Biomedical subjects

G Riethmüller

Publications and source records attributed to G Riethmüller.

At least 145 records · Page 8Linked to original sources

PubMed1990

HLA class-I-transgenic mice as model system to study MHC-restricted antigen recognition in man.

In order to develop a possible animal model to study HLA linked diseases of man, we established HLA-B27 transgenic mice (TGM). As aberrant and overexpression of MHC molecules can be toxic for cells, we aimed at obtaining a physiological expression of the human antigen and used a genomic 25kb Sal I fragment for embryo injection, coding for the HLA-B* 2705 heavy chain. Five independent founder mice were obtained containing varying copies of the fragment (1 to 10). RNA analysis from different tissues showed an expression pattern similar to endogenous H-2 class I genes. HLA-B27 antigen could be detected on lymphocytes derived from all five founder mice, even in the absence of human beta 2-microglobulin (hu beta 2m). It was found that the presence of hu beta 2m strongly enhances HLA-B27 cell surface expression in mice with few copies of the transgene, but was not necessary for efficient and high cell surface presentation in the 10 copy line. In all HLA-B27 TGM lines, the HLA molecule functions as restriction element in anti-viral responses. In addition, we could show that T lymphocytes of the transgenic animals respond to the same HLA-B27 restricted influenza peptide as is recognized by human influenza-specific, HLA-B27 restricted cytotoxic T cell lines.

Animals↗

PubMed1990

Detection, characterization and tumorigenicity of disseminated tumor cells in human bone marrow.

Monoclonal antibodies have been used to detect tumor cells in bone marrow of patients with neuroblastoma, breast cancer, small cell lung cancer, prostatic cancer and gastrointestinal carcinoma. By comparative analysis immunocytology proved to be more sensitive than conventional cytology and histology and had the additional advantage of specificity. A positive correlation exists between the presence of tumor cells in bone marrow and the extent of the primary tumor. The proliferative potential of the micrometastatic cells was assessed by characterization of EGF and transferrin receptors, tumorigenicity was shown by xenotransplantation experiments in nu/nu mice in a few instances. First follow-up studies indicate that the presence of disseminated tumor cells in bone marrow can be taken as predicting the subsequent development of overt metastasis.

Antibodies, Monoclonal↗

PubMed1989

Deposition of complement activation products on plastic-adsorbed immunoglobulins. A simple ELISA technique for the detection of defined complement deficiencies.

The activation of complement components in human serum has been studied using immunoglobulins adsorbed to microtiter plates. The sequential deposition of complement fragments was detected by a series of mono- and polyclonal antibodies in an indirect enzyme-linked immunosorbent assay (ELISA). Antibodies against C1q, C1s, C4b/d, C3b/d, factor B, C5b-9 membrane attack complex (MAC), the regulatory complement proteins C4 binding protein (C4bp) and properdin were reactive. Several lines of evidence suggest that complement activation was via the classical pathway: (1) complement activation was highly isotype-restricted with regard to the adsorbed Igs (human IgG1 and IgG3 as well as mouse IgM, IgG2a and IgG2b isotypes are strong activators in contrast to human IgG2, IgG4, IgA and mouse IgG1); (2) Ca2+ depletion, heat treatment (56 degrees C for 45 min), incubation with 0.5 M KSCN or heat-aggregated immunoglobulins (aggIgG) abrogated serum activity; (3) complement deficient sera (C1q def', C2 def', C6 def' human sera; C2 def', C4 def' guinea pig sera) showed impaired deposition of the complement components that follow the missing component in the cascade of activation. In a clinical study sera from patients with systemic lupus erythematosus (SLE) were investigated in order to measure the effect of hypocomplementemia due to complement consumption. The results obtained suggest that this new and simple assay is well suited for (1) the detection of various inherited complement deficiencies, (2) the semiquantitative evaluation of sera with decreased complement levels, (3) a more detailed study of complement components bound to a solid phase.

Adsorption↗

PubMed1989

Zn++ inhibits both tumor necrosis factor-mediated DNA fragmentation and cytolysis.

