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Biomedical subjects

W Breuer

Publications and source records attributed to W Breuer.

At least 19 recordsLinked to original sources
PubMed2006

[Case report: mast cell leukosis in a neonatal calf].

Multicentric mast cell tumours in a newborn Fleckvieh-calf are described. The calf showed clearly pronounced lesions over the whole body. The lesions were multiple raised, cutaneous, greyisch-red and partially ulcerated. It died three hours after birth. Pathohistological examinations resulted in multiple mast cell tumours within the dermis. In addition multifocal to diffuse mast cell aggregations were observed in several internal organs including the lymph nodes and the bone marrow. No evidence for the presence of bovine leukemia virus was found by both investigating a lymph node homogenate of the calf and a blood sample of the mother cow. In this paper the pathomorphology of this rare disease is described, a possible cause is discussed and a short review of the available literature is presented.

Animals↗
PubMed2004

Comparative morphologic and immunohistochemical investigation of spontaneously occurring thymomas in a colony of European hamsters.

This study documents the characteristics of a large series of spontaneously occurring thymomas in a laboratory colony of European hamsters (Cricetus cricetus). Thymomas are rare organotypic neoplasms originating from the thymic epithelial compartment. Because the hamster thymomas largely resembled their human counterparts, the recent World Health Organization (WHO) classification of human thymic epithelial tumors was used. Forty hamsters of both sexes aged 3-29 months were examined macroscopically and histologically. In 22 (55%) of the 40 animals, necropsy revealed enormous whitish masses in the anterior mediastinum, with a diameter ranging from 0.5 to 4.5 cm and a lobulated structure. The anatomy of the thymus region was normal in the remaining 18 hamsters. Histologically, the tumors presented as thymuslike organoid structures with areas of medullary and cortical differentiation and a predominance of lymphoid cells. A network of epithelial cells in the cortical areas, demonstrated immunohistochemically with a cross-reactive antibody against pancytokeratin, supported the diagnosis of thymoma. Cortical lymphocytes showed positive staining with cross-reacting antibodies against CD3 and terminal deoxynucleotidyl transferase, characteristic of immature T cells. On the basis of these findings, the tumors were classified as B1 thymomas, in some cases with AB or B2 components, according to the new WHO classification for human thymic epithelial tumors.

Animals↗
PubMed2003

Histopathologic and immunophenotypic characterization of extramedullary plasmacytomas in nine cats.

To contribute to the existing body of knowledge in the literature on the apparently rare extramedullary plasmacytoma in cats, lymphoid tumors with plasmacytic cellular morphology taken from nine cats were examined. The paraffin-embedded material was investigated by standard hematoxylin and eosin, and special staining techniques (Giemsa, Congo-red, and periodic acid-Schiff reaction). The tumors also were examined immunohistochemically for the presence of immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin light chains (lambda, kappa), various amyloid proteins, and FeLV-antigen (p27 protein). An immunoglobulin-producing tumor of plasmacellular origin (extramedullary plasmacytoma [EMP]) could be diagnosed in all cases on the basis of immunohistochemical light-chain expression. All but one of the neoplasms occurred in the skin of older, predominantly male cats. As in humans and dogs, the following types could be identified according to their morphologic features: mature type (two), cleaved type (two), asynchronous type (four), and polymorphous type (one). The tumor tissue of three cats revealed amyloid deposits, which were immunohistochemically diagnosed as ALlambda-amyloid in all three cases.

Animals↗
PubMed2003

[Acute herpesvirus-gastritis in a cat].

Gastritis in cats is caused, among other things, by infectious agents, like bacteria, metazoic parasites or viruses. Herpesvirus-gastritis has not as yet been documented in cats. Therefore in this paper such a case will be described. In this case the mucous membrane of the stomach shows multifocal acute necroses with evidence of intranuclear inclusion bodies in epithelial cells of the gastric glands. By means of electron microscopy the causative virus can be specified as herpesvirus.

Acute Disease↗
PubMed2002

Design and applications of methods for fluorescence detection of iron in biological systems.

