Ernst W. Rauterberg

Immunohistochemical detection of 1,25-dihydroxyvitamin D3 receptors and estrogen receptors by monoclonal antibodies: comparison of four immunoperoxidase methods.

We developed an immunohistochemical method for visualization of vitamin D (VDR) and estrogen receptors (ER) in cryostat sections, using monoclonal antibodies (MAb) to the vitamin D receptor and estrogen receptor, respectively. This method is based on an avidin-biotin labeling technique (LAB). To establish a reliable and sensitive method which can be used easily as a routine diagnostic procedure, we systematically compared four different immunoenzymatic methods with respect to their efficiency in detecting vitamin D and estrogen receptors. Compared to the indirect bridged avidin-biotin (IBRAB), the peroxidase- anti-peroxidase (PAP), and the avidin-biotin complex (ABC) methods, the LAB method produced stronger staining intensities and had higher detection efficiency for both vitamin D and estrogen receptors. In addition, the LAB method had a higher spatial resolution compared to the ABC technique in detection of VDR in normal human skin biopsies. In the case of steroid receptors, i.e., nuclear antigens, immunohistochemistry must deal with a relatively low number of antigenic sites per cell, restricted accessibility of the antigens, and slight differences in antigen concentrations among cells. Under these particular conditions, the chemical properties of the conjugates used in the LAB method may explain why it is superior to the other methods. Consequently, the LAB method is recommended for visualization of ER and VDR.

Antibodies to the most tightly bound proteins in eukaryotic DNA

Abstract DNA released from eukaryotic cells by proteases/SDS or by alkali/SDS still contains distinct proteins which are not removed by these cell lysis procedures nor by subsequent phenol treatment. The proteins most tightly bound to DNA can only be isolated by degradation of DNA. In contrast to the protein-DNA complexes, the protein material isolated after degradation of DNA is sensitive to protease treatment. Moreover, the isolated protein material tends to form aggregates which are insoluble in buffers not containing detergents. They are only poorly soluble in buffers containing SDS. The partially solubilized material can be separated by SDS-polyacrylamide gel electrophoresis into two main bands. Antibodies were raised in rabbits against the polypeptides contained in these main bands. Immunofluorescence micrographs are presented of cells treated with the antibodies. The results indicate that the proteins characterized by their involvement in extremely stable protein-DNA complexes also occur independently of DNA in eukaryotic cells.

Distribution of c-myc, c-myb, and Ki-67 antigens in interphase and mitotic human cells evidenced by immunofluorescence staining technique

Using specific antibodies and the immunofluorescence staining technique we found a similar subcellular distribution pattern of the cellular proto-oncogene proteins c-myc and c-myb in interphase and mitotic HL60 and Molt4 cells. Antibodies against c-myc as well as those against c-myb protein gave rise to a nuclear staining excluding the nucleoli. In mitotic cells both proteins are apparently not associated with the chromatin of the condensed chromosomes, but appear diffusely distributed throughout the cytoplasm. In contrast, immunostaining using the proliferation marker antibody Ki-67 yielded in both cell lines several prominent specks in the nucleus and a weak finely dispersed staining throughout the nucleoplasm. No fluorescence was detectable in the cytoplasm. In dividing cells Ki-67 immunofluorescence was found to be associated with the surface of the chromosomes. The functional significance of the different localizations of the proteins is discussed in light of what is currently known about nuclear antigens.

Expression of fetal cytokeratins in epidermai cells and colloid bodies in lichen planus

Clusters of immunoglobulin (Ig)‐coated colloid bodies (CBs) in the dermo‐epidermal zone are a typical immunohistochemical feature in lichen planus (LP)‐lesions. They are considered to represent dyskeratotic basal keratinocytes, yet their composition has not been completely elucidated. In the present study, skin biopsies of 10 LP‐lesions, 3 other dermatoses, and 10 biopsies of normal skin were studied immunohistochemically using monoclonal antibodies (MAbs) against fetal and differentiated epidermal antigens. CBs were identified by FITC‐anti‐Ig. Binding of MAb was visualized by double staining technique. Cytokeratin (CK) 10/11, a marker of epidermal differentiation, was consistently detected in suprabasal keratinocytes and also in up to 95% of Ig‐positive CBs in LP. CK10/11 was additionally detected in basal keratinocytes in 9 LP‐lesions, but not in normal skin. The basal cell‐specific MAb BL7 stained basal layer keratinocytes in all biopsies. In contrast to normal skin, in LP scattered suprabasal keratinocytes and CBs were also positive for BL7 in 10 and 7 cases, respectively. While fetal cytokeratins (CK13 and CK8/18) were completely absent in control skin specimens, both cytokeratins were detected in various numbers of keratinocytes and CBs in all LP‐lesions. Our results support the hypothesis of an epidermal origin of CBs. The cytokeratin profile seems to be severely disturbed in LP. This includes both accelerated differentiation by the expression of suprabasal CK10/11 in basal keratinocytes and dedifferentiation by the expression of fetal epidermal antigens (CK13 and CK8/18). It is tempting to speculate that the observed alterations may trigger T‐cell activation and inflammatory onset in LP.

