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Biomedical subjects

S Mouritsen

Publications and source records attributed to S Mouritsen.

At least 19 recordsLinked to original sources
PubMed2005

Phase I study of TNFalpha AutoVaccIne in patients with metastatic cancer.

We evaluated the safety and immunogencity of a novel vaccine directed against autologous TNFalpha in a Phase I fixed dose escalation trial. The vaccine consisted of two recombinant TNFalpha proteins, with specific peptides replaced by foreign immunodominant T cell epitopes from tetanus toxoid. The main objectives were to establish a safe dose and evaluate the vaccines ability to raise neutralising TNFalpha antibodies. Secondary objectives were improvements in body weight and tumour response. Thirty-three patients were vaccinated with three doses (20, 100, or 400 mug) of TNFalpha vaccine at 2-weekly intervals adjuvanted with aluminium hydroxide. Anti-TNFalpha antibody titres were measured by both a RIA, using soluble native TNFalpha as the antigen, and by an ELISA using immobilized partly denatured TNFalpha. Eleven patients (33%) had mild grade1/2 injection site reactions at the higher doses. In 10 of 20 patients, serum antibodies recognize denatured TNFalpha in the ELISA, whereas, antibody titres against native TNFalpha in the RIA were undetectable. This suggests that the production process had partly denatured the vaccine preventing the formation of cross-reacting antibodies to native TNFalpha. In conclusion, TNFalpha vaccine was able to elicit vaccine specific antibodies. However, since the antibodies were only able to cross-react with partly denatured TNFalpha, evaluation of safety and tumour responses to the TNFalpha vaccine was compromised.

Aged↗
PubMed2001

Active vaccination against IL-5 bypasses immunological tolerance and ameliorates experimental asthma.

Current therapeutic approaches to asthma have had limited impact on the clinical management and resolution of this disorder. By using a novel vaccine strategy targeting the inflammatory cytokine IL-5, we have ameliorated hallmark features of asthma in mouse models. Delivery of a DNA vaccine encoding murine IL-5 modified to contain a promiscuous foreign Th epitope bypasses B cell tolerance to IL-5 and induces neutralizing polyclonal anti-IL-5 Abs. Active vaccination against IL-5 reduces airways inflammation and prevents the development of eosinophilia, both hallmark features of asthma in animal models and humans. The reduced numbers of inflammatory T cells and eosinophils in the lung also result in a marked reduction of Th2 cytokine levels. Th-modified IL-5 DNA vaccination reduces the expression of IL-5 and IL-4 by approximately 50% in the airways of allergen-challenged mice. Most importantly, Th-modified IL-5 DNA vaccination restores normal bronchial hyperresponsiveness to beta-methacholine. Active vaccination against IL-5 reduces key pathological events associated with asthma, such as Th2 cytokine production, airways inflammation, and hyperresponsiveness, and thus represents a novel therapeutic approach for the treatment of asthma and other allergic conditions.

Animals↗
PubMed2001

Functional screening of a retroviral peptide library for MHC class I presentation.

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.

Amino Acid Sequence↗
PubMed2001

Freunds adjuvant alone is antiatherogenic in apoE-deficient mice and specific immunization against TNFalpha confers no additional benefit.

TNFalpha participates in the pathogenesis of atherosclerosis. The effect of immunization against TNFalpha on development of advanced vascular lesions in atherosclerosis-susceptible apoE-deficient mice was investigated. At 5-7 weeks of age, animals received immunization with either Freunds adjuvant and a recombinant antigenic TNFalpha molecule (TNF106), Freunds adjuvant alone, or no immunization. All mice received a Western-type high-fat diet for 12 weeks. Aortic sinus lesion area was determined by microscopic morphometry, the total aortic arch cholesterol content was determined by gas chromatography, and antibodies against TNFalpha, malondialdehyde-modified low density lipoprotein, or heat shock protein 60, were assessed by ELISAs. Immunization with TNF106 induced high-titered circulating antibodies against TNFalpha (n=23), and these antibodies were not detected in mice immunized with Freunds adjuvant alone (n=22), or in non-immunized control animals (n=25). After 12 weeks, the atherosclerotic lesion size was significantly reduced in immunized animals, whether they had been immunized with TNF106 or Freunds adjuvant alone, and the total lesional cholesterol content was decreased in mice immunized with TNF106. There were no correlations between circulating antibody titers and plaque size, total aortic arch cholesterol content, or plasma lipid levels, respectively. Administration of Freunds adjuvant alone can thus reduce formation of mature atherosclerotic lesions in apoE-deficient mice and this response is not modified by specific immunization against TNFalpha.

