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Biomedical subjects

H G Fromme

Publications and source records attributed to H G Fromme.

At least 19 recordsLinked to original sources

Immunocytochemical mapping of IgE/IgG binding sites within the birch pollen grain using serum from a patient undergoing hyposensitization therapy. Comparison with biochemically determined data.

Serum from an atopic patient undergoing a hyposensitization therapy to birch pollen allergens was used to carry out immunocytochemical mapping of specific IgE/IgG binding sites within ultrathin sections of birch pollen grains. There was a distinct rise in the density of specific IgG labelling in the course of therapy, whereas the density of IgE labelling remained fairly constant. However, the patterns of IgG and IgE binding in the pollen grain did not completely coincide since there was only IgE binding to certain pollen structures such as the apertural region. If the widely accepted concept of specific antibodies as 'blocking antibodies' is taken as a basis, the success of therapy must be questioned in this case because no IgG antibodies were formed to some of the allergens localized in the pollen grain and relevant to this patient. Very probably this result must be attributed to an incomplete pollen extract used in hyposensitization therapy. The results of the biochemical measurements (RAST, ELISA) agreed well with the immunocytochemical observations.

Binding Sites, Antibody

Visualization of birch pollen allergens using IgE-containing sera from human atopic individuals in immunogold labelling experiments.

Pollen from birch trees (Betula pendula) was fixed in paraformaldehyde with or without the addition of 0.5% cetylpyridinium chloride, dehydrated and embedded in Lowicryl K4M in the cold. Ultrathin sections were incubated using the following sequence of antibodies and antisera: IgE-containing serum from an atopic human individual allergic to birch pollen allergens, rabbit anti-human IgE antibodies, and colloidal gold-labelled goat anti-rabbit antibodies. Controls were performed by replacing the specific human antiserum by serum from an atopic person with a similar level of IgE antibodies directed against allergens other than birch pollen allergens, or by omitting the human antiserum or the anti-IgE antibody or both. In test experiments, there was a dense specific labelling of the exine and the cytoplasmic matrix of the pollen grain. There was moderate labelling of the apertural regions (poral plugs). There was no labelling of the intine. In pollen grains fixed with the addition of cetylpyridinium chloride, an electron-dense surface coat was precipitated on the outside of the pollen wall. This surface material also remained completely unlabelled.

Antibodies, Anti-Idiotypic

Cross-reactivity of birch anthers and leaves with birch pollen antigens and allergens. A fine-structural immunocytochemical study using the post-embedding protein-A-gold technique.

Ultra-thin sections of vegetative tissues from birch (anthers and leaves) were labeled for pollen antigens and allergens using a commercial rabbit IgG antibody preparation directed against birch pollen antigens and allergens. Antibody binding sites were visualized using the protein A-gold technique. Specific labeling occurred in anther tissue (tape-tum cells, anther wall cells) as well as in the birch leaf (assimilation parenchyma). In both types of tissue, antigens and allergens were detected throughout the living protoplast (including cell organelles such as nuclei, mitochondria, and plastids). The cellulose cell walls were always free from anti-birch-pollen IgG-binding sites. The immunological controls (normal rabbit IgG) showed a low degree of nonspecific labeling. In plant tissues belonging to genera quite different from birch (tulip anther, rhododendron leaves), after incubation with the specific IgG weak labeling was observed. The immunological basis for these results is discussed.

Allergens

Immunoautoradiographic and protein-A/gold labelling experiments for localization of pollen allergens using antisera from atopic human individuals.

Using serum from human atopic individuals with a sufficiently high titre of IgE and IgG antibodies to birch- or hazel-pollen allergens and antigens, the localization of IgE binding sites in birch- and hazel-pollen grains was determined by pre- and post-embedding electron microscopic immunoautoradiography with 125I-anti-IgE, whereas the IgG binding sites were localized in ultrathin sections of birch-pollen grains by the protein-A/gold technique. Concerning the distribution patterns of both IgE/IgG binding sites within the pollen grains, no difference could be observed in the dormant pollen grain: Labelling was found in the exine part of the pollen wall and throughout the highly condensed cytoplasm except for starch grains and lipid droplets. The intine part and the germination pores were almost completely unlabelled. In pollen grains which had been soaked in a hypotonic buffer for 15 min, however, IgE binding sites were predominantly localized within the intine and the germination pores. The specificity of the labelling reactions and the observed differences in the localization patterns are discussed.

Antibodies, Anti-Idiotypic

Connective tissue proteins on the injured endothelium of the rat aorta.

