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W Northemann

Publications and source records attributed to W Northemann.

At least 37 records · Page 2Linked to original sources
PubMed1993

Cytoplasmic islet cell antibodies recognize distinct islet antigens in IDDM but not in stiff man syndrome.

Cytoplasmic islet cell antibodies are well-established predictive markers of IDDM. Although target molecules of ICA have been suggested to be gangliosides, human monoclonal ICA of the immunoglobulin G class (MICA 1-6) produced from a patient with newly diagnosed IDDM recognized glutamate decarboxylase as a target antigen. Here we analyzed the possible heterogeneity of target antigens of ICA by subtracting the GAD-specific ICA staining from total ICA staining of sera. This was achieved 1) by preabsorption of ICA+ sera with recombinant GAD65 and/or GAD67 expressed in a baculovirus system and 2) by ICA analysis of sera on mouse pancreas, as GAD antibodies do not stain mouse islets in the immunofluorescence test. We show that 24 of 25 sera from newly diagnosed patients with IDDM recognize islet antigens besides GAD. In contrast, GAD was the only islet antigen recognized by ICA from 7 sera from patients with stiff man syndrome. Two of these sera, however, recognized antigens besides GAD in Purkinje cells. In patients with IDDM, non-GAD ICA were diverse. One group, found in 64% of the sera, stained human and mouse islets, whereas the other group of non-GAD ICA was human specific. Therefore, mouse islets distinguish two groups of non-GAD ICA and lack additional target epitopes of ICA besides GAD. Longitudinal analysis of 6 sera from nondiabetic ICA+ individuals revealed that mouse-reactive ICA may appear closer to clinical onset of IDDM in some individuals. Mouse-reactive ICAs, however, remained absent in 36% of the patients at diagnosis of IDDM.

Adolescent↗
PubMed1993

Expression of recombinant human thyroid peroxidase by the baculovirus system and its use in ELISA screening for diagnosis of autoimmune thyroid disease.

The cDNAs coding for human full-length and soluble thyroid peroxidase (TPO) were constructed, cloned into a baculovirus transfer vector and used for infection of Spodoptera frugiperda (Sf9) cells. The soluble TPO lacking 87 amino acids of the C-terminal transmembrane and intracisternal domains was designed as a fusion protein with a histidine-hexapeptide as an affinity ligand at its C-terminus. Whereas the recombinant full-length TPO was expressed mainly in an insoluble form in Sf9 cells, the recombinant soluble TPO was almost completely secreted into the culture medium. Both the full-length and the soluble TPO were purified by conventional methods and by a specific affinity chromatography using metal chelating matrix respectively, and tested for their autoantigenicity towards anti-TPO autoantibodies. The ELISA established with the purified recombinant soluble TPO as antigen demonstrated its specificity, practicability and reproducibility in screening of anti-TPO autoantibodies in sera of autoimmune thyroid patients. High correlation (r = 0.89, n = 175) was obtained between the soluble TPO and natural TPO prepared from human thyroid glands. Pathological sera (n = 200) were positively assayed with a significance of 91%.

Animals↗
PubMed1992

Purification of recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen using the expression system pH6EX3 followed by metal chelating affinity chromatography.

A novel plasmid expression vector (pH6EX3) that directs the synthesis of a fusion protein with a histidine hexapeptide at its N-terminus and a foreign protein at its C-terminus was constructed. The fusion gene is controlled by a strong tac promoter, leading to high-level expression of recombinant protein in several bacterial strains; the protein is deposited mainly as an insoluble mass in inclusion bodies. The fusion protein can be purified from the insoluble cell fraction by one-step affinity chromatography based on the selective interaction between the histidine hexapeptide and a metal chelating matrix charged with Ni2+ ions. The principle of this new system was tested by expressing and purifying antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein autoantigen. With the use of column chromatography and pH gradient elution, about 25 micrograms recombinant protein/ml of bacterial culture was obtained.

Base Sequence↗
PubMed1992

Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques.

Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.

Autoantibodies↗
PubMed1992

Production of recombinant rat interleukin-6 in Escherichia coli using a novel highly efficient expression vector pGEX-3T.