Animals↗
PubMed1999

A novel microtiter plate based method for identification of B-cell epitopes.

A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B-cell epitopes in two different proteins using a peptide-based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15-mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor-alpha, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non-covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor-alpha were added to identify potential linear B-cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non-covalent binding although the peptides were in fact present on the non-covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B-cell epitopes in protein antigens.

Amino Acid Sequence↗
PubMed1999

Therapeutic antibodies elicited by immunization against TNF-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is critically involved in the pathogenesis of several chronic inflammatory diseases. Monoclonal antibodies against TNF-alpha are currently used for the treatment of rheumatoid arthritis and Crohn's disease. This report describes a simple and effective method for active immunization against self TNF-alpha. This vaccination approach leads to a T-cell-dependent polyclonal and sustainable anti-TNF-alpha autoantibody response that declines upon discontinuation of booster injections. The autoantibodies are elicited by injecting modified recombinant TNF-alpha molecules containing foreign immunodominant T-helper epitopes. In mice immunized with such molecules, the symptoms of experimental cachexia and type II collagen-induced arthritis are ameliorated. These results suggest that vaccination against TNF-alpha may be a useful approach for the treatment of rheumatoid arthritis and other chronic inflammatory diseases.

Animals↗
PubMed1997

T-cell recognition of tumor-associated carbohydrates: the nature of the glycan moiety plays a decisive role in determining glycopeptide immunogenicity.

Aberrant glycosylation is one of the most constant traits of the malignant cell phenotype. To study T-cell responses to tumor-associated glycans, the mouse hemoglobin-derived decapeptide Hb(67-76), which binds well to the MHC class II molecule E(k) and is nonimmunogenic in CBA/J mice, was either O- or N-glycosylated at its primary T-cell receptor contact residue, position 72, with different glycans attached to either threonine, serine, or asparagine. The carbohydrate moieties included tumor-associated mucins, i.e., the Tn and T antigens, mucin-related glycans, and mucin-unrelated glycans. The side chain of the amino acid in position 72 points away from the MHC binding site when the Hb(67-76) peptide is bound to E(k), so the assumption was that this was also the case for glycans attached to this position. The glycosylated Hb(67-76) peptide analogues were then studied for binding to E(k) and for immunogenicity in CBA/J mice. All 16 glycopeptides bound well to E(k), although those with the more complex carbohydrates bound more weakly than those with monosaccharides. Six of 12 O-glycosylated and 0 of 4 N-glycosylated glycopeptides were able to induce a T-cell proliferative response with a stimulation index above 3.0. Some glycopeptides were not immunogenic, suggesting that there may be holes in the T-cell repertoire due to a lack of T-cell receptor regions accommodating certain glycan structures. The four strongest immunogenic glycopeptides were all O-glycosylated, and interestingly, three of them carried the tumor-associated Tn or T antigen. On the other hand, the Hb(67-76) peptide analogue with the natural mucin Core2 structure attached did not elicit any T-cell response. T cells primed to a glycopeptide with a simple glycan structure such as Tn did not cross-respond significantly to other glycopeptides, indicating a high degree of carbohydrate specificity in T-cell recognition. T cells primed to a glycopeptide carrying the more complex T antigen showed a complicated pattern of cross-responses to glycopeptides with simpler glycan moieties. The fact that it is possible to raise MHC class II-restricted T-cell responses against tumor-associated carbohydrate structures opens new perspectives for the designing of cancer vaccines.

Animals↗
PubMed1997

Carbohydrate and peptide specificity of MHC class II-restricted T cell hybridomas raised against an O-glycosylated self peptide.

