Identification and regulation of 1,25-dihydroxyvitamin D3 receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. Studies in cultured bovine aortic endothelial cells and human dermal capillaries.

Because 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to play roles in both proliferation and differentiation of novel target cells, the potential expression of 1,25(OH)2D3 receptor (VDR) activity was investigated in cultured bovine aortic endothelial cells (BAEC). Receptor binding assays performed on nuclear extracts of BAEC revealed a single class of specific, high-affinity VDR that displayed a 4.5-fold increase in maximal ligand binding (Nmax) in rapidly proliferating BAEC compared with confluent, density-arrested cells. When confluent BAEC were incubated with activators of protein kinase C (PKC), Nmax increased 2.5-fold within 6-24 h and this upregulation was prevented by sphingosine, an inhibitor of PKC, as well as by actinomycin D or cycloheximide. Immunohistochemical visualization using a specific MAb disclosed nuclear localized VDR in venular and capillary endothelial cells of human skin biopsies, documenting the expression of VDR, in vivo, and validating the BAEC model. Finally, additional experiments indicated that BAEC formed the 1,25(OH)2D3 hormonal metabolite from 25(OH)D3 substrate, in vitro, and growth curves of BAEC maintained in the presence of 10(-8) M 1,25(OH)2D3 showed a 36% decrease in saturation density. These data provide evidence for the presence of a vitamin D microendocrine system in endothelial cells, consisting of the VDR and a 1 alpha-hydroxylase enzyme capable of producing 1,25(OH)2D3. That both components of this system are coordinately regulated, and that BAEC respond to the 1,25(OH)2D3 hormone by modulating growth kinetics, suggests the existence of a vitamin D autocrine loop in endothelium that may play a role in the development and/or functions of this pathophysiologically significant cell population.

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.

Phototoxic erythema following PUVA treatment: independence of complement.

The effect of PUVA treatment on normal human serum (NHS), on isolated PMN, or on C3-deficient guinea pigs and congenic (C3-competent) control animals was tested. At a concentration of 0.1 or 1 mM/l 8-MOP and UVA doses of 5-30 J/cm2, PUVA failed to induce any detectable C3-cleavage in NHS. Furthermore, when the complement (C) activation in NHS had been induced before or after PUVA treatment by various methods. PUVA did not modulate the extent of C3-cleavage. PUVA did not affect the viability of isolated PMN, nor did it induce a release of LDH or elastase. No differences between C3-deficient and C-competent guinea pig skin exposed to PUVA were observed in erythema or histologic responses. Immunohistologic examination of specimens from normal guinea pigs revealed C3b and C3d deposits on necrotic keratinocytes, findings restricted to the PUVA-treated areas. Necrosis of keratinocytes was present in skin specimens of C3-deficient animals from PUVA-treated sites to a similar extent. However, deposits of C3-related antigens were completely absent there. From these observations, we suggest that the induction of phototoxic erythema following PUVA treatment is independent of complement.

Expression of 1,25-dihydroxyvitamin D3 receptors in normal and psoriatic skin.

Increasing evidence suggests an immunoregulatory function of the potent steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) which has been successfully applied for treatment of psoriasis. The skin is both a site of production and a target of 1,25(OH)2D3. In vitro, 1,25(OH)2D3 inhibits proliferation and stimulates differentiation of keratinocytes. We investigated the in situ expression of vitamin D-receptors (VDR) in normal and psoriatic skin by immunochemical methods. The VDR were visualized using the monoclonal antibody (MoAb) 9A7g to the VDR and the labeled avidinbiotin technique. Immunoreactivity was consistently confined to nuclei in all skin biopsies. In normal skin specimens (n = 10) VDR antigens were expressed in keratinocytes of all epidermal layers (except those of the stratum corneum) and in cells of the epidermal appendages. Double labeling experiments with MoAb to cluster-defined antigens indicated that melanocytes and approximately 75% of Langerhans cells exhibit 1,25(OH)2D3 receptors in normal skin biopsies (n = 5). Depending on their localization in skin compartments 42-62% of CD11b+ positive macrophages and 45-75% of CD3+ T lymphocytes expressed VDR. Non-lesional psoriatic skin specimens (n = 8) revealed nearly identical staining patterns. Lesional psoriatic skin specimens (n = 8) exhibited a significant increase of VDR expression both in basal and suprabasal epidermal layers as measured by computer-assisted morphometry and showed a remarkable change of the immune cell pattern: the densitity and proportion of VDR positive T lymphocytes and macrophages were higher in the epidermal and the perivascular papillary loop compartment. These in vivo findings strongly support the hypothesis that 1,25(OH)2D3 modulates immune response and cell proliferation/differentiation in human skin.

