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R Ullrich

Publications and source records attributed to R Ullrich.

At least 73 records · Page 4Linked to original sources
PubMed1998

All four putative ligand-binding domains in megalin contain pathogenic epitopes capable of inducing passive Heymann nephritis.

Megalin (gp330) is the main target antigen involved in the induction of Heymann nephritis (HN), a rat model of human membranous nephropathy. Its large extracellular region contains four putative ligand-binding domains separated by spacer regions. Previously, it was reported that the second ligand-binding domain (LBD II) of megalin is involved in the pathogenesis of passive HN because it is capable of binding antibodies in vivo and initiating formation of immune deposits (ID). This study explores the possibility that pathogenic epitopes might also be present in the other putative ligand-binding domains. Recombinant fragments of ligand-binding domains (LBD) I through IV expressed in a baculovirus system were used to generate polyclonal domain-specific antibodies. Antibodies raised against each of the recombinant megalin fragments reacted preferentially with its respective antigen and with whole megalin by immunoblotting. Each of the antibodies also gave a characteristic brush-border staining for megalin by indirect immunofluorescence on rat kidney. When rats were injected with the domain-specific antibodies to test their ability to produce passive HN, glomerular ID were present in kidneys of all injected animals. The staining pattern in glomeruli of rats injected with LBD I, III, or IV was similar to that obtained with antibodies to LBD II. It is concluded that passive HN can be induced with antibodies against LBD I, III, and IV, as well as LBD II, and that each of the ligand-binding domains contains a pathogenic epitope. These findings provide further evidence for the multiple epitope model of HN.

Animals↗
PubMed1998

A differential PCR system for the determination of CCND1 (cyclin D1) gene amplification in head and neck squamous cell carcinomas.

The gene for Cyclin D1 (CCND1) lies within chromosomal region 11q13 and codes for a cell cycle regulator essential for G1 phase progression. This G1-cyclin is a putative protooncogene whose clonal rearrangement and/or amplification and mRNA overexpression occurs in several types of human neoplasias, including head and neck squamous cell carcinomas. Data from the literature suggest that amplification and overexpression of the CCND1 gene could lead to destabilisation of cell cycle control mechanisms and uncontrolled cell proliferation. We developed a differential PCR system for the determination of CCND1 gene amplification in head and neck squamous cell carcinomas. A 115 bp CCND1 fragment and a 150 bp gamma-interferon fragment are amplified simultaneously in the same reaction tube under optimized conditions. Statistical analysis of amplification data obtained by differential PCR revealed excellent correlation with amplification data obtained by conventional Southern hybridization.

Blotting, Southern↗
PubMed1997

Abnormal predominance of IgG in HIV-specific antibodies produced by short-term cultured duodenal biopsy specimens from HIV-infected patients.

The aim of our study was to analyze HIV-specific humoral immunity in the intestinal mucosa at different stages of HIV infection in comparison with serum and saliva. Duodenal biopsy specimens from 30 AIDS patients and 9 HIV-infected patients without AIDS were cultured for 48 hours. Culture supernatants, as well as simultaneously obtained serum and saliva samples, were adjusted to the same immunoglobulin concentrations and tested for HIV-specific IgG and IgA by Western blot. The HIV antigen pattern differed clearly between IgA and IgG but was similar for each isotype independent of its origin (i.e., serum, saliva, or biopsy specimen supernatants). Short-term cultured duodenal biopsy specimens from HIV-infected patients at all stages produced predominantly IgG, which was broadly reactive with HIV antigens. Lower titers of HIV-specific IgA, which recognized few antigens, were found, mostly the glycoprotein gp160. At later stages of the disease compared with earlier stages, the reaction pattern of mucosal IgA from saliva and biopsy supernatants was even more restricted; secretory component was frequently absent. The abnormal predominance of HIV-specific IgG over IgA in mucosal secretions may result from abnormal antibody production in the mucosa rather than from serum leakage. Mucosal inflammation induced by HIV-IgG immune complexes and insufficient immune exclusion by secretory IgA may not only lead to increased mucosal HIV replication but may also contribute to gastrointestinal disease in HIV-infected patients.

Adult↗
PubMed1997

Pathogenic antibodies inhibit the binding of apolipoproteins to megalin/gp330 in passive Heymann nephritis.

