Biomedical subjects
J W Halliday
Publications and source records attributed to J W Halliday.
Desialylated transferrin as a serological marker of chronic excessive alcohol ingestion.
Partly desialylated transferrin was measured in the serum of subjects with chronic alcoholism, of patients with non-alcoholic-related steatohepatitis, diabetes, and other non-alcoholic liver diseases, and of healthy controls. In non-alcoholic patients and controls the maximum desialylated transferrin expressed in relation to total transferrin was 1.5%. This value was exceeded in 18 (90%) of the 20 alcoholics. By contrast, gamma-glutamyl transferase was within the reference range in 9 of the alcoholics.
Genetic hemochromatosis and HLA linkage.
We previously reported five families with primary, genetic (idiopathic) hemochromatosis in whom HLA typing of subjects indicated that a homozygous-heterozygous mating had almost certainly occurred and in whom inheritance of the disease trait was best explained by an autosomal recessive mode of inheritance. However, in one family, two children apparently homozygous for hemochromatosis did not manifest overt evidence of the disease, and alternative explanations were postulated, including autosomal dominant inheritance in this family. Subsequent study of the family members, including repeat HLA-DR serology with more recently defined antisera and DNA genotyping at the HLA-DR locus has, we believe, provided the true explanation for the previous apparent anomaly and adds further evidence for the tight linkage of the disease to the HLA-A locus.
The effect of experimental iron-overload on splenic T cell function: analysis using cloning techniques.
The effect of iron-overload on cell-mediated immunity was examined in C57 mice. Two methods of iron-loading were used: (i) dietary carbonyl iron which produced iron-loading primarily of parenchymal cells or (ii) intraperitoneal administration of iron-dextran which produced iron-loading predominantly of Kupffer cells. Both methods of iron-loading resulted in a diminished capacity of spleen cells to generate an allo-specific cytotoxic response in the absence of exogenous interleukin 2 (IL-2). Exogenous IL-2, however, restored the ability of spleen cells from iron-loaded mice to generate allo-specific cytotoxicity in bulk culture. Clonal assays for the precursor cells of cytotoxic T lymphocytes (CTL-P), performed in the presence of added IL-2, demonstrated that iron-loaded mice contained normal numbers of CTL-P. However, cultures of spleen cells from carbonyl iron-loaded mice generated less IL-2 following Concanavalin A stimulation, apparently as a result of a reduction in the number of IL-2-secreting cells amongst the spleen cell population. This work presents further evidence that iron-overload is associated with defective immunoregulatory control.
The effect of non-transferrin-bound iron on murine T lymphocyte subsets: analysis by clonal techniques.
A number of different immunological properties have been attributed to iron (Fe3+ and Fe2+) and iron-binding proteins. However, in many previous studies, high concentrations of iron were used and cell-cell interactions were not excluded as a possible cause of the observed immunomodulatory effects. In this study, clonal techniques have been used to examine the effect of non-transferrin-bound iron (Fe3+) on the T lymphocyte subsets required for the generation of cytotoxic T lymphocytes (CTL). Concentrations of non-transferrin-bound Fe3+ of 10 microM or greater were shown to inhibit the generation of C57, BALB/c and CBA allo-specific CTL in bulk culture. Limit-dilution analysis revealed that: (i) Fe3+ reduced the cloning efficiency of CTL-precursors (CTL-P) by up to 96% without affecting the rate of clone growth; (ii) Fe3+ did not affect the cloning efficiency of allo-stimulated Ly-2-ve T cell precursors but reduced the rate of clone growth of these cells; (iii) Fe3+ enhanced, by more than 13-fold, the function of clones of Concanavalin A (Con A)-induced suppressor T lymphocytes (STL) which suppressed in vitro the development of CTL from their precursor cells. The data provide further evidence that low concentrations of non-transferrin-bound Fe3+, of the same order as those reported to be present in the serum of patients with iron-overload, have significant immunoregulatory properties.
Improved purification of the human placental transferrin receptor and a novel immunoradiometric assay for receptor protein.
A simplified method for the purification of human placental transferrin receptor is described. The procedure involves chromatography of a detergent extract of placenta on immobilized iron-loaded transferrin. Knowledge of the physiology of the interaction between transferrin and its receptor is applied to enable bound receptor to be eluted under mild conditions and essentially free of containing transferrin. Purified transferrin receptor and a monoclonal antibody to the receptor were used to develop a novel immunoradiometric assay for the receptor in which the monoclonal antibody is the radiolabelled species. A competition between two populations of receptor, one immobilized on a particulate support and the other in solution, provides the basis for the assay. Using this assay we have measured transferrin receptor levels in placental and hepatic tissue and in three cell lines during both the logarithmic and stationary phases of cell growth.