Interleukin-6 (IL-6) is one of the most important mediators of the acute phase reaction in liver. For the production of recombinant rat IL-6 in Escherichia coli, a previously isolated cDNA coding for the rat IL-6 was cloned into the modified novel expression vector pGEX-3T. The IL-6 cDNA was highly expressed as a fusion protein with the glutathione S-transferase (GST) at its C-terminus and rat IL-6 at its N-terminus. The GST-IL-6 fusion protein was controlled by a tac-promoter and could be induced very efficiently by isopropyl-beta-D-thiogalactopyranoside. The synthesized GST-IL-6 fusion protein was insoluble and precipitated intracellularly in E. coli. Using an advanced technique, the insoluble protein was solubilized and purified to homogeneity by affinity chromatography using immobilized glutathione in a one-step procedure.

Amino Acid Sequence↗
PubMed1991

Identification of a glucocorticoid response element contributing to the constitutive expression of the rat liver alpha 1-inhibitor III gene.

alpha 1-Inhibitor III (alpha 1 I3), a broad range plasma proteinase inhibitor, is synthesized with striking tissue specificity in rat livers. The gene is expressed strongly in periportal hepatocytes of healthy adults and less abundantly in regions near the centrilobular vein. This expression pattern is suggestive of a concentration gradient of a blood-borne hormone that enters through the portal vein and diffuses across the lobe toward the centrilobular vein. The alpha 1 I3 gene was known to be regulated both by glucocorticoids and interleukin 6, and therefore the hypothesis was tested that the normal constitutive expression of this gene depended on glucocorticoids. alpha 1 I3 mRNA levels in the livers of hypophysectomized rats with low endogenous glucocorticoid levels were only about 20% of those in control rat livers. Injection of exogenous glucocorticoids reconstituted hepatic alpha 1 I3 mRNA levels up to 64% of their original values in a dose-dependent manner. Similarly, treatment of FAZA rat hepatoma cells with the synthetic glucocorticoid dexamethasone induced alpha 1 I3 mRNA levels in a dose-dependent manner. Taken together these data suggested that glucocorticoids are required for the constitutive high level expression of this gene in normal adult rat livers. A series of 5' deletion constructs and linker scanning mutants of the promoter upstream region were produced and transfected into FAZA cells. A functional glucocorticoid response element was mapped between -168 and -151 base pairs 5' of the transcription start site. This element conforms with an inverted consensus glucocorticoid response element (GRE) but differs in two positions essential for protein DNA interaction between the GRE and the glucocorticoid receptor (GR). The induction of alpha 1 I3 gene promoter region constructs by dexamethasone was abolished by the receptor antagonist RU486, indicating that the GR participated in the activation of the alpha 1 I3 gene. In DNase I footprinting experiments with nuclear protein extracts from untreated and dexamethasone-treated FAZA cells, similar extents of alpha 1 I3 promoter upstream sequences were protected, indicating that proteins capable of binding in the glucocorticoid response-mediating element (GME) region were present before and after arrival of the hormonal signal. However, a purified recombinant fragment of the GR which contained essentially only its DNA binding domain was unable to bind at the GME although it interacted strongly with a consensus GRE sequence.

Acute-Phase Proteins↗
PubMed1991

The expression of interleukin-6 by a rat macrophage-derived cell line.

A stable rat macrophage-derived cell line (RMSV1) was established by transformation of primary peritoneal exudate cells with the SV40 virus. The RMSV1 cell line was used as a model to study the regulation of the interleukin-6 (IL6) gene expression in rat macrophages with respect to lipopolysaccharides (LPS), interleukin-1 (IL1) and glucocorticoids. The IL6 mRNA level in RMSV1 cell lines was induced 20-fold within 4 h by LPS, whereas IL1 had no effect. The glucocorticoids were able to inhibit completely the induction of the IL6 mRNA synthesis by LPS, indicating the negative regulation of the IL6 gene expression by glucocorticoids.

Animals↗
PubMed1991

Gene regulation by interleukin 6.