Repeated affinity chromatography on glass beads with stable capacity: one-step purification of polyclonal IgA or IgM.

Absorption is an essential step in the purification of heterologous anti-idiotypic antibodies. Polyclonal immunoglobulins are preferentially used and have advantages in the absorption step since they have a broader antigenic spectrum than monoclonal immunoglobulin fractions. A method is described for the purification of polyclonal IgA and IgM by repetitive semi-automatic immunoaffinity chromatography using an LKB ultrograd gradient former. Anti-IgA or anti-IgM antibodies, respectively, were covalently bound to controlled pore glass beads. The capacities of the columns were stable during 20--24 absorption/elution cycles. IgA was isolated with a yield of 213 mg and a recovery of 15.2%. The purification factor was 15.2. IgM was isolated in a yield of 178 mg with a recovery of 41.9%. We thus achieved a purification factor of 38. For higher yields the number of cycles can easily be increased.

Comparison of multiple rosetting assays for detecting antibody reactivity of different immunoglobulin classes against surface antigens of benign and malignant tissue culture cells.

Four serological assays for detection of antibody reactivity against surface antigens of benign and malignant cells have been evaluated. In all four assays, antibody attached to the surface of a target cell is detected by a rosette of indicator red blood cells. Of the four assays (antibody mixed hemadsorption assay, immune adherence assay, anti-C3 mixed hemadsorption assay and protein A assay), the anti-C3 mixed hemadsorption assay showed highest sensitivity. By using purified IgM and IgG fractions we could demonstrate that, except for the protein A assay, all assays show reactivity both with IgG and IgM. Protein A assay detected only the IgG fraction, with high sensitivity. The optimization of the four assays is described and the importance of using multiple assays is emphasized.

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.

Ferritin-labeling of antibodies by glutaraldehyde. Comparison of conjugates prepared at different antibody: ferritin: glutaraldehyde ratios.

Optimal molar ratios of antibody: ferritin: glutaraldehyde for the preparation of antibody-ferritin conjugates were investigated. The reaction volumes and the antibody concentration were held constant in twenty different reaction mixtures. The effect of five different antibody: glutaraldehyde ratios ranging from 1:25 to 1:400 and of four different antibody: ferritin ratios (1:1 to 1:01) on the yield of antibody-ferritin conjugates was tested in one-step reactions. The 20 different conjugates were tested by a newly developed gel precipitation technique, the inverse fused line rocket immunoelectrophoresis and by counting relative numbers of ferritin monomers and oligomers under the electron microscope. High concentrations of glutaraldehyde produced large heterogeneous precipitates as visualized in the gel technique. The relatively highest yield of ferritin-labeled antibodies without larger conjugates was produced by reacting antibodies, ferritin and glutaraldehyde at molar ratios of 1:1:100. The FRT-antibody conjugates produced at these molar ratios were partly abe to precipitate with their respective antigens.

Isolation of late complement components by affinity chromatography. II. Purification of the human complement component C6.

We developed a new procedure for the rapid and gentle isolation of the human complement component C6 comparable to that described previously for C9. The procedure is based on affinity chromatography. As a first step, C6 is immunoabsorbed on insolubilized anti-C6 antibodies. These antibodies were derived from C6-defective rabbits (Freiburg strain). C6 was eluted with 3 M thiocyanate, pH 7.2, with a recovery of 15--23% of its hemolytic activity and a more than 270--fold purification. Impurities were removed in a second step by an "anti-impurity" column. The final product yielded a 12% recovery of the hemolytic activity and the purification factor was higher than 1300. The final product was homogeneous in SDS polyacrylamide and immunoelectrophoresis.

A new method for the preparation of anti-idiotypic antibodies against six different human cold agglutinins.