Fluorescence metalosensors provide a means to detect iron in biological systems that is versatile, economical, sensitive and of a high-throughput nature. They rely on relatively high-affinity iron-binding carriers conjugated to highly fluorescent probes that undergo quenching after metal complexation. Metal specificity is determined by probes containing either an iron-binding moiety of high affinity (type A) or of relatively lower affinity (type B) used in combination with a strong specific iron chelator. Due to the heterogeneous nature of biological systems, the apparent metal-binding affinity and complexation stoichiometry ought to be specifically defined. Fluoresceinated moieties coupled to metal-binding cores detect Fe at sub-micromolar concentrations and even sub-microlitre volumes (i.e. cells). Although an ideal probe should also be specific for a particular oxidation state of iron, in physiological conditions that property might be difficult to attain. Quantification of labile iron in cells has relied on the ability of permeant iron chelators to restore the fluorescence of probes quenched by intracellular Fe. Modern design of probes aims to (a) improve probe targeting to specific cell compartments and (b) create probes that respond to metal binding by signal enhancement.

Calibration↗
PubMed2002

Labile iron in parenteral iron formulations and its potential for generating plasma nontransferrin-bound iron in dialysis patients.

BACKGROUND: Labile plasma iron (LPI) associated with iron supplementation has been implicated in complications found in dialysis patients. As LPI can potentially catalyse oxygen radical generation, we determined the presence of labile iron in the parenteral preparations and the frequency of occurrence of LPI in dialysis patients. DESIGN: The capacity to donate iron to apotransferrin (apo-) or to the chelator desferrioxamine (DFO) was measured with fluorescein-Tf (Fl-Tf) and Fl-DFO, respectively. Those probes undergo quenching upon binding to iron. Iron-catalysed generation of oxidant species was determined with dihydrorhodamine. Plasma nontransferrin-bound iron (NTBI), here termed LPI, was determined by mobilization of iron from low-affinity binding sites with oxalate, followed by its quantification with Fl-Tf in the presence of Ga(III). RESULTS: Normal individuals and most (80%) dialysis patients, analysed at least 1 week after iron supplementation showed no detectable (<0.2 microm) LPI. However, approximately 20% of the patients (n = 71) showed significant LPI levels (>0.2 microm), in some cases weeks after iron administration. LPI levels correlated best (r2 = 0.9) with Tf saturation. The iron preparations contained 2-6% low molecular weight and redox-active iron, most of which is chelated by Tf. CONCLUSIONS: Parenteral iron formulations contain a small but significant fraction of redox-active iron, most of which is scavenged by apo-Tf within <1 h. Therefore, oxidant stress associated with iron infusion is likely to be transient. The bulk of the polymeric iron is apparently inaccessible to apo-Tf. Although LPI might return to normal within 2 h of intravenous iron infusion, the long-term persistence of low-level LPI in up to 20% of end stage renal disease (ESRD) patients indicates that complete clearance of the intravenous iron may be more protracted than originally estimated.

Adolescent↗
PubMed2001

A fluorescence-based one-step assay for serum non-transferrin-bound iron.

We introduce a method for monitoring non-transferrin-bound iron (NTBI), a labile and potentially toxic form of serum iron associated with imbalanced iron metabolism. The assay employs fluorescein-labeled apotransferrin (Fl-aTf), which undergoes fluorescence quenching upon binding iron. It has the advantages of simplicity, high sensitivity, and detection of those forms of NTBI that persist in sera with low transferrin saturations. Since NTBI is not readily available for detection, it is mobilized by 10 mM oxalate. Endogenous serum apotransferrin, capable of binding oxalate-mobilized NTBI, is blocked by 0.1 mM gallium(III). This metal, like iron, binds to Fl-aTf, but it neither quenches its fluorescence nor interferes with quenching by iron. Serum and reagent containing oxalate, Ga(Cl)(3), and Fl-aTf are mixed in multiwell plates and fluorescence is determined after 1 h in a microplate reader. To compensate for artifactual fluorescence changes caused by serum color, parallel samples are prepared with excess unlabeled apotransferrin, which scavenges all iron in the sample. Sera from eight hemochromatosis patients were tested for NTBI by the present assay and by an established alternative method, with qualitatively similar results. A potential application of the test is for screening large numbers of samples from patients at risk of developing NTBI.