Interleukin 6 (IL-6) is a central alarm hormone of the mammalian body. During acute and chronic inflammations, it induces acute phase plasma protein synthesis by liver hepatocytes, modulates the immune response and participates in the regulation of body temperature (fever). In addition, it is a growth factor for certain tumor cells, such as myeloma cells. The details of the IL-6 signal transduction mechanism are unknown. We have contributed to this problem at 2 levels: (a), we have mapped an IL-6-response element (IL-6-RE) in the 5' flanking region of the alpha 2-macroglobulin gene (alpha 2M), a prototype rat liver acute phase gene. This element, CTGGGA, serves as a binding site for nuclear factors that facilitate hormone induced transcription. We have begun to characterize these factors from hepatic cells and demonstrated that they undergo characteristic IL-6-induced changes. Similar factors were also discovered in human Burkitt tumor derived cell lines (B cells). These bound at the IL-6-RE of the rat alpha 1M gene and formed indistinguishable protein DNA complexes, as the corresponding hepatic factors. Thus, common elements probably operate in the IL-6 signal transduction cascade in liver cells and B cells; (b), we have cloned the rat liver IL-6 receptor (IL-6-R) and derived its amino acid sequence. It was 53% identical to the human leukocyte IL-6-R and all functional domains were highly conserved. Therefore, the cell-type specific responses to IL-6 in liver cells and lymphocytes were probably not due to cell-type specific forms of the receptor, but to other so far unknown elements of the signal transduction cascade.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Cloning and expression of antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen in Escherichia coli.

Autoantibodies directed against the 68-kDa (U1) ribonucleoprotein antigen are mainly found in sera of patients with mixed connective tissue disease. The corresponding cDNA was fragmented into four regions coding for the major antigenic epitopes A', B', C' and D'. All the epitopes were subcloned and expressed as fusion proteins with the glutathione S-transferase in Escherichia coli using the novel expression system pGEX that allows very high yields of recombinant proteins after a single-step purification. The sera of patients with the autoimmune disease were analyzed for the expressed recombinant proteins by an immunoblotting technique. All positive sera showed a patient-specific behavior and could be divided into four groups regarding recognition of the four antigenic epitopes of the 68-kDa (U1) ribonucleoprotein antigen. The epitope B' was reactive to all patient sera positively tested and classified as the marker antigenic epitope for the mixed connective tissue disease.

Cloning, Molecular↗
PubMed1991

Autocrine activity of interleukin 6 secreted by hepatocarcinoma cell lines.

Among several rat hepatoma cell lines known to secrete interleukin 6 (IL6), the HTC.JZ1 line stands out as a high-level producer. HTC.JZ1 cells were stimulated to secrete up to fourfold increased amounts of IL6 over 24 hours by treatment with lipopolysaccharides (LPS). Both functional IL6 levels, measured as hepatocyte stimulating factor (HSF) activity, and IL6 mRNA concentrations were increased proportionally by exposure to LPS. Similarly, IL6 mRNA was induced by LPS treatment in cultured primary rat hepatocytes. The induction of Il6 mRNA by LPS was inhibited both in primary hepatocyte and hepatoma cell cultures by treatment with the synthetic glucocorticoid dexamethasone, consistent with the known analogous repression of the IL6 gene by dexamethasone in macrophages, monocytes and fibroblasts. IL6 secreted by HTC.JZ1 cells was utilized as an autocrine inducer of endogenous acute phase gene expression: HTC cells expressed constitutive levels of alpha 2-macroglobulin (alpha 2M) mRNA specified by the major rat acute phase gene, the alpha 2M gene, which is known to be regulated by IL6. By contrast, normal rat liver biopsy material and a number of other rat hepatoma cell lines lacked endogenous IL6 production and showed very low to zero expression of endogenous alpha 2M mRNA. Expression of alpha 2M mRNA in HTC.JZ1 cells was inducible by treatment with LPS. The constitutive and the LPS-induced production of alpha 2M mRNA were significantly reduced (up to 50% inhibition) by addition of an anti IL6 serum to the culture medium and removal of the immune complexes. However, complete neutralization of the alpha 2M-inducing HSF activity could not be obtained with anti-IL6 serum alone, probably because HTC.JZ1 cells secrete comparable quantities of a second HSF activity. This activity, the cytokine leukemia inhibitory factor (LIF), is also known to stimulate transcription of the rat alpha 2M gene but was not reactive with anti-IL6 sera. The induction of IL6 mRNA in HTC cells by LPS was regulated at the transcriptional level, as demonstrated by a series of mutagenesis and transfection experiments. Progressive deletion of 5' flanking sequences from the IL6 gene promoter region reduced the basal level, and the LPS-induced promoter activity after transfection into HTC.JZ1 hepatoma cells. IL6 has been shown to act as an autocrine regulator of growth for certain B lymphoid cell lines derived from human multiple myelomas. The results presented here establish that IL6 secreted by certain hepatoma cell lines also acts in an autocrine fashion to induce expression of the endogenous acute phase alpha 2M gene.

