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Biomedical subjects

A Bomford

Publications and source records attributed to A Bomford.

At least 55 records · Page 3Linked to original sources
PubMed1988

Phytohaemagglutinin induced proliferation of lymphocytes from patients with rheumatoid arthritis and iron deficiency.

The response of peripheral blood lymphocytes to stimulation by mitogens such as phytohaemagglutinin (PHA) is commonly depressed in both rheumatoid arthritis and iron deficiency, and as many rheumatoid patients are anaemic with evidence of abnormal iron metabolism it is possible that the same mechanism underlies the observed suppression in both conditions. In the present study the mitogenic response to PHA of lymphocytes from three rheumatoid patients, who were also iron deficient, and two healthy controls has been shown to be significantly less in iron deficient than iron containing media (p less than 0.001). In addition, iron deficient sera from these patients reduced the PHA induced proliferation of lymphocytes from a normal subject (p less than 0.01), an effect which was prevented by prior addition of iron to these serum samples. In iron containing media lymphocytes from five patients and two controls showed no difference in their response to PHA for both the minimum mitogen concentration which enhanced transformation and the peak [3H]thymidine uptake; but patients' lymphocytes showed significantly less response to PHA concentrations of 5 and 10 mg/l (p less than 0.02), resulting in a reduction in the area under the dose response curves up to 20 mg/l (p less than 0.05). These findings show both that iron deficient sera can impair PHA induced lymphocyte transformation and that lymphocytes from iron deficient rheumatoid patients have impaired responsiveness to PHA. Iron is known to be required intracellularly for the enzyme ribonucleotide reductase, which is important for DNA synthesis, and reduced activity of this enzyme could explain these observed effects.

Adult↗
PubMed1988

Nontransferrin-bound iron in plasma from hemochromatosis patients: effect of phlebotomy therapy.

Plasma from patients with iron overload resulting from idiopathic hemochromatosis contains nontransferrin-bound iron, measurable by the bleomycin, assay. During venesection therapy, the concentration of bleomycin iron declines in a way highly correlated with plasma ferritin concentrations. Even when patients had been venesected to give very low total plasma iron concentrations and high transferrin iron-binding capacity, bleomycin-detectable iron was still present at low concentrations. Bleomycin-detectable iron can stimulate damaging free radical reactions, and its persistence in plasma even after prolonged venesection might contribute to the tissue damage that results from iron overload.

Adult↗
PubMed1988

Measurement of ferritin in serum by an indirect competitive enzyme-linked immunosorbant assay.

This indirect competitive enzyme-linked immunosorbant assay (ELISA) for ferritin, unlike other currently available ELISAS, does not require use of an anti-ferritin antibody-enzyme conjugate. Designed for use on microtiter plates, the method has a precision and sensitivity similar to those of other immunoassays. The detection limit is 20 pg of ferritin per test (corresponding to 2.0 micrograms/L in serum samples). Comparison of results obtained on serum from 57 patients by this method with those from a conventional radioimmunoassay gave a correlation coefficient of 0.92.

Enzyme-Linked Immunosorbent Assay↗
PubMed1987

Heterogeneous distribution of transferrin receptors on parenchymal and nonparenchymal liver cells: biochemical and morphological evidence.

To investigate which cells of the liver express the receptor for transferrin, isolated rat liver cells produced by collagenase perfusion were fractionated by repeated differential centrifugation to produce hepatocytes (95% + 1%, mean +/- SD, n = 4) and nonparenchymal cells (97% + 1%, n = 3). Saturable, high-affinity binding of 125I-transferrin was demonstrated on intact cells at 4 degrees C, with average receptor numbers 20,900 +/- 3,160 (mean + SD, n = 4) for hepatocytes and 5,500 + 1,520 (n = 3) for nonparenchymal cells. Total cellular receptors measured in detergent permeabilized hepatocytes were 42,000 +/- 18,330 (mean +/- SD, n = 3) per cell and 14,760 +/- 7,120 (n = 3) per cell in the nonparenchymal fraction. Immunocytochemical demonstration of transferrin using antitransferrin, peroxidase antiperoxidase complex confirmed that both cell types bound transferrin. There was heterogeneity of the staining reaction since there was no detectable staining on 40% of hepatocytes and 60% of nonparenchymal cells. Microdensitometric analysis of the staining product corroborated the biochemical evidence that hepatocytes have, on average, more than three times more transferrin receptors than do nonparenchymal cells. These findings support the concept that the hepatocyte has a central role in the uptake and storage of transferrin iron.

Animals↗
PubMed1986

The effect of desferrioxamine on transferrin receptors, the cell cycle and growth rates of human leukaemic cells.

