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Biomedical subjects

L Fugger

Publications and source records attributed to L Fugger.

At least 37 records · Page 2Linked to original sources
PubMed1999

A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell receptor.

Multiple sclerosis (MS) is a complex chronic neurologic disease with a suspected autoimmune pathogenesis. Although there is evidence that the development of MS is determined by both environmental influences and genes, these factors are largely undefined, except for major histocompatibility (MHC) genes. Linkage analyses and association studies have shown that susceptibility to MS is associated with genes in the human histocompatibility leukocyte antigens (HLA) class II region, but the contribution of these genes to MS disease development less compared with their contribution to disorders such as insulin-dependent diabetes mellitus. Due to the strong linkage disequilibrium in the MHC class II region, it has not been possible to determine which gene(s) is responsible for the genetic predisposition. In transgenic mice, we have expressed three human components involved in T-cell recognition of an MS-relevant autoantigen presented by the HLA-DR2 molecule: DRA*0101/DRB1*1501 (HLA-DR2), an MHC class II candidate MS susceptibility genes found in individuals of European descent; a T-cell receptor (TCR) from an MS-patient-derived T-cell clone specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 4102 peptide; and the human CD4 coreceptor. The amino acid sequence of the MBP 84-102 peptide is the same in both human and mouse MBP. Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination that led to clinical manifestations and disease courses resembling those seen in MS. Spontaneous disease was observed in 4% of mice. When DR2 and TCR double-transgenic mice were backcrossed twice to Rag2 (for recombination-activating gene 2)-deficient mice, the incidence of spontaneous disease increased, demonstrating that T cells specific for the HLA-DR2 bound MBP peptide are sufficient and necessary for development of disease. Our study provides evidence that HLA-DR2 can mediate both induced and spontaneous disease resembling MS by presenting an MBP self-peptide to T cells.

Animals↗
PubMed1999

Transgenic mouse models of rheumatoid arthritis.

A combined analysis of data available in the literature has demonstrated that the strongest association in rheumatoid arthritis (RA) is with DR genes rather than DQ or DP genes. Functional and structural data of RA-associated DR molecules suggest that selective binding of peptides is the molecular basis for this association. The establishment of functional transgenic mice expressing RA-associated HLA class II molecules has proven to be useful in the delineation of the role of these molecules in immune responses possibly related to RA and in the development of humanized models for this disease. Such humanized mice develop arthritis upon immunization with type II collagen (CII), which shows similarities with RA. Interestingly, the immunodominant T-cell determinant in CII is derived from positions 261-273, which overlap with a previously identified CII T-cell epitope restricted by the mouse Aq molecule, which is associated with collagen-induced arthritis. Studies in collagen transgenic mice have shown that recognition of this peptide may lead either to T-cell tolerance or to an arthritogenic response. It is therefore proposed that the T-cell recognition of the CII peptide bound by DR molecules is one of the molecular interactions of critical importance in the development of RA and accordingly also an important target for prevention and treatment of this disease.

Amino Acid Sequence↗
PubMed1999

Linkage and association analysis of susceptibility regions on chromosomes 5 and 6 in 106 Scandinavian sibling pair families with multiple sclerosis.

In the genetically homogeneous Scandinavian population, we have investigated chromosome 5 and the HLA (human leukocyte antigen) region on chromosome 6p21 by applying linkage and association analyses on 106 white sibling pair families with multiple sclerosis. The importance of genes within the HLA region for the susceptibility of multiple sclerosis has previously been reported. More recently, findings have suggested importance of regions on chromosome 5. Half of chromosome 5 was analyzed by using 14 microsatellite markers and a susceptibility region with a maximum LOD score of 1.1 was identified. Chromosome 6 was analyzed by HLA-DR typing and using the TNF alpha microsatellite marker. A peak maximum LOD score of 2.0 was found at the HLA-DR marker. Association studies were made for all the markers, comparing 106 probands from the sibling pairs with 100 unrelated controls. None of the markers on chromosome 5 showed significant association with multiple sclerosis, whereas strong association between multiple sclerosis and DR2 was found, with an odds ratio of 3.7 (p < 10(-5)). It is surprising that association was not seen for any of the TNF alpha alleles including the 121-bp allele, although this allele was in positive linkage disequilibrium with DR2 in both patients and controls. Our results support the existence of multiple sclerosis susceptibility loci on chromosomes 5p and 6p21.