A method based on sequential immunoadsorption for the preparation of a highly specific antibody directed against the idiotypic determinant of human cold agglutinins (CA) is described. In the present study, human cold agglutinins (CA) were used as a model, but the method can easily be adapted for the preparation of anti-idiotypic antibodies against other monoclonal human immunoglobulins. Since regenerable immunoadsorbents were used, the isolation can be accomplished even with rather low amounts of the respective immunoglobulin.

Optimal conditions for the preparation of ferritin-labeled antibodies defined by binding to their antigen in an ELISA.

An ELISA with covalently fixed antigens was used to measure and define ferritin-labeled antibodies. In the presented model, RIG covalently fixed to glass tubes served as antigen. Twenty ferritin-labeled SWaRIG conjugates were prepared with different molar ratios of antibody: ferritin: glutaraldehyde. At various dilutions, their ability to react with the antigen was compared. The amount of FRT in the reactive conjugates was measured using alkaline phosphatase-labeled antibodies against ferritin. The covalent binding of antigens to glass surfaces resulted in a low unspecific binding as shown previously. Besides, these glass tubes with antigens immobilized on their inner surface could be used more than once. This aspect of it renders this test particularly suitable for systems where rare or expensive antigens are used. The range of O.D. values reflecting the amount of reactive SWaRIG/FRT detected in 20 different conjugates (dil 1:1000) was spread over a good range which allowed a specification of optimal conditions for FRT labeling of antibodies. It is concluded that in addition to an electron microscopic evaluation of the conjugates, this assay may be very helpful in defining optimal conditions for the coupling procedure.

A "spontaneous" cold-reactive IgM antibody with anti HLA-B8 specificity in a patient with multiple sclerosis.

A case of a 35-year-old female with multiple sclerosis is reported who developed without apparent prior sensitization a lymphocytotoxic antibody with anti-HLA-B8 specificity. The antibody persisted for several years with the same titer. The cytotoxic activity of the patient's serum was contained within the IgM fraction. The antibody reacted optimally at low temperature, exclusively against lymphocytes homozygous for HLA-B8. In B8-heterozygous cells, cytotoxic reactions were obtained only following enzymatic pretreatment. The antibody's binding avidity was weak; for its complete absorption, many times more B8-positive lymphocytes or platelets were needed than for a "normal" anti-B8 antibody of the same titer. In HLA redistribution and blocking experiments, it was demonstrated that the antigenic determinant recognized by this antibody is carried by the B8 molecule. It is unclear whether "spontaneously" occurring cold-reactive IgM antibodies with HLA specificity are induced by viral agents or whether they reflect "spontaneous" clonal lymphocyte proliferation.

Bioincompatibility--perspectives in 1993.

Bioincompatibility reactions related to the non-physiology of the procedure have plagued dialysis from its early days. Although the problem is certainly multifactorial, the present overview selectively focuses on some aspects of activation of late complement (C) components, the importance of which may have been underappreciated in the past. Dialysis patients are poised for intense C activation because of cumulation of the low molecular weight factor D, an intrinsically active serine esterase which is not inhibited by any known endogenous inhibitor and catalyzes an early step in the alternative pathway. C activation reflects the net balance between activation and inhibition, the latter particularly via factor H binding. Dialyzer membrane characteristics that are related to factor H binding and regulation of initial activation steps include not only membrane surface chemistry but also its microdomain structure. Kinetic studies of the generation of the terminal complement complex (TCC) suggest ongoing generation throughout the duration of a dialysis session (in contrast to the transient release of C-derived anaphylatoxins). Potential consequences of TCC generation include amplification of the non-C-dependent cell activation signals through L-fucose-dependent steps. Efforts to reduce TCC generation by membrane engineering, for example, end group derivatization and optimization of microdomain structure, open perspectives for the development of more biocompatible membranes.

Immunohistology of temporal arteritis: phenotyping of infiltrating cells and deposits of complement components.

Deposition of complement factors, immunoglobulins and infiltrating cells was evaluated by immunohistochemical staining in 30 temporal artery biopsy specimens from patients suffering from temporal arteritis and/or polymyalgia rheumatica and in controls. In the temporal arteritis group infiltrating cells, classic complement, alternative complement and lytic complex activation were detected. In specimens from patients suffering from only polymyalgia rheumatica there was unexpected evidence of classic complement and lytic complex activation. We conclude that immuno-histochemistry provides support for the concept of temporal arteritis and polymyalgia being based on the same pathological process.

