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H Burkhardt

Publications and source records attributed to H Burkhardt.

At least 55 records · Page 3Linked to original sources
PubMed1998

Rat antigen-induced arthritis: cartilage alterations assessed with iodine-123-antileukoproteinase.

UNLABELLED: Imaging of cartilage alterations was attempted in joints of rats with chronic antigen-induced arthritis (AIA) using the cationic 123I-labeled serine proteinase inhibitor antileukoproteinase (123I-ALP; pI > 10), which selectively accumulates in normal cartilage, presumably through interaction with negatively charged proteoglycans. METHODS: Iodine-123-ALP or 123I-myoglobin, a control protein of comparable size but with different isoelectric point (pI=7.3) was injected intravenously into normal or AIA rats. Joint accumulation was followed by scintigraphy for 14 hr. Tissue radioactivity was assessed by well-counter measurements after dissection. The content of charged molecules in articular cartilage was determined by toluidine blue staining; the degree of joint destruction was assessed in parallel by x-ray, ex vivo MRI and histopathology. RESULTS: In intact articular cartilage, ALP accumulated to a significantly higher degree than myoglobin. This preferential accumulation was lost in rats with chronic AIA. The target-to-background ratio for 123I-ALP negatively correlated with the loss of toluidine blue staining in cartilage, which documents depletion of charged matrix molecules (r=-0.92, p < 0.01 at 4 hr; r=-0.97, p < 0.01 at 13 hr). ALP scintigraphy was sensitive in detecting cartilage alterations, even though the degree of joint destruction and inflammatory infiltration was mild, as demonstrated by x-ray, MRI and histopathology. CONCLUSION: In rat AIA, loss of ALP accumulation appears to document proteoglycan depletion in mildly altered arthritic cartilage. ALP scintigraphy may represent a functional assay for early, premorphological cartilage alterations in human arthritis as well.

Animals↗
PubMed1997

Analysis of the DNA-binding activities of Myc/Max/Mad network complexes during induced differentiation of U-937 monoblasts and F9 teratocarcinoma cells.

The bHLHZip protein Max interacts with both the Myc and Mad family proteins forming heterodimers which specifically bind certain E-box DNA recognition sequences, thereby regulating transcription. Whereas Myc proteins actively promote cell proliferation, Mad complexes have the opposite function. Although the main regulation of this network seems to be the control of myc- and mad family gene expression, regulation at the level of DNA-binding and transactivation may also be in operation. Few studies on the DNA-binding activity of native Myc:Max or Max:Mad complexes have been reported mainly due to technical difficulties. To overcome these problems we have developed a specific and sensitive solid phase DNA-binding assay based on partial purification of native Myc, Max and Mad1 complexes by immunological methods. Using this technique we report that the DNA-binding activity of c-Myc-containing complexes is reduced during induced differentiation of U-937 monoblasts and F9 embryonic teratocarcinoma cells. In contrast, the DNA-binding of Mad1-containing complexes increases during monocytic differentiation. In general, the DNA-binding activity of c-Myc and Mad1 correlate with their expression. However, our studies of early kinetics of TPA-induced differentiation of U-937 cells as well as of late events during F9 differentiation suggest that post-translational regulation of Myc and Max DNA-binding may also occur. The solid phase DNA-binding assay may thus provide a tool to study the regulation of DNA-binding in more detail.

Animals↗
PubMed1997

Xenobiotic immunosuppressive agents: therapeutic effects in animal models of autoimmune diseases.

An unprecedented arsenal of new xenobiotic immunosuppressive agents has been developed recently. Most of the new immunosuppressants have been tested primarily in the treatment of allograft rejection in experimental models of transplantation, and some of the new drugs have already proven their safety and efficiency in extensive clinical trials on transplant patients. Another field for their potential application is the treatment of autoimmune diseases. This review will give an overview of the therapeutic potential of the new xenobiotic drugs in different animal models of rheumatoid arthritis, systemic lupus erythematosus, myasthenia gravis, multiple sclerosis, diabetes mellitus, thyroiditis and uveoretinitis. The new xenobiotics are either inhibitors of the de novo synthesis of nucleotides, for example mycophenolate mofetil, mizoribine, leflunomide, and brequinar, or are immunophilin-binding agents (cyclosporin, FK506 and rapamycin) that inhibit signal transduction and cell cycle progression in lymphocytes. A different mode of action is likely to account for the immunosuppressive effects of deoxyspergualin, which may interfere with intracellular chaperoning by the heat shock protein HSP70 and the activation of transcription factor NF-kappa B.