Megalin/gp330 is an endocytic receptor that internalizes multiple ligands including apolipoproteins E (apo E) and B100 (apo B). Megalin is the main antigenic target in passive Heymann nephritis (pHN), where it binds circulating autoantibodies leading to the formation of subepithelial immune deposits (ID)-the hallmark of pHN. Apo E and apo B were found recently to accumulate within these IDs, and evidence was provided that their lipids may undergo peroxidation, causing glomerular basement membrane damage and proteinuria. Here we investigated if ID-forming antimegalin IgG can inhibit the binding and internalization of apo E-betaVLDL (very low density lipoprotein) by megalin, and lead to their accumulation within IDs. By immunoelectron microscopy, apo E and apo B were detected in clathrin-coated pits and multivesicular bodies of podocytes in control rats, suggesting that the uptake of lipoproteins is a constitutive function of the glomerular epithelium. When pHN was induced by intravenous injection of antimegalin IgG, apo E and apo B were found within IDs by immunofluorescence and immunoelectron microscopy. Bound antibodies eluted from glomeruli of rats with pHN were found to inhibit the binding and internalization of apo E-enriched betaVLDL by megalin. These results indicate that pHN-inducing antimegalin IgG is capable of interfering with the uptake of lipoproteins by megalin in vivo during the formation of IDs.

Animals↗
PubMed1997

Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions.

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.

Animals↗
PubMed1997

Detection of antibodies against viral capsid proteins of human herpesvirus 8 in AIDS-associated Kaposi's sarcoma.

Sequences of a new herpesvirus with homology to gammaherpesvirinae were recently identified in AIDS-associated Kaposi's sarcoma (KS). Subsequently this novel virus, called KS-associated virus (KSHV) or human herpesvirus (HHV) 8 was detected in classical KS and AIDS-associated body cavity based lymphomas by polymerase chain reaction. In this report major and minor capsid proteins of HHV-8 were molecularly cloned and produced as recombinant proteins in Escherichia coli. Sera from 69 HIV-1 infected patients with KS, 30 HIV-1 infected patients without KS and 106 control individuals were tested by enzyme-linked immunosorbent assay for anti-HHV-8 capsid IgM and IgG antibodies. Sera from four patients were tested over periods ranging from 18 months to 6 years. IgG antibodies directed against HHV-8 capsid antigens were detected in patients with AIDS-associated KS and in some AIDS patients without KS. Seroconversion with IgM and IgG antibodies directed against HHV-8 capsid proteins occurred more than 1 year prior to diagnosis of KS. In a considerable portion of KS patients no IgM or IgG antibodies against HHV-8 capsid proteins were detected. In these patients there was an inverse relationship between antibodies against HHV-8orf26 and the CD4/CD8 ratio, suggesting that the inconsistency of anti-HHV-8orf26 antibodies is due at least partly to an impaired immune response. No reactivity against HHV-8 capsid antigens was detected in the vast majority of sera from HIV-negative control individuals. Our findings indicate that a specific humoral immune response against capsid proteins is raised in HHV-8 infected individuals, and that anti-capsid antibodies can be used to diagnose HHV-8 infection. The correlation between occurrence of anti-HHV-8 antibodies and KS supports the hypothesis of a causative role of HHV-8.

Amino Acid Sequence↗
PubMed1997

The number and proportion of Vgamma9 Vdelta2 T cells rise significantly in the peripheral blood of patients after the onset of acute Coxiella burnetii infection.

We conducted serial studies on peripheral blood lymphocytes from four patients with acute Coxiella burnetii infection. These studies revealed that the proportion of gammadelta T cells in these patients significantly increased after the onset of disease (mean, 16%; range, 13%-30%) as compared with that in five healthy controls (mean, 4%; range, 0.5%-7%; P < .0055) and that in five controls with pneumonia (mean, 2%; range, 1%-3%; P < .0014). Most of the gammadelta T cells from these patients expressed the Vgamma9 Vdelta2 gene product. During the acute phase of disease, most gammadelta T cells expressed the activation marker human leukocyte antigen DR but not CD25. During this phase, gammadelta T cell activation was higher than alphabeta T cell activation. Our findings indicate that gammadelta T cells are predominantly involved in the acute immune response to C. burnetii.

Acute Disease↗
PubMed1997

Mucosal abnormalities in microsporidiosis.