Chromosome Mapping↗
PubMed1998

Synthetic amino acid copolymers that bind to HLA-DR proteins and inhibit type II collagen-reactive T cell clones.

Copolymer 1 [poly(Y,E,A,K)] is a random synthetic amino acid copolymer of L-tyrosine, L-glutamic acid, L-alanine, and L-lysine that is effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Copolymer 1 binds promiscuously and very efficiently to purified HLA-DR molecules within the peptide-binding groove. In the present study, YEAK and YEAK-related copolymers and type II collagen (CII) peptide 261-273, a candidate autoantigen in rheumatoid arthritis (RA), competed for binding to RA-associated HLA-DR molecules encoded by DRB1*0101 and DRB1*0401. Moreover, these copolymers (particularly YEAK, YAK, and YEK) inhibited the response of DR1- and DR4-restricted T cell clones to the CII epitope 261-273 by >50%. This direct evidence both for competitive interactions of these copolymers and CII peptide with RA-associated HLA-DR molecules and for inhibition of CII-specific T cell responses suggests that these compounds should be evaluated in animal models for rheumatoid arthritis.

Amino Acids↗
PubMed1998

Definition of MHC and T cell receptor contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice.

Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261-273 (CII 261-273). We have defined MHC and T cell receptor contacts in CII 261-273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-Abbeta0 mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints.

Amino Acid Sequence↗
PubMed1998

The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256-270 glycopeptide to H-2Aq and H-2Ap molecules.

The Aq major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2Ap molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2Aq and H-2Ap molecules (referred to as Aq and Ap) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256-270 peptide and restricted to both Aq and Ap molecules were identified. When these clones were incubated with CII protein and either Aq- or Ap-expressing antigen-presenting cells (APC), only Aq-expressing APC were able to induce stimulation. With the use of A(beta) transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256-270 peptide bound with lower affinity to the Ap molecule than to the Aq molecule. Using a set of alanine-substituted CII 256-270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the Aq and Ap molecule, i.e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslationally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256-270 peptide did not change the anchor positions used for binding to the Aq or Ap molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the Aq molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256-270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.

Amino Acid Sequence↗
PubMed1998

Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease.

The action of tissue Transglutaminase (TGase) on specific protein-bound glutamine residues plays a critical role in numerous biological processes. Here we provide evidence for a new role of this enzyme in the common, HLA-DQ2 (and DQ8) associated enteropathy, celiac disease (CD). The intestinal inflammation in CD is precipitated by exposure to wheat gliadin in the diet and is associated with increased mucosal activity of TGase. This enzyme has also been identified as the main target for CD-associated anti-endomysium autoantibodies, and is known to accept gliadin as one of its few substrates. We have examined the possibility that TGase could be involved in modulating the reactivity of gliadin specific T cells. This could establish a link between previous reports of the role of TGase in CD and the prevailing view of CD as a T-cell mediated disorder. We found a specific effect of TGase on T-cell recognition of gliadin. This effect was limited to gliadin-specific T cells isolated from intestinal CD lesions. We demonstrate that TGase mediates its effect through an ordered and specific deamidation of gliadins. This deamidation creates an epitope that binds efficiently to DQ2 and is recognized by gut-derived T cells. Generation of epitopes by enzymatic modification is a new mechanism that may be relevant for breaking of tolerance and initiation of autoimmune disease.

Amino Acid Sequence↗
PubMed1998

Cervical human immunodeficiency virus type 1 shedding is associated with genital beta-chemokine secretion.

Forty human immunodeficiency virus type 1 (HIV-1)-infected women participated in a cross-sectional study of possible correlations between chemokine receptor (CCR5 and/or CCR2B) genotype, HIV-1 RNA and DNA load, and beta-chemokine levels (RANTES, MIP-1alpha, MIP-1beta) in blood and cervix. HIV-1 nucleic acid and beta-chemokines were found in all patient blood samples and in more than half of the cervical samples regardless of CCR5 or CCR2B genotype. High beta-chemokine concentrations were in general associated with high virus loads in blood and cervix. In the blood, the proviral DNA load was significantly correlated with the MIP-1alpha concentration, whereas the DNA load in cervix was significantly associated with the MIP-1beta concentration. The cervical viral RNA load was significantly associated with levels of all three chemokines. Thus, when HIV-1 shedding was highest in the genital tract, it was associated with other combinations of beta-chemokines than virus load in blood, suggesting that local immune reactions strongly influence virus load in the cervical compartment.