Animals↗
PubMed1997

Animal models of autoimmune diseases.

Failure of distinction between self and non-self is regarded a critical event in the pathogenesis of several human diseases such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, uveoretinitis or diabetes mellitus. Autoagressive immune reactions driven by activated autoreactive lymphocytes are a characteristic feature of these autoimmune diseases. The mechanisms by which the pathogenic control of autoreactive lymphocytes deviates from physiology can be studied in appropriate animal models under well-defined experimental conditions. Experimental models of autoimmune diseases in rodent inbred strains allow for the genetic mapping of susceptibility loci and might help to identify candidate genes also relevant to the pathogenesis of human diseases. Finally, the experimental models are valuable tools to develop rational immunotherapeutic strategies. Interesting features of some of the models employed for such research will be introduced in this review.

Animals↗
PubMed1997

[Dextrocardia and Poland syndrome in a 59-year-old patient].

Poland syndrome consists of unilateral absence of the sternal head of the pectoralis major muscle, ipsilateral symbrachydaktylia, and occasionally associated other malformations of the anterior chest wall, mammilla, and mamma. To our knowledge a simultaneous occurrence with dextrocardia was reported in seven patients. We report on an additional patient with this unusual coincidence. A 59-year old man showed mild symbrachydaktylia of the left hand and deformity of the left part of the anterior chest wall, both present since birth. We found an additional dextrocardia with situs solitus, d-loop, and correctly connected great arteries. Besides arterial hypertension there was no remarkable impairment of the patient's condition.

Dextrocardia↗
PubMed1997

The serine proteinase inhibitor antileukoproteinase specifically accumulates in normal but not in arthritic cartilage.

OBJECTIVE: To investigate whether the serine proteinase inhibitor antileukoproteinase (aLP) specifically accumulates in articular and extraarticular cartilage in normal and arthritic rats after intravenous (i.v.) injection. METHODS: [123I] or [125I] radiolabeled aLP and a control protein of comparable size were injected iv into normal rats or rats with chronic, antigen induced arthritis (AIA). Joint accumulation of the 2 proteins was followed by scintigraphy and organ tissue radioactivity was assessed by autoradiography and well counter measurements. Immunoprecipitation of aLP from articular cartilage was also performed and the content of charged molecules in normal and arthritic cartilage determined using toluidine blue staining. RESULTS: The accumulation of both radiolabeled aLP and control protein in the normal joint was clearly detectable by scintigraphy, with significant differences between the 2 proteins. In accordance with the scintigraphic data, direct tissue radioactivity measurements, immunoprecipitation, and autoradiography showed highly specific accumulation of radiolabeled aLP in articular and extraarticular cartilage of normal rats. The specific accumulation of aLP in articular cartilage was lost in rats with AIA in parallel with a loss of charged matrix molecules. CONCLUSION: I.v. injected serine protease inhibitor aLP specifically accumulates in articular and extraarticular cartilage of normal rats. This physiological pathway of cartilage accumulation, lost in proteoglycan depleted arthritic cartilage, may serve to maintain the local balance between proteinase function and inhibition.

Animals↗
PubMed1996

Systemic versus cartilage-specific expression of a type II collagen-specific T-cell epitope determines the level of tolerance and susceptibility to arthritis.

Immunization of mice with rat type II collagen (CII), a cartilage-specific protein, leads to development of collagen-induced arthritis (CIA), a model for rheumatoid arthritis. To define the interaction between the immune system and cartilage, we produced two sets of transgenic mice. In the first we point mutated the mouse CII gene to express an earlier defined T-cell epitope, CII-(256-270), present in rat CII. In the second we mutated the mouse type I collagen gene to express the same T-cell epitope. The mice with mutated type I collagen showed no T-cell reactivity to rat CII and were resistant to CIA. Thus, the CII-(256-270) epitope is immunodominant and critical for development of CIA. In contrast, the mice with mutated CII had an intact B-cell response and had T cells which could produce gamma interferon, but not proliferate, in response to CII. They developed CIA, albeit with a reduced incidence. Thus, we conclude that T cells recognize CII derived from endogenous cartilage and are partially tolerized but may still be capable of mediating CIA.

