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Biomedical subjects

A Bomford

Publications and source records attributed to A Bomford.

At least 37 records · Page 2Linked to original sources
PubMed1994

Antibodies to atypical mycobacteria in primary biliary cirrhosis.

Recent work has shown that there is antigenic similarity between common bacterial proteins and epitopes on biliary epithelial cells. Following evidence that the sera of all of a series of patients with primary biliary cirrhosis contained antibodies to an extract of Mycobacterium gordonae (a ubiquitous environmental organism) and that these antibodies cross-reacted with the major mitochondrial M2 epitopes, the study below has been performed to investigate this phenomenon further. Using Western blotting of membrane extracts of Mycobacterium gordonae and seven other atypical mycobacterial species, no such antibodies were detected in the serum of patients with primary biliary cirrhosis, but antibodies to a 65 kDa mycobacterial protein were found in most patients with primary biliary cirrhosis as well as in normal controls and patients with other chronic liver diseases.

Antibodies, Bacterial↗
PubMed1994

Metal ions catalytic for free radical reactions in the plasma of patients with fulminant hepatic failure.

We propose that the frequency and severity of multi-organ failure (MOF) in fulminant hepatic failure (FHF) involves free radical damage caused by the presence of circulating iron and copper ions, catalytic for free radical reactions. The presence of such metal ions is demonstrated by using the sensitive bleomycin and phenanthroline assays. Antioxidant therapy, e.g., using chelating agents that prevent metal ions from stimulating free radical reactions, may have benefit in the treatment of FHF and its consequences.

Acetaminophen↗
PubMed1993

Reversibility of hypogonadotrophic hypogonadism associated with genetic haemochromatosis.

OBJECTIVE: Recent reports have suggested that hypogonadotrophic hypogonadism in men with genetic haemochromatosis can be reversed by aggressive venesection therapy. We have studied prospectively men with this complication of haemochromatosis in order to document the frequency and completeness of recovery from hypogonadism. PATIENTS AND DESIGN: Six men with symptomatic hypogonadotrophic hypogonadism and genetic haemochromatosis were studied before beginning venesection therapy and again after depletion of body iron stores. MEASUREMENT: Symptoms of hypogonadism, serum gonadotrophins and serum total and free testosterone concentrations. RESULTS: Five men aged 47-66 showed no symptomatic improvement and no change in serum gonadotrophin or testosterone concentrations. The symptoms of one man, aged 33, resolved completely after venesection and this was accompanied by increases in serum LH and FSH. Serum total and free testosterone concentrations increased fourfold, but remained subnormal. CONCLUSION: Only one partial recovery from hypogonadotrophic hypogonadism was seen and this was in the youngest subject. In reviewing the other documented cases of reversal it would appear that the age at diagnosis is critical and there are no proven cases of reversal of hypogonadotrophic hypogonadism in men over the age of 40 at the start of venesection therapy.

Aged↗
PubMed1991

Hip arthropathy in genetic hemochromatosis. Radiographic and histologic features.

Genetic hemochromatosis, a disorder of iron metabolism, results in the deposition of massive amounts of iron in the tissues. Arthropathy is one of a number of clinical features associated with the disease. Characteristic radiographic features in the wrist and hand have been reported, and an increased incidence of severe hip disease has been observed. In this study, hip radiographs of 112 patients with genetic hemochromatosis and arthritis were reviewed, and histologic examination of 2 femoral heads was performed. Twenty-eight of the 112 patients (25%) had evidence of arthritis of the hip joint. In 23 (82%) of the 28 patients, this feature was thought to be associated with osteoarthritis; 2 of these patients had an atypical arthropathy associated with radiolucency of the femoral head and histologic features of atypical stripping of the cartilage from the subchondral bone. These atypical features were not thought to be due to avascular necrosis, pyrophosphate-associated arthropathy, apatite-associated deposition arthritis, or osteoarthritis, but may be typical of genetic hemochromatosis and possibly the result of increased susceptibility to shearing forces at the bone-cartilage interface. In 5 of the 28 patients (18%), chondrocalcinosis was the sole abnormal finding on radiography. Ten of the 28 patients eventually required hip surgery, which confirms the severity of the hip disease associated with genetic hemochromatosis.

Adult↗
PubMed1990

Determinants of the interaction between the iron-responsive element-binding protein and its binding site in rat L-ferritin mRNA.