N-deglycosylation of human complement component C9 reduces its hemolytic activity

The effect of enzymatic deglycosylation of human complement component C9 on its hemolytic activity was investigated. Treatment of native C9 (Mr 71,000) with glyocpeptidase F (PNGase F) results in a stepwise decrease of the mol. wt. The formation of an Mr 67,000 peptide which is further converted to Mr 63,000 suggests that there are two N-linked carbohydrate chains per C9 polypeptide. Removal of approximately 88% of the N-linked oligosaccharides results in 80% reduction of the hemolytic activity (CH50). The completely N-deglycosylated Mr 63,000 peptide contains a remaining amount of 25% of the total carbohydrates of native C9. These glycans are assumed to be O-linked and predominantly attached to the C9a part of C9. The electrophoretic mobility of C9 is not affected by endoglycosidase F or H treatments revealing that the two N-linked glycans are of the tri- or tetra-antennary complex type. Cleavage of terminal sialic acids from native C9 by neuraminidase results in an Mr 67,000 product with nearly unaltered hemolytic activity. In contrast to other glycoproteins in which deglycosylation remained without major effects on their functional activity, our findings suggest that the N-linked carbohydrates are required for full expression of hemolytic activity of C9.

Bioincompatibility of dialysis membranes: factor H binding correlates inversely with complement activation indicating a local imbalance of involved proteases/anti-proteases.

The problem of bioincompatibility reactions has plagued those who applied hemodialysis from its very beginning. Although other mechanisms for triggering of adverse effects during dialysis, i.e. anaphylactic reactions against the sterilizer ethylenoxyde or against substances extracted from the membranes, from the potting or the tubings have recently come into focus, complement (C) activation is one potentially important factor in bioincompatibility. In the present report, we try to summarize evidence for an involvement of the C system in bioincompatibility of dialysis membranes. We add some recent findings of our laboratory relating to the roles of factor H, the regulatory antiprotease and inhibitor of the alternative pathway C3 convertase.

Detection of a human autoantibody against intercalated cells of kidney-collecting tubule

Serum from a 24-year-old woman with a history of habitual abortions was examined for autoantibodies by indirect immunofluorescence microscopy. Fluorochrome-labelled antibodies to IgG revealed cytoplasmic staining of single cells in rat kidney collecting and connecting tubules. An identical staining pattern was reproducibly obtained in human and rabbit kidney, pointing to a cytoplasmic antigen concentrated in the apical pole of these cells. Lectin-binding histochemistry and immunohistochemical experiments using markers for different cell populations of the renal collecting tubule by double immunofluorescence technique, identified the cells recognized by the autoantibody as intercalated cells (ICC). Remarkably, this autoantibody reacted with a different antigen from those described to date in ICC, as evidenced by distribution and intracellular localization. The pattern of immunostaining suggests that all ICC are recognized in total rather than only one subpopulation. The connection between habitual abortions and this novel autoantibody are discussed.

Häufigkeit von Autoantikörpern gegen Doppelstrang-DNS bei negativem ANA-lmmunfluoreszenztest

Es gilt einerseits, die -Testmethode insbesondere hinsichtlich der physikalischen Parameter der immunfluoreszenzmikroskopischen Untersuchung besser zu standardisieren und sensibler zu machen. Durch Angabe der Titerstufen über einem Normalserum-Grenzwert anstelle der tatsächlichen Verdünnungen könnte weiterhin die Vergleichbarkeit der -Ergebnisse zwischen den Labors verbessert werden. Andererseits sollte der Nachweis von anti-ds-DNS-Antikörpern etwa durch Entwicklung und Einführung neuer Testmethoden so verändert werden, daß eine Reaktion mit Einzelstrang-DNS unterbleibt.