Type V collagen (TVC), fibronectin (FN), and laminin (LAM) were detected on the endothelial surface of mechanically injured rat aortas with the help of monospecific antisera and protein A - gold conjugates, carbon film surface replicas, and conventional embedding techniques. Deendothelialized tracks were produced in the thoracic aorta, and the presence of the connective tissue proteins on the luminal surface of the endothelium was studied. The changes in the distribution of the proteins during repair of the endothelial surface was followed for up to 6 days after injury. From 1 to 3 days after injury small numbers of gold particles, indicating the presence of TVC, were found between the adherent platelets on the freshly deendothelialized subendothelial matrix and in higher amounts on cell debris and collagen fibers. On the sixth day after injury, however, the amount of TVC between the sparsely distributed platelets on the deendothelialized areas was significantly higher than it was previously. FN and LAM were readily detectable on the subendothelial matrix and on the damaged marginal endothelial cells. These proteins were especially obvious on both margins of the tracks even from the first day after treatment. FN was found also in connection with fibrin precipitations as well as on the surface of some platelets and monocytes. The amount of FN and LAM present on the damaged area decreased slightly up to the sixth day. Monocytes and leukocytes adhered mostly at the margin of the wound area in the vicinity of the lesions on the endothelium. FN and LAM were often detectable under and around these adherent cells. Little of the connective tissue proteins was found on the uninjured and on the regenerated endothelial cells. The results showed subtle transitory changes in the surface pattern of the subendothelial connective tissue matrix of the injured intima. The adhesion of blood-borne cells may have been induced by FN and LAM on the endothelial surface near the lesions, and later partly prevented by increasing amounts of TVC on the surface.

Animals

Immunoelectron-microscopic localization of diffusible birch-pollen antigens in ultrathin sections using the protein-A/gold technique.

Pollen grains from Betula pendula were fixed in a mixture of p-formaldehyde and cetylpyridinium chloride (CPC) for the precipitation of soluble pollen glycoproteins. After dehydration and embedding at low temperatures in the water-soluble resin, Lowicryl K4M, ultrathin sections of the pollen grains were incubated using specific antibodies against birch-pollen extract and protein-A/gold complexes. Antigen activity was found in the CPC-precipitated surface material and within the exine (bacular cavities) and the cytoplasm (except for starch grains and lipidic droplets). There was no labelling within the intine. The region of the germinal aperture also showed a very low degree of antigen activity. The control sections were almost completely free of background staining.

Antigens

Ultrastructural demonstration of a glycoproteinic surface coat in allergenic pollen grains by combined cetylpyridinium chloride precipitation and silver proteinate staining.

In allergenic birch pollen grains, highly watersoluble surface substances were precipitated by the cationic detergent cetylpyridinium chloride (CPC) during aqueous fixation. After processing the pollen for electron microscopy, ultrathin sections of pollen grains were subjected to the periodic acid - thiocarbohydrazide - silver proteinate (PA-TCH-SP) procedure according to Thiery (1967) for the detection of vicinal glycol groups. It was found that the material precipitated by CPC on the surface and within the exine cavities of the pollen wall strongly reacted with the PA-TCH-SP reagent thus indicating the presence of polysaccharides on the surface of birch pollen grains. In samples which had not been treated with the cationic detergent, PA-TCH-SP reactivity was reduced to thin linings on the surface and within the exine cavities. In both cases the exine proper did not stain whereas the intine showed moderate staining. Within the aperture region of the intine, PA-TCH-SP reactivity is preferably associated with fibrillar or reticular structures. The results are discussed with special reference to biochemical findings on allergenic birch pollen proteins.

Cetylpyridinium

Presence of connective tissue proteins on the endothelium of the rat aorta.

The microdistribution of type V collagen, fibronectin, and laminin on the luminal surface of perfusion-fixed normal rat aortic endothelium has been studied by an immunoelectron microscopic method using monospecific antibodies and a protein A-gold complex. Gold particles indicating the presence of these biologically active connective tissue proteins were localized in groups on and in the vicinity of the interendothelial border. They were also found on the small flaps of cell junctions as well as on certain cell projections and scattered on the cell surface. Correlative transmission electron-microscopic examinations proved the specificity of these localizations. The endothelial cells of the aorta differed markedly in the amount of scattered connective tissue proteins on their surface, suggesting that there are several types of aortic endothelial cells with distinct functional differences. The findings provide evidence that connective tissue proteins may contribute to the surface pattern of the normal endothelium, especially on cell borders. It is likely that these proteins influence functions such as the permeability and chemotactic activity of the endothelium pertinent to the development of vascular disease.

Animals

Assessment of magnesium and aluminum in erythrocytes by the laser microprobe mass analyzer (LAMMA).

The erythrocyte magnesium content was determined using two different analytical systems (atomic absorption spectrometry and the laser microprobe mass analyzer) with specimens from 18 patients with normal renal function and from 10 patients with chronic renal insufficiency. There was a good correlation (r = 0.73) between the two analytical systems. Additionally, the laser microprobe mass analyzer was used to determine the aluminum content of plasma and erythrocytes in specimens from patients who ingested moderate quantities of aluminum hydroxide for several months. Results showed a significant elevation of the plasma aluminum concentration, but a normal erythrocyte aluminum content. The laser microprobe mass analyzer may be useful in studies of mineral metabolism in humans.

Adult

Electron microscopic localization of concanavalin A receptor sites in pollen surface material after fixation with glutaraldehyde-cetylpyridinium chloride.