Ferritin messenger RNA has been shown to be translationally inactivated by the binding of a cytosolic protein to a 28-nucleotide iron-responsive element (IRE) located in the 5'-untranslated region of the mRNA. This interaction has been studied using quantitative receptor-ligand binding methods with gel retardation and nitrocellulose filter binding assays for the separation of bound complex from free RNA. In competition assays the entire 5'-untranslated region and the isolated IRE bound identically. The specificity of the RNA binding was studied using IRE variants. Two IREs from transferrin receptor mRNA and several variants with single base substitutions in the stem or loop had similar affinities. RNAs which could not form a stem-loop structure bound 1000-fold less well. These studies demonstrate the importance of the RNA conformation and the relative insensitivity of binding to much of the primary sequence. Saturation assays with increasing concentrations of 32P-IRE resulted in a binding hyperbola characteristic of mass action binding to a single class of sites with a KD = 0.09 nM. At 37 degrees C the dissociation rate is 0.04 min-1 (t 1/2 = 17 min). This rate is fast enough to account for the shift of ferritin RNA from the ribonucleoprotein pool to polysomes after rats are injected with iron. Determination of the concentration of the repressor requires accounting for three interconverting pools: free active repressor, mRNA-bound protein, and inactive (low affinity) repressor. Rat liver cytosol has a concentration of free active repressor of about 1 pmol/mg protein. Protein bound to endogenous mRNA can be measured by pretreatment with micrococcal nuclease or by separation with DEAE-Sepharose chromatography; it is present at a level similar to that of the free active protein. Inclusion of high levels of thiol reductants in the binding incubations reduces the inactive or low affinity repressor, forming unstably activated protein which has the same KD as the endogenous active protein; this inactive or low affinity protein is 2-4 times more abundant. A mechanism for iron regulation is proposed which accounts for the kinetics, the multiple protein pools, and the characteristics of the protein in these pools.

Animals↗
PubMed1990

Molecular analysis of the human MHC class I region in hereditary haemochromatosis. A study by pulsed-field gel electrophoresis.

The unknown allele that predisposes to the development of haemochromatosis in man has been localized to the HLA class I region on the short arm of chromosome 6. We have utilized pulsed-field gel electrophoresis in conjunction with probes that map within, or in the vicinity of, this region to search for structural lesions that may further define the disease locus. Using the enzyme Mlu I, fragments that associated specifically with the HLA-A23, A31 and B8 alleles were identified. However, in members of three pedigrees affected by haemochromatosis, and in six unrelated patients with the disorder, no disease-specific differences were detected in the DNA fragments with four restriction enzymes and eight probes when compared with healthy individuals. These data suggest that the lesion responsible for hereditary haemochromatosis lies beyond the resolution of this technique and does not involve large structural deletions or extensive re-arrangements in this highly polymorphic region of the genome.

Alleles↗
PubMed1990

Mechanisms of inhibition of mononuclear cell activation by the iron-chelating agent desferrioxamine.

Iron-withholding by the chelating agent desferrioxamine abrogates the proliferative response of human peripheral blood mononuclear cells (PBMC) to phytohaemagglutinin (PHA). The present study investigated whether desferrioxamine operates late in the activation process or, as recently suggested, at an early stage, by inhibiting the appearance of the interleukin-2 (IL-2) receptor. Human PBMC were stimulated with PHA (10 micrograms/ml) and [3H]thymidine ([3H]TdR) incorporation determined after 66 hr of culture. Greater than 90% inhibition was achieved by concentrations of desferrioxamine as low as 5 mumol/l present throughout culture, while IL-2 receptor expression (anti-Tac), analysed by FACS, was maintained at up to 75% of control levels. 300 mumol/l desferrioxamine present throughout culture abrogated [3H]TdR incorporation and additionally suppressed IL-2 receptor to 10-15% of control levels. In contrast, the same high dose of desferrioxamine when added for 2 hr to cells previously cultured for 66 hr produced 80% inhibition of [3H]TdR incorporation but failed to inhibit expression of the IL-2 receptor. Desferrioxamine rapidly achieved equilibrium across the cell membrane (within 60 min) and chelated 59Fe delivered to activated cells by the transferrin endocytic cycle. These results indicate that desferrioxamine can inhibit T-cell activation either early or late in the process by chelating iron and independently of an effect on the IL-2 receptor. In support of a dual effect of the drug is the finding that at 50 mumol/l, desferrioxamine-enhanced expression of the transferrin receptor occurred, an adaptive response made to intracellular iron depletion, while IL-2 receptor expression was inhibited.