Influence of alcohol and caffeine consumption on caffeine elimination.
Ten healthy male volunteers were each studied on four separate occasions to assess the role of regular caffeine and alcohol intake on caffeine elimination. Antipyrine disappearance was also studied as an established quantitative test of hepatic microsomal function. Regular caffeine intake in high doses for 1 week failed to alter either antipyrine or caffeine pharmacokinetics. In contrast, alcohol intake of 50 g/day significantly prolonged caffeine half-life by 72% (P less than 0.005) and diminished caffeine clearance by 36% (P less than 0.0005). However, antipyrine kinetics were unaltered. These results demonstrate that alcohol, in amounts commonly consumed, is a strong inhibitor of caffeine metabolism.
Simple radioimmunoassay for transferrin using insolubilized antitransferrin antibodies: its application to cultured cells.
A radioimmunoassay (RIA) for transferrin is described. The assay uses antitransferrin antibodies covalently coupled to the particulate support Matrex Pel 102, and is simple, sensitive, reproducible, and rapid. Transferrin measurement with this assay is independent of the degree of iron saturation of the protein. The RIA was applied to the measurement of transferrin concentrations in a variety of cultured human cells. Each of 11 cell lines studied contained endogenous transferrin, but the greatest concentration was found in Chang liver cells. Red blood cells were used as a negative control.
The effect of iron (Fe3+) on the cloning efficiency of human memory T4+ lymphocytes.
The effect of iron (Fe3+) and normal human liver ferritin on the proliferative response of normal human lymphocytes to tetanus toxoid was examined. This proliferative response involved memory T4+ lymphocytes as shown by a selective depletion study. Limit dilution analysis revealed that iron, present as ferric citrate, affected the initiation of clone development, and that concentrations of ferric citrate from 30 microM to 1 nM were able to reduce significantly the cloning efficiency of precursor T cells (up to 90% reduction). The reduced cloning frequency was not due to immunological suppression. Clone size was also reduced when iron was present during culture. In contrast, the presence of normal human liver ferritin during culture (concentration range: 300 micrograms/1-10,000 micrograms/1) had no effect on lymphocyte proliferation. The data indicate that low molecular weight iron (as ferric citrate) in concentrations similar to those which have been reported in the serum of patients with iron overload diseases, can interfere with antigen-specific lymphocyte responses and this may have implications for the development of infections and neoplasia in diseases of iron-overload.
Characterization of the binding of ferritin to the rat liver ferritin receptor.
The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.
A method for analysing the clonal precursors of concanavalin A-induced suppressor cells.
Spleen cells from 3 different strains of mice (C57 (H-2b), CBA (H-2k) and BALB/c (H-2d] were stimulated in vitro with different concentrations of concanavalin A (CA) for 48 h. This resulted in the production of cells capable of inhibiting the generation of alloantigen-specific cytotoxic T lymphocytes (CTL) in a mixed lymphocyte culture (MLC). 1 microgram/ml was an effective concentration of CA to induce C57 and BALB/c suppressor cells (SC), but 5 micrograms/ml CA was required to induce CBA SC. SC precursors (SC-P) were shown to be radiosensitive and the results suggest that SC themselves may be radiosensitive. SC were effective in the presence of added interleukin-2 (IL-2). SC were then induced at limit dilution in microwells in a volume of 25 microliter. A MIC (200 microliter) was then (after 48 h) added to each microwell. This resulted in a dilution of the concentration of CA to a level below which it was effective at inducing suppression. Cytotoxicity was then assessed 7 days later. It was thus possible to analyse SC-P at the clonal level and estimate their frequency. The frequency of C57 splenic SC-P (active against a C57 anti-BALB/c MLC) was 14.4 X 10(-6), the frequency of CBA splenic SC-P (active against a CBA anti-BALB/c MLC) was 92.3 X 10(-6), and the frequency of BALB/c splenic SC-P (active against a BALB/c anti-CBA MLC) was 15.8 X 10(-6). It was possible to analyse SC-P at a clonal level whether or not the MLC contained added IL-2. SC and SC-P were shown to be sensitive to anti-Thy-1 and complement.
Apoptosis as the mode of cell death in antibody-dependent lymphocytotoxicity.