OBJECTIVE: To determine the prevalence of microsporidiosis in HIV-infected patients with and without diarrhoea and to characterize alterations in mucosal architecture and brush border enzyme activities in patients with microsporidiosis. PATIENTS: A total of 259 HIV-infected patients undergoing oesophago-gastroduodenoscopy because of diarrhoea (n = 123) or other symptoms (n = 136) were studied. METHODS: Patients were evaluated for the presence of microsporidia by electron microscopy of duodenal biopsies. Brush border enzyme activities were measured by histochemistry and mucosal architecture was determined by three-dimensional morphometry in biopsies from patients with microsporidiosis and compared with biopsies from a subgroup of HIV-infected patients with or without other enteropathogens. RESULTS: Enterocytozoon bieneusi was detected in 17 patients and Encephalitozoon intestinalis was detected in two patients. Microsporidiosis was significantly more frequent in patients with chronic diarrhoea (19.1%; P < 0.0001) or in patients with acute diarrhoea (7.2%; P = 0.04) than in patients without diarrhoea (1.5%). Microsporidiosis was associated with lactase deficiency (P = 0.03) and a reduced activity of alkaline phosphatase (P = 0.028) and alpha-glucosidase (P = 0.025) at the basal part of the villus compared with brush border enzymes in patients without enteropathogens. Patients with microsporidia had reduced villus height (P = 0.043) and a villus surface reduced by 40% (P = 0.004) compared with patients with enteropathogens other than microsporidia. CONCLUSIONS: Our study confirms the association between microsporidia and diarrhoea. The pathophysiologic mechanism by which microsporidia cause diarrhoea appears in part to be malabsorption, caused by a reduction of absorptive mucosal surface and impairment of enterocyte function.

AIDS-Related Opportunistic Infections↗
PubMed1997

Effects of ciprofloxacin and ofloxacin on adult human cartilage in vitro.

Chondrocyte toxicity and necrosis were seen with electron microscopy after incubation of human adult cartilage biopsy specimens in ciprofloxacin or ofloxacin. In vitro exposure of chondrocytes to fluoroquinolones did not affect apoptosis as determined by flow cytometry. While the immediate clinical significance of this finding remains unclear, the possibility of long-term cartilage damage after fluoroquinolone treatment cannot be excluded.

Adult↗
PubMed1997

Comparative evaluation of modified trichrome and Uvitex 2B stains for detection of low numbers of microsporidial spores in stool specimens.

At present, the laboratory diagnosis of intestinal infections caused by microsporidia depends on the detection of the typical spores either with a modified trichrome stain (MTS) or by staining with fluorochromes. The purpose of the present study was (i) to compare staining with MTS (MTS method) and the staining with the fluorochrome Uvitex 2B (U2B method) with respect to their sensitivities and specificities, particularly in the presence of low numbers of spores, and (ii) to evaluate their reliabilities under routine laboratory conditions. First, 30 negative human stool specimens as well as 30 specimens enriched with a low concentration of microsporidial spores were examined. The U2B and MTS methods detected 27 and 30, of the positive samples, respectively (95% confidence intervals for sensitivity, 0.73 to 0.98 for the U2B method and 0.88 to 1.00 for the MTS method) without yielding false-positive results (95% confidence intervals for specificity, 0.88 to 1.00 for the MTS and U2B methods). In addition, analysis of serial dilutions of 17 stool specimens from AIDS patients containing microsporidia revealed comparable detection thresholds (P = 0.52) for both methods. Finally, 40 slides prepared from one stool specimen containing very few microsporidia and 40 negative slides were included in the routine diagnostic program during 1 month in order to monitor laboratory handling and run-to-run variations. Again, both methods exhibited comparable sensitivities (95% confidence intervals, 0.83 to 0.99 for the MTS method and 0.91 to 1.00 for the U2B method) and specificities (95% confidence intervals, 0.91 to 1.00 for the MTS and U2B methods). In conclusion, MTS and U2B methods are equally useful in the diagnosis of microsporidiosis. However, since detection thresholds for both methods differed considerably in all diluted stool specimens, performance of a combination of both methods may be more sensitive than the performance of only one procedure in the event of very low numbers of microsporidial spores.

AIDS-Related Opportunistic Infections↗
PubMed1997

The HELLP syndrome.

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Analgesia, Obstetrical↗
PubMed1996

Receptor-associated protein and members of the low density lipoprotein receptor family share a common epitope. An extended model for the development of passive Heymann nephritis.

Heymann nephritis is an experimental rat model for human membranous glomerulonephritis. Two target antigens have been identified in the proximal tubule brush border of rat kidneys. One of them is megalin, a 600-kDa membrane protein that belongs to the family of low density lipoprotein receptor (LDLR)-related proteins. The other one is receptor-associated protein (RAP), a polypeptide of 40 kDa that associates with members of the LDLR family. Here we show that antibodies produced against recombinant human RAP strongly cross-react with the chicken oocyte receptor for very low density lipoprotein and vitellogenin (LR8), and with two other members of the LDLR family, LDLR-related protein and megalin. The interaction of this antibody with LR8 showed binding characteristics exactly as those demonstrated for the physiological ligands of this receptor, in that binding of the antibody: (i) is Ca2+-dependent; (ii) is abolished by unfolding of the cysteine-rich binding domain by reduction; and (iii) interferes with the binding of very low density lipoprotein and vitellogenin. Immunopurification of the LR8-specific subpopulation of the polyclonal antiserum yielded an IgG fraction strongly reacting with LR8 as well as with RAP. Using recombinant fragments of RAP and peptide mapping, the cross-reacting epitope(s) could be narrowed down to three short sequences (5-7 residues) in the COOH-terminal part of the protein. After immunization with RAP, anti-LR8 antibodies and anti-RAP antibodies arise simultaneously, indicating that the receptor-specific activity is not due to anti-idiotypic antibodies. These findings suggest the existence of a common epitope(s) on RAP and members of the LDL receptor family. Based on these results, we present an extended molecular model for the development of passive Heymann nephritis.