MHC class II E(k)-restricted, IL-2 secreting T cell hybridomas were raised against the synthetic glycopeptide Hb(67-76)-alpha-GalNAc, (T72(Tn)), in CBA/J mice (H-2(k)). The fine specificity of the hybridomas against the glycan moiety was investigated by testing their response against a panel of Hb(67-76)-derived glycopeptides, all with a glycan attached to serine or threonine at the position 72 in the peptide, but with different glycans. The hybridomas showed a high degree of specificity for the alpha-GalNAc moiety with few and faint cross-responses to the glycopeptides having other glycans attached even though some of these were structurally very similar to alpha-GalNAc. The fine specificity of the hybridomas for the peptide moiety was investigated by testing their responses to a panel of Hb(67-76)-alpha-GalNAc glycopeptides with alanine substitutions at all positions except at the two MHC binding anchor positions, I68 and K76, and the T72 to which the alpha-GalNAc was attached. Glycopeptides substituted with alanine at positions where the amino acid side chain pointed toward the TCR did not stimulate the hybridomas, whereas glycopeptides substituted with alanine at positions orientated down into the MHC binding groove stimulated many of the hybridomas. These results indicate that the glycan attached to the peptide as well as solvent-accessible parts of the peptide are recognized with a high degree of specificity by the T cells, whereas the parts of the peptide buried in the MHC binding site are less important or totally ignored by the T cells.

Animals↗
PubMed1997

Constrained glycopeptide ligands for MPRs. Limitations of unprotected phosphorylated building blocks.

A new methodology for the synthesis of cyclic and phosphorylated glycopeptide templates was developed. First, fully protected building blocks containing mannose and mannose disaccharides with bis-trichloroethyl phosphate on Fmoc-Thr-OPfp were synthesized. These were used in solid-phase assembly through side chain anchoring of glycosylated hexa- and octa-peptides protected at the C-terminal carboxylate as the allyl ester. Selective allyl ester cleavage and head-to-tail cyclization under pseudodilution conditions gave a high yield of pure cyclic peptide templates. Unprotected phosphate in the building block was evaluated as an alternative to the problematic trichloroethyl group. It was found that one unprotected phosphate is readily incorporated, whereas the second unprotected phosphorylated building block react very slowly due to electrostatic repulsion in the solid-phase synthesis. For comparison with previous binding studies modified glycopeptide templates containing only phosphorylated mannose monosaccharides or templates modified in the peptide part were synthesized. All the structures were tested for their binding to the mannose 6-phosphate receptor, and it was found that although mannose disaccharides are required for optimal interaction, the detailed structure of the peptide template has a strong influence on binding to the receptor. The restricted conformations of the cyclic peptides decreased the binding considerably.

Chromatography, High Pressure Liquid↗
PubMed1996

Breaking of B cell tolerance toward a highly conserved self protein.

Self proteins are processed and presented by APCs in the same way as foreign proteins. Presentation of fragments derived from self proteins does not, however, lead to Th cell stimulation because of T cell tolerance. In this study, a novel approach was used to investigate whether B cell tolerance toward a self Ag could be due to the absence of this Th cell recognition. The highly conserved nonimmunogenic protein ubiquitin was used as a model protein. Two modified ubiquitin molecules were constructed with ubiquitin segments exchanged either with the T cell epitope, OVA(325-336), which binds to the mouse A(d) MHC class II molecule, or with the T cell epitope, hen egg lysozyme(50-61), which binds to the A(k) molecule. Mice were immunized with the resulting proteins. Both modified proteins elicited strong autoantibody responses toward soluble native ubiquitin, demonstrating that insertion of a single foreign T cell epitope can overcome the B cell nonresponsiveness. The T cell regulatory role of one of the inserted foreign T cell epitopes in ubiquitin was studied, and at least two different Th cell specificities were found to operate in the response. The T cells were directed against: 1) the inserted epitope, and 2) a combination of the inserted epitope and parts of the neighboring ubiquitin regions. Therefore, the absence of T cell help seems to be an important reason for B cell tolerance toward self proteins.

Amino Acid Sequence↗
PubMed1996

T cell recognition of Tn-glycosylated peptide antigens.