Amino Acid Sequence↗
PubMed1996

Differential effects of parathyroid hormone fragments on collagen gene expression in chondrocytes.

The effect of parathyroid hormone (PTH) in vivo after secretion by the parathyroid gland is mediated by bioactive fragments of the molecule. To elucidate their possible role in the regulation of cartilage matrix metabolism, the influence of the amino-terminal (NH2-terminal), the central, and the carboxyl-terminal (COOH-terminal) portion of the PTH on collagen gene expression was studied in a serum free cell culture system of fetal bovine and human chondrocytes. Expression of alpha1 (I), alpha1 (II), alpha1 (III), and alpha1 (X) mRNA was investigated by in situ hybridization and quantified by Northern blot analysis. NH2-terminal and mid-regional fragments containing a core sequence between amino acid residues 28-34 of PTH induced a significant rise in alpha1 (II) mRNA in proliferating chondrocytes. In addition, the COOH-terminal portion (aa 52-84) of the PTH molecule was shown to exert a stimulatory effect on alpha1 (II) and alpha1 (X) mRNA expression in chondrocytes from the hypertrophic zone of bovine epiphyseal cartilage. PTH peptides harboring either the functional domain in the central or COOH-terminal region of PTH can induce cAMP independent Ca2+ signaling in different subsets of chondrocytes as assessed by microfluorometry of Fura-2/AM loaded cells. These results support the hypothesis that different hormonal effects of PTH on cartilage matrix metabolism are exerted by distinct effector domains and depend on the differentiation stage of the target cell.

Animals↗
PubMed1995

Differential effects by Mad and Max on transformation by cellular and viral oncoproteins.

c-Myc is an essential component of the regulatory mechanisms controlling cell growth. Max is the obligatory partner of c-Myc for all its biological functions analysed to date. Recently two Max interacting proteins, Mad and Mxi1, have been identified. It has been suggested that these two proteins modulate c-Myc function, in the simplest model by competing with c-Myc for the interaction with Max. We have analysed different aspects of Mad function in comparison to Max. Native Mad/Max heterodimers bound specifically to a c-Myc/Max consensus DNA binding site. Furthermore Mad inhibited efficiently c-Myc, mutant p53, adenovirus E1a, or human papilloma virus type 16 transformation of rat embryo cells in cooperation with activated Ha-Ras. Myc transformed clones showed an increased cell cycle time and a reduced immortalization frequency after cotransfection with either mad or max. In contrast to Mad, Max did not inhibit E1a/Ha-Ras cotransformation but repressed c-Myc/Ha-Ras transformation efficiently. Mad delta N, an N-terminal deletion mutant of Mad, was as efficient in repressing c-Myc/Ha-Ras cotransformation as full length Mad but showed little inhibitory activity when assayed on E1a/Ha-Ras. Unlike wt Mad, Mad delta N had little effect on cell growth. Our data suggest that Mad affects cell growth at least in part by a c-Myc independent mechanism.

Animals↗
PubMed1995

Characterization of the binding region for the Yersinia enterocolitica adhesin YadA on types I and II collagen.