Cell Line↗
PubMed1998

Distinct determinants of human immunodeficiency virus type 1 RNA and DNA loads in vaginal and cervical secretions.

The relationship between human immunodeficiency virus type 1 (HIV-1) viral RNA and proviral DNA load in vagina and cervix and that found in the plasma and peripheral blood mononuclear cells (PBMC) was investigated in 28 HIV-1-infected women. Of the patients, 64% had > or = 1 HIV-1 RNA-positive genital sample, while 71% had > or = 1 DNA-positive sample. The higher the cervical HIV load, the more widespread was the virus in the genital tract. A strong correlation was found between viral RNA load in plasma and the genital tract, whereas the association between proviral DNA load in PBMC and the genital tract was less evident. Cervical HIV-1 DNA correlated with a viral RNA load > or = 50,000 copies/mL. Cervical HIV-1 RNA levels ranged from 10% to 100% of the plasma levels. Thus, a continuous transmission risk from untraumatized genital epithelium exists in the majority of HIV-1-infected women at all stages of infection.

Blood Transfusion↗
PubMed1998

Functional characterization of HLA-DRA1*0101/DRB1*0401 molecules expressed in Drosophila melanogaster cells.

We have expressed the human MHC class II molecule, HLA-DRA1*0101/-DRB1*0401 (DRB1*0401), in Drosophila melanogaster Schneider 2 cells under control of the Drosophila metallothionein promoter. Upon induction with CuSO4, flow cytometry revealed expression of DRB1*0401 on the surface of the Drosophila cells at high levels. The membrane-bound class II molecules could present peptides specifically to DRB1*0401-restricted T cells. Drosophila-expressed DRB1*0401 molecules revealed a decreased N-linked glycosylation as compared to DRB1*0401 molecules purified from a human B-cell line. The purified DRB1*0401 molecules from Drosophila cells were dissociated into subunits in SDS-PAGE but could be stabilized with a peptide known to bind DRB1*0401 with a high affinity, indicating that the recombinant class II molecules from Drosophila cells are either empty or occupied by low affinity endogenous peptides. This assumption was further substantiated by the observation that the class II molecules from Drosophila cells had a much higher peptide-binding capacity than DRB1*0401 molecules derived from the human B-cell line. Otherwise, the two species of DRB1*0401 had similar peptide-binding specificities and affinities. The purified recombinant DRB1*0401 molecules also showed biological activity because immobilized complexes of DRB1*0401 and synthetic peptides specifically stimulated DRB1*0401-restricted T cells.

Amino Acid Sequence↗
PubMed1997

Identification of immunodominant T cell epitopes of human glutamic acid decarboxylase 65 by using HLA-DR(alpha1*0101,beta1*0401) transgenic mice.

Glutamic acid decarboxylase isoform 2 (GAD65; EC 4.1.1.15) has been identified as a key target autoantigen of insulin-dependent diabetes mellitus (IDDM). IDDM is genetically associated with the major histocompatibility complex (MHC), and particular alleles from the HLA-DQ and HLA-DR loci contribute to disease. Among DR4 subtypes, HLA-DRB1*0401, HLA-DRB1*0402, and HLA-DRB1*0405 alleles lend susceptibility, while HLA-DRB1*0403 confers protection. We have utilized HLA-DR(alpha1*0101,beta1*0401) (hereafter referred to as DR0401), human CD4, murine class II null triple transgenic mice and recombinant GAD65 to generate T cell hybridomas, and we have used overlapping sets of peptides to map the immunodominant epitopes of this autoantigen. We have identified 10 immunogenic regions for GAD65, of which 6 are recognized by multiple hybridomas. These epitopes are also generated by human antigen-presenting cells and their presentation is DR0401 restricted, as shown by the use of typed human lymphoblastoid cell lines and antibody blocking experiments. Immunodominant GAD65 epitopes defined in transgenic mice correspond to GAD65 regions previously shown to elicit T cell responses specifically in DR0401 IDDM patients, underscoring the validity of this approach. Interestingly, although the major epitopes contain DR0401 binding motifs, one of the epitopes contains a DR0405 motif.