The cellular events involved in direct tumor-cell destruction by tumor necrosis factor (TNF) are still incompletely understood, but a role of endonucleases has been suggested. In the present study we have analyzed in detail the effect of Zn++, an inhibitor of endonucleases, on TNF-mediated DNA-fragmentation and on cytolysis in Actinomycin-D-pre-treated WEHII 64-S cells. Beginning 2 hr after addition of TNF, a rapid degradation of cellular DNA is observed, as evidenced by release of 3H-Thymidine (TdR) label from nuclei into cytoplasm. TNF-mediated lysis of WEHII64-S cells begins at 3 hr and reaches plateau levels at 7 hr. Addition of Zn++ to TNF-treated WEHII64 cells completely abrogates DNA fragmentation at ImM. Of greater importance is the fact that Zn++ treatment also completely blocks TNF-mediated cytolysis of the target cells. Concentrations-between 0.1 and I mM ZnSO4 prevent cell death, as assessed by chromium-release and Trypan blue dye exclusion. In addition, ZnCl2, but not other divalent cations like CaCl2, MgSO4 and CuSO4 in the same concentration range, prevents cell death as well, demonstrating that the effect in fact is mediated by Zn++. Zn++ added 2 hr after TNF treatment, still effectively inhibits cell lysis, indicating that it acts at a late stage after binding of TNF to its receptor. Our data suggest that activation of endonucleases is not an accompanying effect but an essential step in TNF-mediated tumor-cell destruction.

Animals↗

PubMed1989

[Bone marrow micrometastases in colorectal cancers].

To date, the detection of individual micrometastases or disseminated tumor cells has not been possible with conventional diagnostic procedures. With the aid of new immunocytochemical investigation, however, histogenetically different cells can now be detected in the bone marrow. The monoclonal antibody, cytokeratin component 18, is most suitable for the detection of micrometastases from colorectal carcinoma. In 22 out of 82 patients with colorectal carcinoma, we were able to detect CK2-positive cells. The incidence of these "micrometastases" is represented as a function of primary tumor size, lymph node involvement, distant metastasis, and degree of malignancy of the tumor, age and sex of the patient, and tumor localization.

Adult↗

PubMed1989

Downregulation of tumor necrosis factor expression in the human Mono-Mac-6 cell line by lipopolysaccharide.

Mono-Mac-6 cells, but not U937 cells, can be induced to rapidly express tumor necrosis factor (TNF) mRNA and protein when triggered with lipopolysaccharide (LPS) at 1 microgram/ml. Preincubation of the cells for 3 d with low amounts of LPS (10 ng/ml) results in nearly complete suppression of TNF secretion. This downregulation appears to occur at the pretranslational level since specific mRNA is virtually undetectable under these conditions. By contrast, the same preincubation with 10 ng/ml LPS results in enhanced phagocytosis (28.6-67.2% for Staphylococcus aureus), demonstrating that not all monocyte functions are suppressed. While these results show that only stringent exclusion of LPS from culture media allows for induction of TNF in the Mono-Mac-6 cell line, the pronounced effect of LPS preincubation may also provide a suitable model with which to study the mechanisms of LPS-induced desensitization.

Blotting, Northern↗

PubMed1989

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on collagen synthesis of human skin fibroblasts in vitro.

The effect of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) on collagen metabolism by human diploid fibroblasts in confluent monolayer culture was examined. Recombinant TNF alpha reduced collagen mRNA levels 2-fold and stimulated collagenase mRNA levels 5-fold, while recombinant IFN gamma affected only collagen mRNA levels. The combination of TNF alpha (10 ng/ml) and IFN gamma (100 ng/ml) resulted in a much stronger (about 30-fold) reduction of collagen mRNA levels indicating that the two cytokines act synergistically. In contrast no such synergism was observed with respect to collagenase mRNA levels. The effect of TNF alpha and IFN gamma on collagen metabolism reported here indicates a complex interaction of different cytokines in the control of tissue remodeling that occurs during inflammation, repair, or atrophy.

Adult↗

PubMed1989

The use of fusion proteins to study HLA-B27-specific allorecognition.

Potential antigenic regions of the various external domains of the HLA-B27 antigen were expressed as fusion proteins in bacterial hosts and analyzed for their ability to induce humoral and cellular responses. Monoclonal antibodies directed against the proteins recognized monomorphic determinants of denatured HLA-antigens, but not B27-antigens expressed by intact lymphocytes. T-cell proliferation and IL-2 secretion were induced with a fusion protein representing regions of the first and second domains around amino acid residue 114. None of the fusion proteins stimulated cytotoxic T-lymphocytes (CTL) in an HLA-specific manner, although several included those amino acid sequences thought to be important for CTL recognition.

Animals↗

PubMed1989

Identification of a sulfoglycolipid epitope shared by cells of neuroectodermal and hematopoietic origin.

Monoclonal antibody (mAb) SNH.1 detects an epitope which is restricted to cells of neuroectodermal and hematopoietic origin. The mAb was obtained by immunization of a mouse with liposomes containing a crude extract of human melanoma acidic glycolipids. The SNH.1 antigen isolated from melanoma was identified as a sulfated glycolipid, closely related or identical to sulfogalactosyl-ceramide. When tested with different lipids, mAb SNH.1 reacted as well with other sulfoglycolipids. The staining of mAb SNH.1 is restricted to the cytoplasm and often localized to the perinuclear region. Therefore, the SNH.1 mAb epitope may be detectable only during the biosynthesis of sulfoglycolipids.