OBJECTIVE: The plasmid-encoded adhesin YadA confers pathogenic functions on Yersinia enterocolitica, a microorganism associated with reactive arthritis. While emerging evidence has indicated that the persistence of the bacteria in individuals with reactive arthritis is a prerequisite for the development of the disease, the tissue specificity of this immunologic disease sequela remains elusive. The present study was undertaken to investigate YadA-mediated binding of Y enterocolitica to the most abundant collagens in joints, types I and II collagen. METHODS: Binding studies were performed with recombinant Y enterocolitica strains and highly purified type II collagen and the alpha 1(I) chain of type I collagen, or fragments of these collagens generated by various enzymatic and nonenzymatic cleavage procedures. Interactions of bacteria with the proteins were determined in binding assays with radiolabeled proteins. RESULTS: Binding regions for YadA were identified at the 181-amino acid fragment alpha 1(I)78CBN of type I collagen and the CB10 fragment of type II collagen. From binding and blocking experiments with alpha 1(I) fragments, cyanogen bromide-derived or mammalian collagenase-derived type II collagen fragments, and synthetic peptides with collagen-like structures, it was concluded that the binding site for YadA on collagen is determined by a restricted amino acid sequence and is defined within a highly homologous 134-amino acid region. Furthermore, the binding site is not affected by mammalian collagenase digest. Binding of YadA-positive yersiniae to collagen could be inhibited by an antiserum specific for YadA. CONCLUSION: This study provides the first evidence of a binding site for bacterial proteins on collagens which is not determined by the repetitive sequence Gly-X-Y of collagens. We speculate that the binding region is conserved between types I and II collagen, the most abundant collagens in the joints. Specific binding of Yersinia products to joint collagens might contribute to the arthritogenic potential of enteropathogenic yersiniae.

Adhesins, Bacterial↗
PubMed1995

Macrophages, but not dendritic cells, present collagen to T cells.

Dendritic cells, such as epidermal Langerhans cells, play a crucial role for the antigen-specific priming of T cells. We have addressed the question whether dendritic cells present collagen, a major protein component in tissues through which dendritic cells migrate, i.e. the basement membrane, dermis, and synovial tissue. Langerhans cells, spleen cells and peritoneal macrophages were compared for antigen-presenting capacity using a panel of mouse T cell hybridomas reactive with different determinants on type II collagen, myelin basic protein, ovalbumin and pepsin. Langerhans cells did not present any of the type II collagen determinants, unless the antigen was administered as a 15-mer peptide, but did present myelin basic protein, ovalbumin and pepsin. Spleen cells and peritoneal macrophages, in contrast, presented all type II collagen determinants. This biased antigen presentation was also observed when Langerhans cells were pulsed with antigen in vivo. The inability to present type II collagen is related to the collagen sequence as such, since both native type II collagen, type II collagen alpha chains, as well as a type II collagen determinant incorporated in type I collagen, were not presented by Langerhans cells. In addition, granulocyte/macrophage colony-stimulating factor-expanded blood dendritic cells displayed the same biased antigen presentation, suggesting that the inability to present collagen is not restricted to dendritic cells localized in epidermis. B cell-deficient mice could prime a type II collagen-reactive T cell response, thus excluding B cells as obligatory antigen-presenting cells for the priming of collagen-reactive T cells. We suggest that neither Langerhans cells nor B cells, but macrophages are the primary antigen-presenting cells in the immune response towards type II collagen.

Amino Acid Sequence↗
PubMed1994

Interaction of enteropathogenic Yersinia enterocolitica with complex basement membranes and the extracellular matrix proteins collagen type IV, laminin-1 and -2, and nidogen/entactin.

The plasmid-encoded virulence factor yersinia adhesin A (YadA) contributes to pathogenicity of Yersinia enterocolitica which might be related to its adhesive potential. Therefore, we have investigated the interaction of Y. enterocolitica with basement membrane (BM) and with the major BM proteins collagen type IV, laminin, and nidogen/entactin. Recombinant YadA-positive but not YadA-negative yersiniae bound specifically to lens capsule BM tissue, as well as purified collagen type IV and the laminin-1 and -2 (formally known as merosin) isoforms. Binding sites are located on the alpha 1 chain of the 58-nm amino-terminal 7sL fragment of collagen type IV and on the elastase-fragment E1 of laminin-1. YadA-mediated binding of yersiniae to collagen type IV was rapid and saturable, it was independent of divalent cations, stable over a wide pH range, and not influenced by higher salt concentrations. D-Glucose and D-galactose did not interfere with binding, indicating a protein-protein interaction. In contrast, adhesion of yersiniae to the laminin-2 isoform occurred also independent of YadA expression and no binding was observed to nidogen/entactin. The results support the hypothesis that adhesion of Y. enterocolitica could contribute to pathogenicity of enteropathogenic yersiniae. Further definition of binding sites for YadA on BM proteins might allow determination of the relevance of Yersinia-BM interactions to infection.