Pollen from birch trees (Betula pendula) was fixed in glutaraldehyde containing 0.5% cetylpyridinium chloride (CPC), incubated with concanavalin A (Con A)-ferritin, postfixed in osmium, dehydrated, and embedded in Epon. On ultrathin sections, ferritin particles were observed closely associated with the electron-dense material precipitated by CPC on the surface of the pollen grains. Controls for CPC, which were fixed in glutaraldehyde alone, showed no electron-dense material on the surface. In controls for Con A, which were incubated in Con A-ferritin in the presence of the inhibitory sugar (alpha-methyl-D-mannopyranoside), no ferritin particles were observed. The above-described procedure thus allows the localization of sugar residues in highly soluble pollen wall glycoproteins.

Cetylpyridinium

Localisation of acetyl-CoA: alpha-glucosaminide N-acetyltransferase in microsomes and lysosomes of rat liver.

Subcellular fractions of rat liver were obtained by differential centrifugation. The fractions enriched in lysosomes or microsomes were further fractionated in discontinuous sucrose density gradients or continuous iso-osmotic gradients made of modified colloidal silica. The fractions were analyzed for marker enzymes of the different subcellular organelles and for acetyl-CoA: alpha-glucosaminide N-acetyltransferase. The acetyl-CoA: alpha-glucosaminide N-acetyltransferase activity showed a bimodal distribution. About one fourth of the activity was associated with lysosomes, whereas the greater part of the activity was recovered in the microsomal fraction. Plasma membrane-enriched fractions contained only trace amounts of acetyl-CoA: alpha-glucosaminide N-acetyltransferase. The lysosomal and microsomal acetyl-CoA: alpha-glucosaminide N-acetyltransferases are membrane-bound and can be solubilized with Triton X-100. The pH dependence and sensitivity to various ions was similar for the lysosomal and microsomal enzyme.

Acetyltransferases

[Element standards for quantitative X-ray microanalysis of biological specimens in the scanning transmission electron microscope].

Quantitation in biological X-ray microanalysis usually depends on the reference to one or more element standards. This paper deals with the general characteristics of such standards and gives a survey of the various methods of standard preparation reported in literature. Their advantages and disadvantages are briefly discussed. Practical experiments were carried out to prepare a potassium, calcium, and sulfur standard for X-ray microanalysis in the scanning transmission electron microscope.

Calcium

LAMMA--investigations of biological and medical specimens.

One feature of the Laser Microprobe-Mass-Analyzer (LAMMA) is the analysis of biomedical specimens. This method allows to obtain qualitative and quantitative information about the inorganic and--in a qualified sense--about the organic constituents of microvolumes of thin histological sections or of single cells (e.g. erythrocytes) with a relative sensitivity going down to the sub-ppm range. The paper describes the technical structure and features of the laser-microprobe, its advantages and possibilities, but also its limits in the investigation of biomedical specimens. This will be pointed out by some examples: 1) Analysis of blood from patients suffering from renal insufficiencies (A1 transportation, Fe/Mg ratio of erythrocytes). 2) Pb detection in fetal tissues after maternal Pb intoxication. 3) Analysis of trace elements (detection limits and superposition of organic background from embedding material). 4) Analysis of isotopes and molecules. The present contribution is not so much a discussion of medical problems, but mainly a discussion about the methodical and practical use of the laser microprobe.

Aluminum

Metabolism of sulfated glycosaminoglycans in cultured endothelial cells and smooth muscle cells from bovine aorta.

The glycosaminoglycan metabolism of cultured endothelial cells and of cells grown from the intima and from the media layer of bovine aorta thoracia was investigated in a comparative study. The following results were obtained: 1. Endothelial cells have in common with intima and media cells the distribution of newly formed sulfated glycosaminoglycans into extracellular, pericellular and intracellular compartments. Endothelial cells, however, synthesize lower amounts of glycosaminoglycans and distribute them in a different ratio into the three pools. 2. Though all the various cell lines synthesize chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparan sulfate and small amounts of keratan sulfate, endothelial cells exhibit a unique distribution pattern of sulfated glycosaminoglycans in each of the three compartments. Generally, a high proportion of heparan sulfate and chondroitin 6-sulfate and a very low dermatan sulfate content was detected. 3. Heparan sulfate produced by endothelial cells has a higher N-sulfonate content when compared with that from other sources. The cell membrane-associated heparan sulfate, especially, exhibits some heparin-like features as judged by nitrous acid degradation and susceptibility towards heparitinase.

Animals

Electron microscopic studies in cultivated plants. I. Green pods of Phaseolus vulgaris var. nanus L.

Scanning (SEM) and transmission (TEM) electron microscopic studies in the legumen of Phaseolus vulgaris var. nanus L. (kidney beans) were carried out. In this work emphasis was laid on the analysis of the morphological structure of the parenchyma tissue, being the chief component of the pod at edible maturity. The leaf character of the pod becomes especially evident by the occurence of stomata, trichomes, and typical cuticle structures on the outside of the pod (SEM). The cells of the mesophyll-like parenchyma tissue are distinguished by a great range of variation of their plastids (chloroplasts--chloroamyloplasts--amyloplasts) and some special cytological features such as a strongly developed rough ER, big nuclei, and numerous plasmodesmata (TEM).

Cell Nucleus