The effect of the iron chelator, desferrioxamine, on transferrin binding, growth rates and the cell cycle was investigated in the human leukaemic cell line, K562. At all concentrations of the chelator (2-50 microM) binding of 125I-transferrin was increased by 24 h and reached a maximum at 72-96 h. Maximum binding (6-8-fold increased) occurred in cells treated with 20 microM-desferrioxamine, in contrast with control cells which, at 96 h, showed a 50% decrease over initial binding. Scatchard analysis at 4 degrees C showed that this increased binding was due to an increase in the number of receptors, as the Kd was similar in induced (1.8 nM) and control (1.5 nM) cells. After 96 h cells, cultured with 20 and 50 microM-desferrioxamine accumulated 59Fe from bovine transferrin at over twice the rate found with control cells, reflecting the increase in transferrin receptors. Although iron uptake was unimpaired by the chelator there was a dose-dependent inhibition of cell growth, with control cells completing three divisions in 96 h and those in 10 microM-desferrioxamine only two divisions. At the highest concentration (50 microM), cell division was abrogated although cell viability was maintained (85%). In contrast, DNA synthesis was not markedly affected, except at 50 microM-desferrioxamine when incorporation of [3H]thymidine was 52% of that in control cells. Flow cytometry revealed that there was a progressive accumulation of the cells in the active phases of their cycle (S, G2 + M). Desferrioxamine may increase transferrin receptors in two ways: by chelating a regulatory pool of iron within the cell, and by arresting cells in S phase when receptors are maximally expressed.

Cell Cycle↗
PubMed1986

Intracellular forms of iron during transferrin iron uptake by mitogen-stimulated human lymphocytes.

Transferrin receptors expressed by mitogen stimulated human lymphocytes mediate the uptake of transferrin iron into haem, ferritin and a non-haem, non-ferritin component. In spite of different rates of iron uptake by cells from different individuals, the proportional incorporation of 59Fe into these components was similar, suggesting that there was an obligatory relationship between the different forms of iron in the cells. By 3 h over 60% of the iron taken up was incorporated into ferritin while less than 10% was found in haem. Initially (10 min) non-haem, non-ferritin iron comprised 70% of total iron and this diminished to 30% by 3 h. At 10 min 80% of iron in the non-haem, non-ferritin component was retained by anti-transferrin affinity columns indicating it was transferrin-bound. The proportion retained fell to reach a steady state level of 50% by 60 min. These results indicate that 10-20% of the iron in the cells was not recognized as transferrin, ferritin or haem iron. The finding that iron incorporation into this unidentified pool reached equilibrium while that into haem and ferritin increased suggests the iron may act as a precursor for functional and storage compounds.

Chromatography, Affinity↗
PubMed1986

Wash off liver cytology: a complementary diagnostic tool to liver biopsy.

Parenchymal and non-parenchymal cells were harvested by washing the liver tissue core and needle after percutaneous biopsy. The cytological material obtained was suitable for morphological analysis, including showing the presence of surface and cytoplasmic antigens using labelled antibody techniques. This technique provides a combined cytological and histological approach to the diagnosis of liver disease.

Antigens↗
PubMed1986

Treatment of rheumatoid arthritis with desferrioxamine.

Desferrioxamine was used to treat six patients who had rheumatoid arthritis refractory to conventional treatment, on the basis that levels of intra-articular iron would be reduced and inflammation lessened. After a period of initial intensive treatment which was limited by side effects, five patients continued on once-weekly maintenance doses. Two patients had temporary improvement in their symptoms, but relapsed in spite of continuing treatment. The remaining three patients completed six months of treatment with no improvement in their rheumatoid disease. There were no significant changes in rheumatological parameters, immunological markers of disease activity nor radiological evidence of improvement. Treatment did lead to significant falls in haemoglobin concentration (p less than 0.01), mean corpuscular volume (p less than 0.05) and serum ferritin levels (p less than 0.02). Therefore, in spite of a reduction in iron available for haem synthesis and a fall in tissue storage iron the rheumatoid inflammatory process persisted.

Adult↗
PubMed1985

Intracellular processing of transferrin and iron by isolated rat hepatocytes.

Transferrin bound by isolated rat hepatocytes is rapidly endocytosed and enters a compartment of low density. Little was found associated with the lysosomes, even though the protein was subsequently lost from the cells. Iron entering the cells on transferrin was subsequently found in a number of intracellular components: transferrin, haem, ferritin and a residual fraction. After 2 h incubation with 59Fe-transferrin almost 70% of the iron was in ferritin, and this proportion increased to 80% during a 'chase' experiment. Residual iron, because of its rapid increase at the start of the incubation and its decline during the 'chase', probably represents an intracellular transit pool, which at steady state was present at 23 pg/10(6) cells.

Animals↗
PubMed1985

Release of iron from the two iron-binding sites of transferrin by cultured human cells: modulation by methylamine.