Calibration↗
PubMed2001

Oligosaccharyltransferase is highly specific for the hydroxy amino acid in Asn-Xaa-Thr/Ser.

Pig liver oligosaccharyltransferase (OST), which is involved in the en bloc transfer of the Dol-PP-linked GlcNAc(2)-Man(9)-Glc(3) precursor on to asparagine residues in the Asn-Xaa-Thr/Ser sequence, is highly stereospecific for the conformation of the 3-carbon atom in the hydroxy amino acid. Moreover, substitution of the hydroxy group by either SH as in cysteine, or NH(2) as in beta,gamma-diamino-butanoic acid as reported previously [Bause, E. et al., Biochem. J. 312 (1995) 979-985], followed by the determination of the pH optimum for enzymatic activity, indicates that neither a negative nor a positive charge in the hydroxy amino acid position is tolerated by the enzyme. Binding of the threonine beta-methyl group by OST is also specific, with serine, L-threo-beta-hydroxynorvaline and L-beta-hydroxynorleucine containing tripeptides all bound much less efficiently than the threonine peptide itself. The data are interpreted in terms of a highly stereospecific hydrophobic binding pocket for the threonine CH(3)-CH(OH) group.

Animals↗
PubMed2001

ICL670A: a new synthetic oral chelator: evaluation in hypertransfused rats with selective radioiron probes of hepatocellular and reticuloendothelial iron stores and in iron-loaded rat heart cells in culture.

ICL670A (formerly CGP 72 670) or 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]- benzoic acid is a tridentate iron-selective synthetic chelator of the bis-hydroxyphenyl-triazole class of compounds. The present studies used selective radioiron probes of hepatocellular and reticuloendothelial (RE) iron stores in hypertransfused rats and iron-loaded heart cells to define the source of iron chelated in vivo by ICL670A and its mode of excretion, to examine its ability to remove iron directly from iron-loaded myocardial cells, and to examine its ability to interact with other chelators through a possible additive or synergistic effect. Results indicate that ICL670A given orally is 4 to 5 times more effective than parenteral deferoxamine (DFO) in promoting the excretion of chelatable iron from hepatocellular iron stores. The pattern of iron excretion produced by ICL670A is quite different from that of DFO and all iron excretion is restricted to the bile regardless of whether it is derived from RE or hepatocellular iron stores. Studies in heart cell cultures have shown a favorable interaction between DFO and ICL670A manifested in improved chelating efficiency of ICL670A, which is most probably explained by an exchange of chelated iron between ICL670A and DFO. These unique chelating properties of ICL670A may have practical implications for current efforts to design better therapeutic strategies for the management of transfusional iron overload.

Animals↗
PubMed2001

Desferrioxamine-chelatable iron, a component of serum non-transferrin-bound iron, used for assessing chelation therapy.

This study introduces a method for monitoring a component of serum non-transferrin-bound iron (NTBI), termed "desferrioxamine-chelatable iron" (DCI). It is measured with the probe fluorescein-desferrioxamine (Fl-DFO), whose fluorescence is stoichiometrically quenched by iron. DCI was found in the serum of most patients with thalassemia major (21 of 27 tested, range 1.5-8.6 microM), but only in a minority of patients with hereditary hemochromatosis (8 of 95 samples from 39 patients, range 0.4-1.1 microM) and in none of 48 controls. The method was applied to monitoring the appearance of iron in the serum of patients under chelation therapy. Short-term (2 hours) follow-up of patients immediately after oral administration of deferriprone (L1) showed substantial mobilization of DCI into the serum (up to 10 microM within 30-60 minutes). The transfer of DCI from L1 to Fl-DFO was observed in vitro with preformed L1-iron complexes, and occurred even at L1/iron ratios exceeding 3:1. Simultaneous administration of oral L1 and intravenous DFO to patients abrogated the L1-mediated rise in DCI, consistent with the shuttling of iron from L1 to DFO in vivo. A similar iron transfer from L1 to apo-transferrin was observed in vitro, lending experimental support to the notion that L1 can shuttle iron in vivo to other high-affinity ligands. These results provide a rationale for using chelator combinations, with the highly permeant L1 acting as an intracellular chelator-shuttle and the less permeant DFO serving as an extracellular iron sink. Potential applications of the DCI assay may be for studying chelator action and as an index of patient chelation status.