Animals↗
PubMed1990

IL-6 functions as an exocrine hormone in inflammation. Hepatocytes undergoing acute phase responses require exogenous IL-6.

We have previously shown that IL-6 is the major monocyte- and fibroblast-derived regulator of acute phase protein gene expression and synthesis in hepatocytes in inflammation. Recently, we and others have shown that rat and human hepatoma cells express IL-6 mRNA, and the question arose as to whether normal hepatocytes express IL-6 and whether any such expression occurs under normal physiologic conditions or is seen in inflammation. Poly A+ mRNA of liver from normal rats and from rats undergoing an acute phase response was not positive when probed with cDNA for rat IL-6 under conditions in which macrophage mRNA was strongly positive. We then compared poly A+ mRNA from purified hepatocytes freshly isolated from normal rats--from rats that were undergoing an acute inflammatory response and from freshly isolated normal hepatocytes that had been cultured for 24 h in the presence or absence of dexamethasone (microM). Only the mRNA from normal hepatocytes cultured for 24 h in the absence of any glucocorticoid was obviously positive for IL-6. The increased expression of gamma-fibrinogen mRNA indicated the presence of inflammation. These results confirm the identification of IL-6 as an exogenous hormone for regulating normal hepatic acute phase protein synthesis in inflammation and rules out an autocrine mechanism being active in the liver in normal homeostasis.

Acute-Phase Reaction↗
PubMed1990

Interleukin 6 is an autocrine growth factor for mesangial cells.

Interleukin 6 (IL-6) induces the acute phase response, differentiation of B cells, proliferation of T cells, thymocytes, hematopoietic progenitors, hybridoma and plasmacytoma cells. Monocytes, T cells, fibroblasts, epithelial and endothelial cells secrete IL-6. Since IL-6 responsive cell-types may participate in the pathogenesis of glomerular inflammation, we studied the secretion of IL-6 by rat MCs, using the IL-6 dependent hybridoma cell line B9. The results of our studies indicate that MCs secrete IL-6 with a molecular weight of 17-42 kDa and isoelectric point of 4.0 to 5.3 MC-IL-6 activity could be blocked by a polyclonal antimurine-IL-6 antibody. MC express IL-6 mRNA as determined by Northern blot. Furthermore, our data demonstrate that IL-6 acts as an autocrine growth factor for MC. Incubation of subconfluent MC with recombinant IL-6 results in a dose-dependent increase of 3H-thymidine incorporation and number of MCs. Moreover, reverse phase HPLC fractions of MC-CM containing IL-6 activity increase 3H-thymidine incorporation by MC. In addition to its possible paracrine role in mediating the immune response in the glomerulus, MC-IL-6 may also be one of the autocrine signals leading to mesangial cell proliferation in vivo.

Animals↗
PubMed1990

Acute-phase reaction induces a specific complex between hepatic nuclear proteins and the interleukin 6 response element of the rat alpha 2-macroglobulin gene.