We have investigated the effect of increasing concentrations of methylamine (5, 10, and 25 mM) on the removal of iron from the two iron-binding sites of transferrin during endocytosis by human erythroleukemia (K562) cells. The molecular forms of transferrin released from the cells were analyzed by polyacrylamide gel electrophoresis in 6 M urea. Endocytosis of diferric transferrin was efficient since greater than 10% of surface-bound protein escaped endocytosis and was released in the diferric form. Although transferrin exocytosed from control cells had been depleted of 80% of its iron and contained 65-70% apotransferrin, iron-bearing species were also released (15% C-terminal monoferric; 10% N-terminal; 10% diferric). The ratio of the two monoferric species (C/N) was 1.32 +/- 0.12 (mean +/- SD; n = 4), suggesting that iron in the N-terminal site was more accessible to cells. In the presence of methylamine there was a concentration-dependent increase in the proportion of diferric transferrin release (less than 80% at 25 mM) and a concomitant decrease in apotransferrin. Small amounts of the iron-depleted species, especially apotransferrin, appeared before diferric transferrin, suggesting that these were preferentially released from the cells. The discrepancy between the proportions of the monoferric transferrin species noted with control cells was enhanced at all concentrations of methylamine, most markedly at 10 mM when the C/N ratio was 2.4. The N-terminal site of transferrin loses its iron at a higher pH than the C-terminal site, and so by progressively perturbing the pH of the endocytic vesicle we have increased the difference between the two sites observed with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1984

The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin.

Animals↗
PubMed1984

Abnormal in vitro function of a variant human transferrin.

Normal and variant transferrins have been isolated from the plasma of an individual heterozygous for the abnormal protein. The normal and variant proteins were separated, saturated with 59Fe, labelled with 125I and then compared in their interaction with PHA-stimulated human lymphocytes in vitro. At 4 degrees C the variant protein bound to the transferrin receptors on these cells with an association constant (6.25 +/- 2.53 X 10(7) l mol-1, mean +/- SD; n = 4) which was an order of magnitude lower than that of the normal diferric transferrin (6.31 +/- 1.82 X 10(8) l mol-1, mean +/- SD; n = 4). This low association constant was reflected in a much reduced rate of iron donation to the cells at 37 degrees C (22.2 +/- 8.3 pg/10(6) cells/h compared with 48.1 +/- 15.6 pg/10(6) cells/h). As the variant transferrin has both abnormal iron-binding properties (Evans et al, 1982) and an abnormal interaction with the transferrin receptor, it would appear that these two functions may be closely interdependent.

Humans↗
PubMed1983

Dual pathways for the uptake of rat asialotransferrin by rat hepatocytes.

Studies of the interaction of rat asialotransferrin with rat hepatocytes at 4 degrees C showed that the protein binds to two types of receptor on these cells--to 31,000 transferrin receptors with a Ka of 1.9 x 10(7) liters . mol-1 and to 110,000 asialoglycoprotein receptors with a Ka of 1.4 x 10(6) liters . mol-1. The uptake of the protein by the cells at 37 degrees C is mediated by both receptors simultaneously. Binding to the asialoglycoprotein receptor was abolished by the addition of excess asialoorosomucoid and, under these conditions asialotransferrin, taken into the cells via the transferrin receptor, was subsequently released from the cells undegraded, an event also observed when the uptake of holotransferrin was examined in similar studies. Addition of excess unlabeled transferrin prevented uptake of asialotransferrin via the transferrin receptor and internalization was then mediated solely by the asialoglycoprotein receptor. Ligand subsequently released from the cells after endocytosis via this receptor was found to be degraded, with 80% soluble in trichloroacetic acid, a proportion similar to that observed during release of asialoorosomucoid from the cells. These results indicate that rat asialotransferrin is processed by the asialoglycoprotein uptake pathway in the same way as observed with other ligands. Release from the cells of undegraded asialotransferrin occurred only after endocytosis via the transferrin receptor.

Animals↗
PubMed1983

Uptake and release of transferrin and iron by mitogen-stimulated human lymphocytes.

Phytohaemagglutinin stimulation of human peripheral blood lymphocytes resulted in the expression of transferrin receptors and the uptake of iron into the cells. As assessed from the resistance of the 125I label to pronase, transferrin was rapidly bound and internalized at 37 degrees C, while at 4 degrees C 85% of the 125I label remained on the cell surface and was degraded by the pronase. Over 94% of the 125I label associated with and subsequently released from the cells was acid precipitable, indicating that transferrin was not degraded during its uptake and release. After reincubation for 1 h in fresh medium 70% of the cell associated 125I-transferrin was released. In contrast, less than 30% of the 59Fe was released, showing that iron was removed from transferrin and retained by the cells. Concentration dependent binding of 125I-transferrin estimated at 37 degrees C occurred with an apparent Ka of 5.7 +/- 1.1 X 10(7) l mol-1 (mean +/- SD, n = 4) indicating little variation between cells from different individuals, although the number of transferrin molecules associated with the cells varied greatly from 6.2 X 10(4)/cell to 1.4 X 10(5)/cell. The rate of iron uptake from 59Fe and 125I labelled transferrin at 37 degrees C by the cells from different subjects was also very variable, with a range between 0.46 and 2.27 pg Fe/min/10(6) cells (n = 6). However, iron uptake did not correlate with the amount of transferrin bound. This suggests that transferrin uptake and the release of iron from the transferrin to the interior of the cell are controlled independently.

Cells, Cultured↗