Adhesins, Bacterial↗
PubMed1994

T cell recognition of carbohydrates on type II collagen.

A critical event in an immune response is the T cell recognition of peptides bound to major histocompatibility complex (MHC) molecules on the surface of an antigen presenting cell (APC). Although the majority of eukaryotic proteins are glycosylated, it has not yet been shown that T cell recognition of such proteins involves recognition of the bound carbohydrates. Type II collagen (CII), the major protein constituent of joint cartilage, is posttranslationally modified by hydroxylation and glycosylation of lysines. In this report we show that posttranslational modifications of the immunodominant peptide CII(256-270) generate a structural determinant that is distinct from the determinant represented by the corresponding synthetic peptide. Elimination of carbohydrates, present on CII, by two different biochemical methods revealed that the carbohydrates, O-linked to the hydroxylysines within the CII(256-270) determinant, were crucial for the reactivity towards the posttranslationally modified peptide. Furthermore, a T cell hybridoma specific for the glycosylated determinant was stimulated by tryptic CII-peptides presented by fixed APCs, thus showing that the carbohydrates are involved in the trimolecular complex T cell receptor/peptide/MHC. Finally, the importance of the bound carbohydrates for the arthritogenicity of CII was investigated by comparing the development of arthritis after immunization with carbohydrate-depleted and glycosylated CII, respectively. Incidence, time of onset, and severity of the disease were significantly affected by the elimination of carbohydrates, whereas no significant difference in anti-CII antibody titers was seen.

Amino Acid Sequence↗
PubMed1994

Serum collagenase activity in chronic liver diseases.

To examine whether serum collagenase activity reflects the amount of hepatic collagenase in the fibrotic liver, we measured serum collagenase activity in 67 patients with chronic liver disease and in 26 healthy controls. Collagenase activity in serum was measured after reactivation by denaturing and dissociating the inhibitors with 3 M KSCN and 1 mM aminophenylmercuric acetate. Serum collagenase activity was 35% lower than control in chronic persistent hepatitis, 48% lower in chronic active hepatitis, 56% lower in liver cirrhosis and 68% lower in hepatocellular carcinoma. To interpret this finding of low serum collagenase activity, we measured serum concentration of TIMP (Tissue Inhibitor of Metallo-Proteinases). Serum TIMP concentration was increased as liver disease developed, and it was inversely correlated with serum collagenase activity. These results suggest that in this assay condition serum collagenase activity is influenced by TIMP, and thus may not reflect the amount of hepatic collagenase in patients with chronic liver disease.

Adult↗
PubMed1994

Regulation of transcription factors c-Myc, Max, and c-Myb by casein kinase II.

A number of transcription factors have been shown to be phosphorylated by casein kinase II (CKII). We have identified CKII phosphorylation sites in c-Myc, Max, and c-Myb which are phosphorylated in the cell. Whereas little evidence to any functional significance of the CKII sites in c-Myc has been obtained, phosphorylation of its heterodimeric partner Max alters DNA binding properties. CKII phosphorylation of Ser-2 and -11 in Max resulted in enhanced DNA binding kinetics of both Max/Max homo- and Myc/Max heterodimers without altering steady state binding. Replacing these serine by alanine residues and comparing the wild type with the mutant Max proteins in transactivation assays did not reveal any significant differences. For c-Myb mutational analysis of the CKII phosphorylation sites showed altered steady state DNA binding. Replacing Ser-11/12 by alanine residues resulted in increased DNA binding compared to wt c-Myb or Myb Asp-11/12 as demonstrated by up to 10-fold differences in the dissociation constants. In transactivation assays, the Ala mutant showed consistently an increased activity both on a synthetic and on the mim-1 promoter. A potential CKII phosphorylation site in c-Fos was not phosphorylated in vitro. Analysis with peptides demonstrated that a proline residue at position +1 relative to the acceptor serine was inhibitory.

Amino Acid Sequence↗