[Neonatal screening for congenital hypothyreoidism in Germany. The development of concerned children in retrospect analysis using the federal state "Hessen"]. / Neugeborenenhypothyreosescreening in Deutschland. Retrospektive Analyse der Entwicklung von betroffenen Kindern am Beispiel des Bundeslandes Hessen.

BACKGROUND: Since widespread screenings for hypothyreosis were started in 1981 in Germany, the numbers of mental and physical handicaps due to hypothyroidism are reduced markedly. The aim of this study is to evaluate the actual efficiency of the newborn screenings in Germany using in the federal state "Hessen". METHODS/SUBJECTS: All children born between 1988 and 1992 with suspicions laboratory results in the screening examination were contacted personally and statements concerning the screening itself and the physical and mental development were gathered from their parents, doctors and teachers. RESULTS: 99.1% of all hessian newborns born between 1988 and 1992 were included into the screening for hypothyreosis. The incidence of congenital hypothyreosis in general was 1:3 313. An etiological classification was possible in 77% of the patients which is divided as follows: 40% athyreosis, 24% hypoplasia of the thyroid, 8% dyshormonogenesis, 5% ektopia of the thyroid. In 67% of the cases hormone substitution was initiated during the first 14 days of life. In 23.9% it was started in the third week, in 6.8% in the fourth week and only in 2.3% of the patients treatment was started later on. The physical development of the children with congenital hypothyreosis can be regarded as widely normal. The school achievement was moderately retarded even when treatment was started in the early neonatal period. CONCLUSIONS: The screening for hypothyreosis is well established in Hessen concerning tracking, organisation and analysis. There are short comings concerning the follow ups of children with suspicions findings, which shall be overcome by creating a new position. The long-term-follow-up according to the guidelines of the "Arbeitsgemeinschaft Pädiatrische Endokrinologie" is of central interest. Furthermore compliance is improved by regular personal counselling with the parents.

Inside-out vesicles prepared from complement lysed sheep red blood cells.

Antibody sensitized sheep red blood cells were lysed to 68% by diluted human serum. The ghosts were shown to undergo vesiculation induced by low ionic strength buffer and gentle shearing forces like osmotically lysed ghosts. About 45% of the vesicles derived from complement lysed ghosts (as referred to vesicle protein) remained floating on top of a linear 3-18% dextran T110 gradient during ultracentrifugation. Using antibodies as probes for sideness about 90% of the floating vesicles displayed an inside-out orientation. Floating inside-out vesicles bear a slightly reduced but significant number of C9 molecules on their inverted extracellular side of the membrane as compared to the (leaky or collapsed) vesicles banding in the gradient. This finding suggests that the floating is not due to a lack of C5b-9 (under the assumption that all C9 was bound to C5b-9). It is concluded that lysis with diluted human serum may result partly in ineffective C5b-9 complexes and/or in lesions with limited permeability which do not impair vesicle floating on top of polymer gradients.

Characterisation and specificity of glomerular basement membrane antigens identified by sera of patients with anti-GBM nephritis.

The sera of 21 patients positive for antibodies against GBM in indirect immunofluorescence tests were examined by immunoblotting. We demonstrated antibodies against 50, 48, 43 and 29 kD molecular weight peptides in 20 of 21 sera using collagenase-digested GBM, in 19 of 21 using trypsin-digested GBM, and in 10 of 21 using elastase-digested GBM. Although the spectrum of molecular weights of the antigenic proteins was similar in all three digests, they differed with respect to preservation of antigenicity upon reduction with mercaptoethanol. Many of the sera of patients and controls reacted with proteins unrelated to GBM, e.g. albumin and prealbumin. Furthermore, some control sera reacted with one single peptide of the above-mentioned specific GBM peptides. Our results suggest that the highly purified 29 kD peptide of the collagenase digest or the 50 kD peptide of the trypsin digest provide the best antigens to develop a screening test for antibodies against GBM. However, serum antibodies against these antigens will not be absolutely specific for anti-GBM antibody-mediated nephritis, as shown by the immunoblot experiments.