Amino Acid Sequence↗
PubMed1997

Peptide-specific killing of antigen-presenting cells by a recombinant antibody-toxin fusion protein targeted to major histocompatibility complex/peptide class I complexes with T cell receptor-like specificity.

Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complex (MHC) complexes by the T cell receptor. Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently, phage display was used to isolate an antibody that has T cell receptor-like specificity. It recognizes mouse MHC class I H-2Kk molecules complexed with a H-2Kk-restricted influenza virus-derived hemagglutinin peptide (Ha255-262) but does not bind to class I H-2Kk alone, peptide alone, or H-2Kk complexed with other peptides. We have used this antibody to make a recombinant antibody-toxin fusion protein (immunotoxin) and show herein that it specifically kills antigen-presenting cells in a peptide-dependent manner and with T cell receptor-like specificity. We find a striking correlation between the fine specificity of binding of the antibody and the cytotoxic activity of the recombinant immunotoxin. We also show specific killing of influenza virus-infected target cells. The results suggest that it should be possible to develop novel immunotherapeutic strategies against human cancer by making recombinant antibodies that will recognize cancer-related peptides complexed with MHC class I molecules on the surface of cancer cells and using these to deliver toxins, radioisotopes, or cytotoxic drugs to the cancer cells.

ADP Ribose Transferases↗
PubMed1997

Sequencing-based typing of HLA-A locus using mRNA and a single locus-specific PCR followed by cycle-sequencing with AmpliTaq DNA polymerase, FS.

The large number (59) of alleles now known at the HLA-A locus is a serious challenge to the existing methods for HLA typing, including many of the DNA based methods. Here, we describe a sequencing-based typing (SBT) protocol for typing of HLA-A alleles using a single A-locus-specific PCR. This reaction amplifies an 824 base pair product from cDNA, prepared from mRNA, covering exons 1-3 and most of exon 4. This product allows identification of all possible combinations of two alleles from this locus. The sequencing strategy used for allele assignment contains several improvements compared to those previously published. The enzyme AmpliTaq DNA Polymerase, FS, used, combines high-quality sequencing, i.e. long reads, low background, and uniform peak heights making the identification of heterozygous positions very reliable in a fast and easy protocol developed by determining the optima for a number of variables. Thus, this strategy meets most of the requirements for the use of sequencing in HLA typing. Furthermore, this method is very flexible. The use of a PCR primer-pair tailed with the recognition sites for two different sequencing primers allows the application of the same sets of fluorescent-labelled sequencing primers regardless of the amplified locus. Thus, the protocol can very easily be extended to cover the B- and C-locus too, simply by adding PCR reactions specific for these loci to the protocol. Using this protocol, we investigated a total of 65 cell lines and clinical samples, many of the latter chosen from samples difficult to type by serology. Our method gave in all cases unambiguous results and proved functional for work requiring the highest resolution.

Alleles↗
PubMed1997

The HLA-DQ7 and -DQ8 associations in DR4-positive rheumatoid arthritis patients. A combined analysis of data available in the literature.

Several different lines of evidence have demonstrated that inherited susceptibility to rheumatoid arthritis (RA) is associated with the DRB1 genes encoding the HLA-DR4 and HLA-DR1 molecules. A contrasting hypothesis has recently been proposed, suggesting that, in general, the DRB1 locus is associated with protection to RA and that the RA-associated DRB1 alleles are not responsible for the primary disease association but merely permissive for the susceptibility conferred by the HLA-DQ alleles with which they are in linkage disequilibrium. We have performed a critical review of the literature on the HLA association in RA with special emphasis on studies in which both an HLA-DR and -DQ association has been investigated. Our analyses provide strong evidence against the hypothesis that HLA-DQ molecules play a major role in the general susceptibility to RA. Thus, the strongest association in rheumatoid arthritis is with DRB1 genes rather than DQB1 genes.

Alleles↗
PubMed1996

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide.

A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.

Carcinoma, Squamous Cell↗
PubMed1996

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.

Amino Acid Sequence↗
PubMed1996

A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells.

Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.

Amino Acid Sequence↗