Cell Division↗
PubMed1989

Biochemical studies of the iron cores and polypeptide shells of haemosiderin isolated from patients with primary or secondary haemochromatosis.

Haemosiderin isolated from different iron-loading syndromes, primary haemochromatosis (PHC) and secondary haemochromatosis (SHC) biochemically exhibited differences in both their iron core and peptide composition. The rate of release of iron from PHC haemosiderin to oxalate was 3-fold greater than that from SHC haemosiderin. The major peptides separated by SDS-PAGE showed a major band at Mr 20,000 for PHC haemosiderin and at Mr 15,000 for SHC haemosiderin.

Deferoxamine↗
PubMed1989

Occupancy of the iron-binding sites of human transferrin in sera obtained from different anatomical sites.

Transferrin is the major iron-transport protein in serum. Four molecular species (apo-, two mono-, and diferric transferrins) can be distinguished on the basis of their occupancy with iron. These species differ physicochemically and in the affinity with which they bind to the transferrin receptor. To elucidate the possible role of the four molecular species in directing the flow of iron between the major anatomical sites of iron release and utilization we have analyzed sera from eight stable multiorgan donors. The samples were obtained from veins draining the spleen, gut, and liver, and from the periphery. Employing polyacrylamide-gel electrophoresis in combination with crossed immunoelectrophoresis we were able to identify the four molecular species in all samples. Apo-transferrin was the predominant molecular species while diferric transferrin was the least abundant (P less than 0.01). The monoferric species were dominated by the acid-stable form (iron loading on the C-terminal end of the molecule) with ratios C/N from 1.2 (splenic and posthepatic serum) to 1.5-1.6 in mesenteric or peripheral samples respectively. We conclude from our study that it is unlikely that the monoferric transferrin species play a role in directing internal iron exchange and that in accordance with most of the literature the acid-stable form is preferentially loaded under physiological conditions of iron metabolism.

Adolescent↗
PubMed1989

Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy.

The nature of non-transferrin-bound iron in the plasma or serum of iron-overloaded hemochromatosis patients was studied by high performance liquid chromatography (HPLC) and high resolution nuclear magnetic resonance (NMR). 500-MHz proton Hahn spin-echo NMR spectra of plasma or serum, combined with the use of the iron chelator desferrioxamine, suggests complexation of iron ions with citrate and a possible involvement of acetate. Addition of FeCl3 to hemochromatosis samples broadened the NMR signals from citrate. HPLC analysis rigorously confirmed the presence of an iron-citrate complex in ultrafiltrates of plasma or serum studies with added FeCl3 or desferrioxamine supported this conclusion. It is proposed that non-transferrin-bound iron in the plasma of iron-overloaded patients exists largely as complexes with citrate and possibly also as ternary iron-citrate-acetate complexes. The presence of such complexes would account for the ability of non-transferrin-bound iron to be measurable by the bleomycin assay and for its rapid clearance from the circulation by the liver.

Acetates↗
PubMed1989

Regulation of the hepatic transferrin receptor in hereditary hemochromatosis.

The liver is the main site of iron accumulation and pathologic sequelae in hereditary hemochromatosis. Whether this is a result solely of inappropriately increased absorption of iron by the gastrointestinal tract or a more generalized regulatory failure of iron balance is unknown. Using immunohistochemical techniques, we have examined the effects of therapeutic changes in liver iron stores on the expression of the hepatic transferrin receptor in hereditary hemochromatosis. Ten patients with untreated hereditary hemochromatosis had no detectable staining for transferrin receptor in their liver biopsies. All had increased hepatic ferritin (mean = 19.9 micrograms per mg protein, range = 1 to 31.7 micrograms per mg protein) and hepatic iron levels (mean = 36.2 micrograms per mg protein, range = 3.6 to 69.9 micrograms per mg protein). In contrast, hepatocyte transferrin receptor was detected in seven patients in whom hepatic iron stores were markedly depleted by venesection (hepatic ferritin mean = 0.32 microgram per mg protein, range = 0.16 to 0.53 microgram per mg protein; hepatic iron mean = 0.98 microgram per mg protein, range = 0.3 to 2.1 micrograms per mg protein). Sequential data from one patient confirmed the reexpression of receptor in response to therapeutic iron depletion, whereas data from another patient studied during treatment illustrated a reciprocal relationship between liver tissue distribution of iron and expression of transferrin receptor. The finding that appropriate physiologic regulation of the hepatic transferrin receptor operates in hereditary hemochromatosis does not support the concept of a generalized defect in receptor-mediated uptake of transferrin-bound iron.