Animals↗

PubMed1989

Anti-CD4 antibody treatment of patients with rheumatoid arthritis: I. Effect on clinical course and circulating T cells.

Eight patients with arthritis (seven with rheumatoid, one with psoriatic arthritis) were treated for 7 d with a daily injection of 10 mg of mouse monoclonal anti-CD4 antibodies (three with VIT4, five with MT151). With the exception of a short-lasting low-grade fever in one patient, no side effects were observed. Clinical symptoms (morning stiffness, number of swollen joints, pain assessment and Ritchie articular index) improved in all patients within 7 d of treatment. Improvement lasted from 3 weeks to greater than or equal to 5 months (mean approximately 11 weeks). Rheumatoid factors, immune complexes and other laboratory parameters did not change during or after treatment. Skin reactivity to recall antigens was suppressed in four out of six patients during treatment but returned to pretreatment levels within 6 weeks. Immunofluorescent analysis revealed a short-lasting drop of T cells, mainly of the CD4+ CDw29+ subset, but monocytes were also affected. The injected antibody was detectable on circulating cells for about 10 h. Within 20-24 h, the cell distribution returned to pretreatment levels. In six out of eight patients an anti-mouse-Ig response was seen. We conclude that mouse anti-CD4 monoclonal antibody (MoAb) treatment is well tolerated and that the cellular immunological changes observed are short-lasting. The low incidence of side effects may justify further clinical studies to evaluate the clinical efficacy of such treatment.

Antibodies, Anti-Idiotypic↗

PubMed1989

Anti-CD4 antibody treatment of patients with rheumatoid arthritis: II. Effect of in vivo treatment on in vitro proliferative response of CD4 cells.

In treating rheumatoid arthritis (RA) patients with anti-CD4 antibodies (MT151 10 mg/day, 7 d) we observed diminished skin-test response to recall antigens and reduced proliferative response to antigens and mitogens (in vitro) 2-4 h following the injection. To investigate whether this diminished response is due to functional impairment of CD4+ cells, we analyzed the proliferative response of CD4+ cells to various stimuli, in conditions where the CD4 structure was either cross-linked or not. We found that in vivo anti-CD4-antibody-coated cells could be induced to proliferate if the anti-CD4 antibody was cross-linked with an anti-CD3 antibody [BMA030-F(ab')2] added in vitro and per se non-mitogenic. Also, without cross-linking the cell-bound anti-CD4 antibodies, no impairment of the proliferative capacity of CD4-antibody-coated cells could be detected; double fluorescence analyses of phytohaemagglutinin (PHA)- or CD3-stimulated cells showed that a similar proportion of CD4+ cells proliferated before and after anti-CD4-antibody treatment. The diminished proliferation observed following anti-CD4 treatment correlated to the reduced number of CD3+ and CD4+ cells in culture, which suggests that altered cell distribution might be one factor contributing to the impaired immune response.

Antibodies, Monoclonal↗

PubMed1989

Classical pathway of complement activation in normal and diseased human glomeruli.

Monoclonal antibodies (mAb) reactive against complement components involved in the classical activation pathway were applied in an indirect immunoperoxidase technique for the histological study of normal and diseased human renal tissues. Prominent staining with antibodies against the C4d fragment was seen in all glomeruli and some renal arteriolar walls. The C4d staining was mesangial with light microscopy, whereas the subendothelial site of the glomerular basement membrane (GBM) also appeared to be positive in immunoelectron microscopy. In similar localization, albeit with distinctly weaker intensity, IgM and C4 binding protein (C4bp) were detected. In kidney biopsies from patients with various types of glomerulonephritis, C4d reactive antibodies stained the glomerular structures in a strong, diffuse or granular pattern in contrast to the more segmental distribution and weaker staining intensity in normal kidney specimens. Increased amounts of C4d, occasionally also of C4b, were paralleled in diseased kidney tissues by distinct deposits of IgM and/or IgG in the presence of C4bp. This study suggests that the C4d fragment in normal human glomeruli is indicative of a continuous, local complement activation via the classical pathway induced by the physiological deposition of IgM-containing immune complexes.

Animals↗

PubMed1989

De novo expression of intercellular-adhesion molecule 1 in melanoma correlates with increased risk of metastasis.

