cultures of these three cell types, containing known quantities of cellular protein per flask, were incubated with 0 or 2 mg ethanol/ml for 72 h and the culture supernatants subjected to affinity chromatography on blue sepharose CL-6B. Pure albumin fractions obtained in this way were adjusted to the same optical density and tested for cytotoxicity against A9 cells. The data showed that the albumin fractions obtained from ethanol-containing macrophage cultures were considerably more cytotoxic than those obtained from ethanol-containing cultures of PLC/PRF/5 and Hep G2 cells. It appeared that, for a given quantity of ethanol metabolised, considerably more acetaldehyde was released extracellularly by macrophages than by the two hepatoma cell lines and that this acetaldehyde bound to albumin to form cytotoxic acetaldehyde-albumin complexes. The data raise the possibility that macrophages are an important source of extracellular acetaldehyde and circulating acetaldehyde-albumin complexes in vivo.

Acetaldehyde↗
PubMed1991

Congenital dyserythropoietic anaemia with novel intra-erythroblastic and intra-erythrocytic inclusions.

A hitherto undescribed form of congenital dyserythropoietic anaemia is reported. The patient was severely anaemic and hydropic at birth and is now 8 years old. She has a moderate normochromic normocytic anaemia. HbF level of 50%, reticulocyte count of 5-12% and hyperbilirubinaemia. Bone marrow smears showed intense normoblastic erythroid hyperplasia with morphological evidence of dyserythropoiesis; the most common dysplastic features were basophilic stippling of polychromatic erythroblasts and erythrocytes and marked abnormalities of nuclear shape in polychromatic erythroblasts. Electron microscope studies showed that some polychromatic erythroblasts and several erythrocytes contained inclusions which were rounded, elongated or irregular in outline or were doughnut-shaped. These inclusions consisted of compact masses of tubules and saccules which may represent smooth endoplastic reticulum together with Golgi cisternae. The ultrastructural studies also revealed peculiar membrane-bound cylindrical structures in a rare late erythroblast, and phagocytosed erythroblasts within some macrophages. The technique of combined Feulgen microspectrophotometry and 3H-thymidine autoradiography demonstrated a pile-up of early polychromatic erythroblasts in the G1 and G2 phases of the cell cycle, indicating a prolongation of, or an arrest at, these phases. Furthermore, nearly a quarter of all erythroblasts failed to incorporate 3H-leucine into protein. Thus the anaemia appeared to be due to a combination of disordered erythroblast function, increased ineffectiveness of erythropoiesis and peripheral haemolysis. The primary defect may be an excessive synthesis or impaired degradation of intracytoplasmic membranes.

Anemia, Dyserythropoietic, Congenital↗
PubMed1991

Natural autoantibodies may play a role in ineffective erythropoiesis during megaloblastic haemopoiesis.

Marrow aspirates from 11 patients with megaloblastic haemopoiesis and from 14 healthy individuals with normoblastic haemopoiesis were studied for antibodies associated with polychromatic/orthochromatic erythroblasts, using an 125I-labelled anti-human immunoglobulin reagent and autoradiography. In addition, the expression on these cells of receptors for FcIgG (FcR) and of the type I receptor for fragments of the third complement component (CR1) were investigated with receptor-specific monoclonal antibodies, 125I-labelled anti-mouse immunoglobulin and autoradiography. The percentages of immunoglobulin-positive erythroblasts were significantly greater in the megaloblastic than in the normoblastic marrows. Abnormally high percentages of labelled erythroblasts were present in patients without any manifestations of an autoimmune disorder. The percentage of labelled erythroblasts in the marrows of the patients correlated well with the degree of anaemia. FcR were absent on the majority of megaloblasts or normoblasts while the expression of CR1 was similar in both types of cell. The difference between the percentage labelling of megaloblasts and normoblasts was therefore unlikely to be due to greater binding of immune complexes with or without associated complement to megaloblasts than normoblasts. The megaloblast-bound immunoglobulin is, therefore, likely to have recognized abnormally expressed epitopes on the surface of megaloblasts. The results suggest that natural autoantibodies play a role in the destruction of erythroblasts during megaloblastic haemopoiesis.

Antibodies, Monoclonal↗