A light and electron microscopic study of antibody-dependent lymphocytotoxicity was carried out with the object of elucidating the mechanisms responsible for the cell killing, the basis for the research being the relationship that has recently been shown to exist between the morphology of cell death and its pathogenesis. Chang liver cells coated with a rabbit anti-human antibody were used as targets and normal human peripheral-blood lymphocytes as effector cells. Cytotoxicity assays using release of 51Cr demonstrated extensive K-cell killing, thus validating the suitability of the model for morphological studies. Cell death displaying the features of apoptosis correlated with K-cell activity. A small amount of cell death by classical necrosis was observed, but its extent appeared to be unrelated to the presence of lymphocytes, to pre-treatment of the target cells with antibody, or to the magnitude of 51Cr release. The results support evidence indicating that lymphocytotoxicity depends on activation of a self-destruct program within the target cell. They do not favour a mechanism involving the production of plasma membrane lesions analogous to those responsible for complement-mediated immune cytolysis.
Cohort study of internal malignancy in genetic hemochromatosis and other chronic nonalcoholic liver diseases.
The risk of hepatocellular carcinoma (HCC) and other internal malignancies was examined in patients with genetic hemochromatosis (GH) by following 208 patients from the time of diagnosis to June 1983 and by comparing the numbers of cancers they developed with expected values constructed from cancer registry incidence data by means of actuarial methods. In addition, cancers occurring in a comparison group of 148 subjects with other chronic nonalcoholic liver diseases (CLD) were determined. Among the GH group, 16 new cases of HCC occurred subsequent to the diagnosis of GH, together with 8 other malignancies. The 16 cases of HCC reflect a 200-fold excess risk, which from all indications represents the first quantitation of the risk of this tumor in GH. There appears to be no increased risk of other malignancies in this disease. Among the CLD group only 1 HCC and 1 other malignancy occurred.
Alterations in the liver with ageing.
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The effect of acute liver damage on circulating ferritin levels in vivo and in the isolated perfused rat liver.
The effects of minimal acute liver injury on circulating ferritin levels have been examined in the rat both in vivo and in the isolated perfused liver. Liver damage produced by 6 mmol/kg of D-galactosamine (GalN) in vivo resulted in a marked rise in plasma ferritin levels 4 h after administration, 2 h before any significant increase in plasma aspartate transaminase. In the isolated perfused liver, damage produced by 5mM GalN introduced into the perfusate also produced an early increase in circulating ferritin before any evidence of release of intracellular enzymes, or alteration in liver histology as assessed by light microscopy was apparent. It is concluded that minimal acute liver damage results in a pronounced increase in circulating ferritin levels before other evidence of liver dysfunction. This is unlikely to be due solely to increased release from damaged cells but may rather result from an alteration in the mechanism responsible for ferritin homeostasis.
Use of chromatofocusing to detect a transferrin variant in serum of alcoholic subjects.
We describe a technique for detecting an abnormal (pl 5.7) transferrin component in serum, which appears after prolonged heavy consumption of alcohol. Serum transferrin was purified by chromatography on DEAE-Affi-Gel Blue and analyzed by chromatofocusing on an ion-exchange column (Mono P). The abnormal transferrin component was detected in 17 of 20 patients (85%) with a history or prolonged consumption of alcohol (100 g per day), and in control subjects who ingested up to 80 g of alcohol per day for seven days, but not in 14 normal control subjects or 14 patients with liver disease unrelated to alcohol. The variant consistently disappeared from the serum within three weeks of cessation of alcohol consumption. It is apparently produced by desialylation of ordinary human transferrin. We find that chromatofocusing on an ion-exchange column is a sensitive and reliable technique for its identification and conclude that detection of this desialylated transferrin indicates recent prolonged alcohol ingestion.
The detection of IgG anti-hepatocyte antibodies by ELISA in sera of patients with chronic active hepatitis: correlation with disease activity.
Anti-hepatocyte membrane IgG antibodies were detected in serial dilutions of sera from patients with chronic active hepatitis using an enzyme linked immunosorbent assay with isolated rat hepatocytes as the antigenic substrate. The assay is rapid, reliable and reproducible. Antibodies to hepatocyte membrane antigens were detected in 24 out of 31 (77%) patients with autoimmune chronic active hepatitis. Four of these patients were negative in a 1/10 dilution but became positive on progressive dilution. In 12 patients, liver biopsy was performed at the same time as serum was obtained. In these patients, the titre of antibodies to hepatocyte membrane antigen correlated significantly with the overall biopsy score of disease activity and particularly with the degree of portal tract infiltration. In 2 patients followed serially, titres of anti-hepatocyte membrane antibodies fell progressively in parallel with clinical, biochemical and histological evidence of improvement in the liver disease. The estimation of titres of antibodies to hepatocyte membrane antigens using isolated rat hepatocytes as the antigenic substrate may assist in the diagnosis, assessment of disease activity and follow-up of individual patients with chronic active hepatitis.
Ferritin metabolism and the liver.
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