Antibodies, Monoclonal↗
PubMed1989

Optimizing the immunohistochemical signal from the transferrin receptor in liver tissue.

Cellular expression of the transferrin receptor is determined by the proliferative state and iron requirements of the cell. Previous immunohistochemical studies using a number of anti-(transferrin receptor) monoclonal antibodies confirmed the biochemical evidence that hepatocytes express the receptor, although the distribution shown was patchy with only a small number of cells showing positive staining. In the present study, a number of techniques have been compared to optimize detection of the immunohistochemical signal from the transferrin receptor in human liver tissue. Using an alkaline phosphatase detection system, widespread expression of this receptor with both cytoplasmic and membrane staining was found in all parenchymal and non-parenchymal cells.

Alkaline Phosphatase↗
PubMed1989

Hypogonadism and sexual dysfunction in hemochromatosis: the effects of cirrhosis and diabetes.

The contribution of diabetes and cirrhosis to sexual dysfunction and hypogonadism was evaluated by two-way analysis of variance in a group of 30 men with idiopathic hemochromatosis. The prevalence of severe sexual dysfunction was significantly higher in men with hemochromatosis than in a control group matched for prevalence of diabetes and age (P less than 0.001). In both controls and hemochromatosis patients the presence of diabetes was significantly associated with sexual dysfunction (P less than 0.005), but the more severe symptoms in the hemochromatosis patients were related to the additive effects of hypoandrogenism (P less than 0.01). Sexual dysfunction was a common early complaint in hemochromatosis patients, but these symptoms were frequently overlooked, leading to diagnostic delay. Mean testicular volume was a useful measure of gonadal status, being significantly correlated with indices of serum free testosterone (rs = 0.83; P less than 0.01) and LH (rs = 0.71; P less than 0.001). The presence of cirrhosis did not contribute significantly to symptomatology, but had an effect independent of and additive to hypogonadotropic hypogonadism in reducing serum free testosterone (P less than 0.02) and estradiol (P less than 0.002), an effect apparently mediated through central rather than testicular mechanisms. Hypoandrogenism was associated with an increase in serum sex hormone-binding globulin (SHBG) concentrations (P less than 0.005), but cirrhosis also had an independent effect in raising SHBG (P less than 0.005), which could not be accounted for by changes in circulating sex hormone concentrations. Thus, the evaluation of sexual dysfunction or hypogonadism in men with hemochromatosis requires consideration of the effects of both diabetes and cirrhosis. Because of the greater variance in SHBG some estimate of free testosterone rather than total testosterone is preferable.

Adult↗
PubMed1989

Non-caeruloplasmin copper and ferroxidase activity in mammalian serum. Ferroxidase activity and phenanthroline-detectable copper in human serum in Wilson's disease.

It is thought that 5-10% of human serum copper consists of copper ions complexed with histidine, amino acids or albumin. The phenanthroline assay developed by Gutteridge (Biochem. J. 218, 983-985; 1984) is shown to measure all these forms of copper down to a sensitivity of 0.1 mumol/dm3, yet it does not detect any copper ions in freshly-prepared serum or plasma from rats, mice, rabbits or guinea-pigs, or freshly-prepared serum from humans. It is concluded that the "non-caeruloplasmin copper pool" is much smaller than has previously been supposed. No phenanthroline-detectable copper could be measured in serum freshly prepared from four patients with uncomplicated Wilson's disease, but it could be measured in serum from a patient with fulminant hepatic failure. After liver transplantation, concentrations of phenanthroline-detectable copper in this patient fell to zero within two days. Studies on the ferroxidase activity of freshly-prepared serum or plasma samples shows that little ferroxidase II activity is present in samples from healthy adults or from the patient with Wilson's disease and fulminant hepatic failure. In all the patients, ferroxidase I activities are sub-normal. Freshly-prepared plasma or serum samples from several animal species generally show lower ferroxidase I and greater ferroxidase II activities than do human samples, but only in rabbits does ferroxidase II account for a high proportion of total plasma ferroxidase activity. Storage of biological fluids can cause release of copper from caeruloplasmin and a rise in ferroxidase II activity; these events may have confused some earlier studies.