Adolescent↗
PubMed2001

Immunohistological demonstration of erythroid cells in canine bone marrow.

In an immunohistological/cytological study of canine bone marrow, the aim was to demonstrate canine erythroid cells with the help of various commercially available antibodies against human antigens (monoclonal antibody against glycophorin A, polyclonal antibodies against haemoglobin and spectrin). In order to preserve possible cross-reacting epitopes various fixation methods (cross-linking, precipitating and dehydrating fixing agents, partly in combination with unmasking measures), decalcification techniques [acid or ethylenediaminetetraacetic acid (EDTA) decalcification] and tissue-embedding methods (paraffin embedding, cryostat sectioning technique) were used. Alternative methods, such as the preparation of cell smears and immunoblotting, were also employed. The only result that was of use for routine diagnostic procedures (paraffin sections) was that obtained by using polyclonal antibodies against haemoglobin. Best results were achieved when tissue was fixed in a formaldehyde-glutaraldehyde mixture, decalcified in EDTA and treated with microwave irradiation. The primary antibody was used in a dilution of 1:500 and incubated for 16 h. With the exception of mature red blood cells and proerythroblasts, different stages of erythrocytopoietic cells in canine bone marrow were shown to be arranged in erythrons. The polyclonal antibody against spectrin also showed clear cross-reactivity, but was only employable in other systems (immunoblotting). The monoclonal antibody against glycophorin A reacted only when used on human tissue or cells.

Animals↗
PubMed2001

Exploring the "iron shuttle" hypothesis in chelation therapy: effects of combined deferoxamine and deferiprone treatment in hypertransfused rats with labeled iron stores and in iron-loaded rat heart cells in culture.

Although iron chelation therapy results in a significant improvement in well-being and life expectancy of thalassemic patients with transfusional iron overload, failure to achieve these goals in a substantial proportion of patients underlines the need for improved methods of treatment. In the present studies we used selective radioactive iron probes of hepatocellular and reticuloendothelial (RE) iron stores in hypertransfused rats and iron-loaded heart cells to compare the source of iron chelated in vivo by deferoxamine (DFO) or by deferiprone (L1) and its mode of excretion, to examine the ability of DFO and L1 to remove iron directly from iron-loaded myocardial cells, and to examine the mechanism of their combined interaction through a possible additive or synergistic effect. Our results indicate that L1 given orally is 1.6 to 1.9 times more effective in rats, on a weight-per-weight basis, than parenteral DFO in promoting the excretion of storage iron from parenchymal iron stores but shows no advantage over DFO in promoting RE iron excretion. Simultaneous administration of DFO and L1 results in an increase in chelating effect that is additive but not synergistic. The magnitude of this additive effect is identical to an increase in the equivalent (weight or molar) dose of DFO alone rather than the sum of the separate effects of L1 and DFO. This finding is most probably the result of a transfer of chelated iron from L1 to DFO. These observations may have practical implications for current efforts to design better therapeutic strategies for the management of transfusional iron overload.

Animals↗
PubMed2001

Analysis of structural signals conferring localisation of pig OST48 to the endoplasmic reticulum.

Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the co-translational transfer of GlcNAc2-Man9-Glc3 from Dol-PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ER-targeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man9-mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the C-terminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48AK) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ER-residency on OST48. The double-lysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48AK-3, when co-expressed in COS-1 cells together with myc-tagged ribophorin 1, was quantitatively retained in the ER. By contrast, co-expression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMOdeltaK-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the C-terminus of the Man9-mannosidase luminal domain. Thus ER-localisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the double-lysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in re-capture of molecules which have escaped from the ER.

Amino Acid Motifs↗
PubMed2000

The assessment of serum nontransferrin-bound iron in chelation therapy and iron supplementation.