Animals↗
PubMed1996

Mapping rat megalin: the second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis.

Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.

Amino Acid Sequence↗
PubMed1996

Induction of passive Heymann nephritis with antibodies specific for a synthetic peptide derived from the receptor-associated protein.

Passive Heymann nephritis (pHN) is an experimental rat model for human membranous glomerulopathy. In pHN, the formation of subepithelial immune deposits (ID) involves as antigenic targets the membrane glycoprotein gp330/megalin and the 44-kD receptor-associated protein (RAP). A single binding site for ID- inducing antibodies (Abs) was previously mapped to the 86 NH2-terminal amino acids of RAP (RAP1-86). To further narrow this epitope, Abs eluted from the glomeruli were immunoblotted on membranes that were loaded with overlapping synthetic peptides representing the amino acid sequence of RAP (SPOTs system). Two adjacent Ab-binding domains with the sequences PVRLAF, (amino acids 39-44) and HSD-LKIQE (amino acids 46-53), which were separated by a single L residue at amino acid 45, were detected. Rabbit Abs raised against synthetic peptides containing these domains individually (P31-44 and P46-53) failed to procedure glomerular IDs. By contrast, Abs raised against a larger composite peptide (P31-53) induced IDs within 3d that were firmly cross linked to the glomerular basement membrane. These data suggest that Ab binding in vivo depends on the conformation of the antigenic target sequence that is preserved in the synthetic peptide P31-53, which covers the entire Ab-binding domain of RAP but not in its subdomains, P31-44 and P46-53. Collectively, these results locate the sole ID-inducing epitope of RAP to amino acids 39-53.

Amino Acid Sequence↗
PubMed1996

Intestinal T-cell lymphoma: a reassessment of cytomorphological and phenotypic features in relation to patterns of small bowel remodelling.

Intestinal T-cell lymphoma (ITCL) is an uncommon entity among primary gastrointestinal lymphomas. In this study we evaluated tumours from 20 patients presenting with (n = 8) or without (n = 12) a history of coeliac disease (CD). Neoplastic lesions were composed of predominantly small (n = 4), small-to-medium (n = 2), medium/mixed-to-large (n = 7) or large and anaplastic (n = 7) cells. Different patterns of tumour growth and remodelling of the small bowel wall were observed. Pattern a (n = 4) was characterized by an intramucosal spread of small tumour cells with a small growth fraction. This pattern resembles mucosal inflammation in CD. In pattern b (n = 2), ulcerated solitary or multiple tumours composed of small to medium-sized cells were observed. The adjacent or distant mucosa showed a nearly normal architecture. In pattern c (n = 7), ulcerated lesions were composed of medium-sized to large cells. Mucosal flattening occurred in all segments infiltrated by lymphoma. In pattern d (n = 7), bowel remodelling was observed along the small intestine even at sites not affected by lymphoma. The main neoplastic lesions were composed of pleomorphic large or anaplastic cells frequently expressing the CD30 molecule. Intramucosal spread of a small epitheliotropic T-cell population was observed in the vicinity or even at distant segments of the small bowel. The demonstration of clonal rearrangements of T-cell receptor genes helped to trace widespread occurrence of this small intraepithelial neoplastic component. We suggest that different features of tumour cells such as the expression of activation antigens may contribute to the remodelling of small bowel mucosa. The addition of immunophenotyping data to macroscopic and microscopic features of specimens provided evidence that this uncommon lymphoma exhibits a spectrum in cytological composition and growth patterns. However, despite the considerable heterogeneity of the cases analysed, most of them shared a characteristic immunohistochemical profile (CD3+, CD8+/-, CD103+), further substantiating the view that ITCL is the neoplastic equivalent of an intraepithelial T-cell subset of the small intestine. This phenotype and the intraepithelial accumulation of lymphoma cells observed in the surviving mucosa are clues to the diagnosis of this clinicopathological lymphoma entity characterized by a broad range of morphological expressions.

Adult↗