Adult↗
PubMed1988

Ferritin iron kinetics and protein turnover in K562 cells.

The binding, incorporation, and release of iron by ferritin were investigated in K562 cells using both pulse-chase and long term decay studies with 59Fe-transferrin as the labeled iron source. After a 20-min pulse of labeled transferrin, 60% of the 59Fe was bound by ferritin with the proportion increasing to 70% by 4 h. This initial binding was reduced to 35% when the cells were exposed to the chelator desferrioxamine (5 mM) for an additional 30 min. By 4 h the association of 59Fe with ferritin was unaffected by the presence of the chelator, and levels of 59Fe-ferritin were identical to those in control cells (70%). Between 4-10h there was a parallel decline in 59Fe-ferritin in both control and desferrioxamine-treated cells. When incoming iron was bound by ferritin it was, therefore, initially chelatable but with time progressed to a further, nonchelatable compartment. In turnover studies where ferritin was preloaded with 59Fe by overnight incubation, 50% of the label was released from the protein by 18 h, contrasting with a t 1/2 for cellular iron release of approximately 70 h. The half-time of 59Fe release from ferritin was accelerated to 11 h by the presence of desferrioxamine. The half-time for ferritin protein turnover determined by [35S]methionine labeling was approximately 12 h in the presence or absence of the chelator. Thus, when the reassociation of iron with ferritin was prevented by the exogenous chelator there was a concordant decay of both protein and iron moieties. The direct involvement of lysosomes in this turnover was demonstrated by the use of the inhibitors leupeptin and methylamine which stabilized both 59Fe (t 1/2 = 24 h) and 35S (t 1/2 = 25.6 h) labels. We conclude that in this cell type the predominant mechanism by which iron is released from ferritin is through the constitutive degradation of the protein by lysosomes.

Animals↗
PubMed1988

Bleomycin-detectable iron in serum from leukaemic patients before and after chemotherapy. Therapeutic implications for treatment with oxidant-generating drugs.

Patients with acute myeloid leukaemia show elevated plasma iron and, in 2/6 cases studied, low-molecular-mass iron complexes capable of stimulating radical reactions were present in the plasma. Shortly after the onset of chemotherapy, there is a sharp rise in transferrin saturation and all patients studied showed low-molecular-mass iron in their plasma. It is proposed that such iron could interact with oxidants generated by certain drugs (e.g. adriamycin or daunorubicin) to facilitate tissue damage, and that some of the side-effects of chemotherapy might be ameliorated by careful co-administration of small doses of desferrioxamine.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1988

Chelation of transferrin iron by desferrioxamine in K562 cells. The partition of iron between ferrioxamine and ferritin.

In this study we have determined whether desferrioxamine can chelate iron delivered to human leukaemic cells by the transferrin endocytic cycle. The cellular uptake of desferrioxamine was investigated by an indirect method in which the conversion of repeated pulses of [59Fe]transferrin to [59Fe]ferrioxamine was determined at two concentrations of the drug. Maximum generation of [59Fe]ferrioxamine occurred in cells exposed to either 100 microM- or 500 microM-desferrioxamine after 40-60 min. Thereafter (up to 180 min) [59Fe]ferrioxamine levels remained steady with 20% of a 59Fe pulse partitioning to chelator at 100 microM and 50% at 500 microM. Of the cellular [59Fe]ferrioxamine loss 50% occurred within 90-120 min. In cells preloaded with desferrioxamine for 1 or 4 h the partitioning of iron during a 3 h incubation with [59Fe]transferrin was dependent upon the extracellular concentration of the chelator. Above 1 mM more than 80% of entering iron was converted to ferrioxamine and less than 5% partitioned to ferritin. Below this concentration (50-500 microM) a proportion of the iron became ferritin associated (7-41%). There was a linear increase in the total amount of intracellular [59Fe]ferrioxamine in accordance with cellular iron uptake showing that transferrin continued to cycle in the presence of high concentrations of desferrioxamine. The uptake of iron and generation of ferrioxamine were markedly reduced by 5 mM-methylamine, which prevented endosome acidification and uncoupling of iron from endocytosed transferrin.

Animals↗