Interleukin 6 (IL-6) was established as a transcriptional inducer of the rat alpha 2-macroglobulin gene, a prototype liver acute-phase gene. Maximum induction occurred when the 5' flanking sequences of this gene (position -209 to -43) directed expression from the gene's own TATA box and transcription start site. Removal of the hexanucleotide CTGGGA (position -164 to -159) abolished 60-70% of the hormonal induction in FAO1 rat hepatoma cells. This hexanucleotide was defined as the IL-6 response element (IL-6-RE). The IL-6-RE is well conserved in the cytokine-responsive regions of other acute-phase genes and serves as a binding site for nuclear proteins. A characteristic DNA-protein complex (complex I) was formed with nuclear proteins from normal rat livers. A different, hormone-inducible complex (complex II) was assembled specifically with nuclear proteins from acute-phase rat livers or from IL-6-treated human Hep 3B hepatoma cells. Complex II was competitively inhibited by oligonucleotides representing the conserved IL-6-RE sequence from other acute-phase genes. Thus, the proteins building complex II likely participate in a general signal transduction mechanism mediating the transcriptional activation by IL-6 of several acute-phase genes.

Animals↗
PubMed1990

Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements.

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.

Acute-Phase Proteins↗
PubMed1990

Simplified northern blot hybridization using 5% sodium dodecyl sulfate.

We describe a simplified Northern blot hybridization procedure. This procedure eliminates the need for many reagents commonly used in RNA hybridization and replaces them with two buffers containing 5% sodium dodecyl sulfate for pre-hybridization, hybridization and all post-hybridization washes.

Animals↗
PubMed1990

Production of interleukin 6 by hepatoma cells.

Five rat hepatoma cell lines were shown to secrete hepatocyte-stimulating factors (HSFs) capable of inducing a characteristic spectrum of acute phase genes. Three of these lines, but not normal rat livers or livers from rats with an experimentally induced acute inflammation, produced interleukin 6 (IL6) mRNA. An anti-rat IL6 serum was prepared against synthetic peptides derived from the rat IL6 cDNA sequence. This antiserum cleared authentic rat IL6 and a fraction of the HSF activities secreted by the hepatoma cell lines. After concentration of culture supernatants from FTO2B hepatoma cells, IL6 was detected with the anti-IL6 serum by immuno-blot analysis. Biosynthesis of IL6 in the HTC line was demonstrated by metabolic labeling and immunoprecipitation. Secretion of HSF activities by hepatoma cells was increased by serum factors. These data suggest that different rat hepatoma lines each secrete different characteristic sets of HSF activities and establish unambiguously that IL6 is secreted by at least some of these lines.

Acute-Phase Proteins↗
PubMed1989

Structure of the rat interleukin 6 gene and its expression in macrophage-derived cells.

Rat peritoneal exudate cells produce two interleukin 6 (IL6) messenger RNA species, a major 1200 nucleotide and a 5-fold less abundant, 2400-nucleotide species. A cDNA clone representing the major species was isolated, and sequenced. The 1055-base pair insert covered the 3'-nontranslated region, the 211 triplet coding region and most of the 5'-nontranslated region. The derived rat IL6 amino acid sequence was 93 and 58% identical, respectively, with mature murine and human IL6. Rat IL6 lacks N-glycosylation sites but contains a fifth cysteinyl residue in addition to the 4 residues shared in conserved positions with murine and human IL6. Stable murine L cell and human HeLa-derived cell lines were established by cotransfection with rat IL6 cDNA and a selectable neomycin resistance marker. These lines secrete 9-fold increased amounts of functional IL6 over their respective parental cells. A stable rat macrophage-derived cell line, RM-SV1, was established by transformation with simian virus 40. IL6 and Il1 mRNA levels are inducible 20- and 3.5-fold, respectively, in this line by treatment with lipopolysaccharide with kinetics characteristic of macrophages. A set of three overlapping genomic DNA clones was isolated and a 10-kilobase DNA segment was sequenced containing the rat IL6 gene plus 2.9 kilobases of 5'-flanking and 1.3 kilobases of 3'-flanking sequences. The two transcription start sites used in RM-SV1 cells were mapped within 5 base pairs of each other. The exon/intron boundaries are conserved with the murine and human IL6 genes. The two IL6 mRNA species are generated by alternative polyadenylation at sites separated by a distance of 1.2 kilobases. The intervening region contains a repetitive element 72-80% identical with the rat and murine consensus L1 family sequences.

Amino Acid Sequence↗