The 89-kDa cell surface glycoprotein, P3.58, is detectable on advanced human melanomas in situ but not on benign melanocytes or early melanomas. cDNA cloning of P3.58 from melanoma cells was accomplished by screening a lambda zap expression vector library with monoclonal antibodies produced against the denatured antigen. Nucleotide sequencing of the clones revealed that P3.58 is identical to the intercellular-adhesion molecule 1. No qualitative differences in P3.58 mRNA species could be seen between melanoma cells and hematopoietic cells and no differences in gene organization were observed between peripheral blood leukocytes and melanoma cells. Inspection of the deduced amino acid sequence of P3.58 indicated the presence of the consensus sequence characteristic for complement-binding proteins. The acquisition of this cell-adhesion molecule during the process of tumor progression is speculated to contribute to the development of metastasis in melanoma.

Amino Acid Sequence↗

PubMed1989

MUC18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily.

The MUC18 antigen is an integral membrane glycoprotein of 113 kDa whose expression on primary human melanomas correlates with poor prognosis and the development of metastatic disease. MUC18 is expressed only sporadically in benign melanocytic nevi and thin primary melanomas that have a low probability of metastasizing. However, with increasing tumor thickness, MUC18 expression becomes more frequent and it is found on 80% of advanced primary tumors and metastases. MUC18-encoding cDNA clones were obtained by screening a human melanoma phage lambda expression library with monoclonal antibodies produced against the denatured antigen. The deduced sequence of 603 amino acids consists of a signal peptide, five immunoglobulin-like domains, a transmembrane region, and a short cytoplasmic tail. The highest sequence similarity is with a group of nervous system cell adhesion molecules, which includes neural cell adhesion molecule (N-CAM). The close structural relationship with these molecules suggests that MUC18 may also be a developmentally regulated cell adhesion molecule.

Amino Acid Sequence↗

PubMed1989

Monoclonal antibody MACG1 distinguishes between different molecular species of the ganglioside GM3.

The present study investigates the chemical structure of a ganglioside, detected by monoclonal antibody (MAb) MacG1, which reacts with intracytoplasmic granules of tumor-infiltrating macrophages. The results obtained by enzymatic hydrolysis and fast-atom bombardment-mass spectrometry reveal that MAb MacG1 reacts with a subcomponent of the ganglioside GM3 found in melanoma and bovine brain. MAb MacG1 might be a powerful tool to distinguish among GM3 species and could help to define their possibly different biological functions.

Animals↗

PubMed1989

Expression and immunogenicity of HLA-B27 in high-transfection recipient P815: a new method to induce monoclonal antibodies directed against HLA-B27.

The immunization of a (BALB/c x C57BL/6) FI mouse with murine transfectants expressing the HLA-B27 antigen resulted in a panel of polymorphic monoclonal antibodies with specificity for HLA-B27 and some additional HLA-antigens. Specificity of the antibodies was defined by cytofluorometric analysis on a panel of lymphoblastoid cell lines (LCL) derived from HLA typed individuals. Three of these antibodies are cytotoxic, and one of them inhibits B27-specific T cell cytotoxicity. Our results indicate that HLA-class I transfectants could be used to generate polymorphic antibodies, and that these antibodies may be helpful for HLA typing and for definition of epitopes recognized by T cells.

Animals↗

PubMed1989

Biosynthesis of complement C4 messenger RNA in normal human kidney.

Complementary DNA (cDNA) probes were used to investigate the extrahepatic production of the major histocompatibility complex (MHC)-linked complement components C4, factor B and C2 in various normal human tissues. The presence of the corresponding messenger RNA (mRNA) was tested by Northern blot analysis. Complement C4 mRNA was found in liver, and with high intensity also in normal kidneys. In contrast, no C2 mRNA and only very low amounts of factor B mRNA could be detected in the kidney. Slot blot hybridization was performed to quantitate the amount of C4 mRNA, and the intensity of C4 mRNA hybridization in the kidney samples was about 25% compared with liver RNA. C4-specific transcripts were not present in isolated glomeruli but in the renal interstitium. Other human tissues, such as tonsil, spleen, thymus, brain, lung and peripheral mononuclear cells, contained no C4 mRNA. Low amounts of C4 mRNA were found in colon, thyroid gland, lymph node and breast carcinoma. The results obtained with lung, where C2 mRNA was found but no C4 mRNA, further indicate an independent, tissue-specific regulation of the class III gene expression. The results, showing that the complement C4 genes are transcribed very efficiently in normal human kidney, suggest a direct role of complement C4 in renal pathogenesis.

Adult↗

PubMed1989

[Step-wise expression of melanoma-associated antigens in tumor progression].

Selection of monoclonal antibodies for differential reactivity with benign and malignant melanocytic lesions has led to the identification of molecules which may be involved in the development of metastases. Based on the observed alterations in the antigenic profile we propose a scheme representing the tumor progression of melanocytes to metastatic melanoma.

Antigens, Neoplasm↗