Nontransferrin-bound iron (NTBI) appears in the serum of individuals with iron overload and in a variety of other pathologic conditions. Because NTBI constitutes a labile form of iron, it might underlie some of the biologic damage associated with iron overload. We have developed a simple method for NTBI determination, which operates in a 96-well enzyme-linked immunosorbent assay format with sensitivity comparable to that of previous assays. A weak ligand, oxalic acid, mobilizes the NTBI and mediates its transfer to the iron chelator deferoxamine (DFO) immobilized on the plate. The amount of DFO-bound iron, originating from NTBI, is quantitatively revealed in a fluorescence plate reader by the fluorescent metallosensor calcein. No NTBI is found in normal sera because transferrin-bound iron is not detected in the assay. Thalassemic sera contained NTBI in 80% of the cases (range, 0.9-12.8 micromol/L). In patients given intravenous infusions of DFO, NTBI initially became undetectable due to the presence of DFO in the sera, but reappeared in 55% of the cases within an hour of cessation of the DFO infusion. This apparent rebound was attributable to the loss of DFO from the circulation and the possibility that a major portion of NTBI was not mobilized by DFO. NTBI was also found in patients with end-stage renal disease who were treated for anemia with intravenous iron supplements and in patients with hereditary hemochromatosis, at respective frequencies of 22% and 69%. The availability of a simple assay for monitoring NTBI could provide a useful index of iron status during chelation and supplementation treatments. (Blood. 2000;95:2975-2982)

Chelating Agents↗
PubMed2000

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency.

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.

Alleles↗
PubMed2000

The importance of non-transferrin bound iron in disorders of iron metabolism.

The concept of non-transferrin bound iron (NTBI) was introduced 22 years ago by Hershko et al. (Brit. J. Haematol. 40 (1978) 255). It stemmed from a suspicion that, in iron overloaded patients, the large amounts of excess iron released into the circulation are likely to exceed the serum transferrin (Tf) iron-binding capacity (TIBC), leading to the appearance of various forms of iron not bound to Tf. In accordance with this assumption, NTBI was initially looked for and detected in patients with > or = 100% Tf-saturation. As techniques for its detection became more sophisticated and sensitive, NTBI was also found in conditions where Tf was not fully saturated, leading to a revision of the original view of NTBI as a simple spillover phenomenon. In this review, we will discuss some of the properties of NTBI, methods for its detection, its significance and potential value as an indicator for therapeutic regimens of iron chelation and supplementation.

Humans↗
PubMed1999

H-ferritin subunit overexpression in erythroid cells reduces the oxidative stress response and induces multidrug resistance properties.

The labile iron pool (LIP) of animal cells has been implicated in cell iron regulation and as a key component of the oxidative-stress response. A major mechanism commonly implied in the downregulation of LIP has been the induced expression of ferritin (FT), particularly the heavy subunits (H-FT) that display ferroxidase activity. The effects of H-FT on LIP and other physiological parameters were studied in murine erythroleukemia (MEL) cells stably transfected with H-FT subunits. Clones expressing different levels of H-FT displayed similar concentrations of total cell iron (0.3 +/- 0.1 mmol/L) and of reduced/total glutathione. However, with increasing H-FT levels the cells expressed lower levels of LIP and reactive oxygen species (ROS) and ensuing cell death after iron loads and oxidative challenges. These results provide direct experimental support for the alleged roles of H-FT as a regulator of labile cell iron and as a possible attenuator of the oxidative cell response. H-FT overexpression was of no apparent consequence to the cellular proliferative capacity. However, concomitant with the acquisition of iron and redox regulatory capacities, the H-FT-transfectant cells commensurately acquired multidrug resistance (MDR) properties. These properties were identified as increased expression of MDR1 mRNA (by reverse transcription polymerase chain reaction [RT-PCR]), P-glycoprotein (Western immunoblotting), drug transport activity (verapamil-sensitive drug efflux), and drug cytotoxicity associated with increased MDR1 or PgP. Although enhanced MDR expression per se evoked no significant changes in either LIP levels or ROS production, it might be essential for the survival of H-FT transfectants, possibly by expediting the export of cell-generated metabolites.

ATP Binding Cassette Transporter, Subfamily B, Mem↗