The mouse hemoglobin-derived decapeptide Hb (67-76), VITAFNEGLK, which binds well to Ek and is non-immunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn (alpha-D-N-acetylgalactosamine, or alpha-D-GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn-glycosylated peptides were then studied for binding to Ek and for immunogenicity in CBA/J mice. All of those glycopeptides which had the Tn attached to serine or threonine at a position in the peptide where, according to the crystal structure determinations, the amino acid side chain was oriented downwards into the binding site of the major histocompatibility complex (MHC) molecule, completely lost their capacity for binding to Ek. This was the case for the glycopeptides with Tn attached at position 68 and 76, which are the major anchor residues and for those with Tn attached at position 71 and 73, which function as secondary anchor residues. Those glycopeptides which had Tn attached to serine or threonine at positions where the side chain pointed away from the binding site maintained their capacity for binding to Ek, except for those with Tn attached at position 70 and 74. Furthermore, some of the MHC-binding glycopeptides were immunogenic. In particular, this was the case for the glycopeptide with Tn attached to the central position 72 in the decapeptide. From previous studies, this is known to be the dominant T cell receptor contact residue of Hb (67-76). The results suggest that T cells may be capable of recognizing epitopes which are partially defined by a small glycan group.

Amino Acid Sequence↗
PubMed1996

Methylcholanthrene-induced sarcomas in nude mice have short induction times and relatively low levels of surface MHC class I expression.

In order to study the role of the T-cell-mediated immune defense in tumor development, a total of 93 sarcomas were induced using different doses (8 micrograms (0.1%), 40 micrograms (0.5%) and 400 micrograms (5%)) of 3-methylcholanthrene in athymic nude Balb/c mice and phenotypically normal immunocompetent Balb/c mice. A shorter tumor induction time and a higher tumor incidence after treatment with low doses of methylcholanthrene were seen in nude mice than in immunocompetent mice, indicating that they have a lower resistance to the carcinogen. Contrary to expectations we found that the MHC class I expression of tumors from nude mice was lower than that of tumors from normal mice. Higher surface expression of MHC class I was demonstrated on high dose tumors from normal mice than on low dose tumors from normal mice. The cellular composition of the individual tumors raised in nude mice was more heterogeneous with respect to MHC class I expression. Since the mice differ genetically only with respect to the nu gene, these results indicate that a lack of T-cell-mediated defense mechanisms may confer upon the bearer a lower resistance to 3-methylcholanthrene and a different MHC profile of the ensuing tumor.

Animals↗
PubMed1995

Hydrocoating: a new method for coupling biomolecules to solid phases.

Solid-phase immunoassays such as enzyme-linked immunosorbent assays require one of the assay components to be immobilized. Most frequently this is achieved by passive adsorption of the antigen or antibody to a hydrophobic polymer surface composed of, e.g., polystyrene. Alternatively the biomolecule can be bound indirectly via passively adsorbed carrier proteins or directly via functional groups on the solid phase using cross-linking agents. Here we describe a new technique--hydrocoating--for covalent immobilization of biomolecules, such as peptides, in highly hydrophilic surroundings. Peptides were immobilized on microtiter plates via covalent bonds to an activated hydrophilic polymer. Soluble dextran was activated using 2,2,2-triflouroethanesulphonyl chloride (tresyl chloride) leading to activation of hydroxyl groups on the dextran polymer. This activated dextran molecule was immobilized on a surface containing amino groups leaving a sufficient number of active groups for secondary binding of other biomolecules. Peptides, that were either undetectable or poorly recognized when adsorbed on polystyrene, were readily recognized when immobilized by the hydrocoating technique. Furthermore, peptides immobilized by this method were recognized 5-10-fold better compared to the same peptides immobilized covalently on a surface containing secondary amino groups. The technique appears to provide an alternative to passive adsorption of biomolecules on solid phases and may be useful in the future development of immunoassays.

Amino Acid Sequence↗
PubMed1994

Attachment of oligosaccharides to peptide antigen profoundly affects binding to major histocompatibility complex class II molecules and peptide immunogenicity.

To investigate the immunogenicity of glycopeptides, a peptide fragment from hen egg lysozyme, HEL(81-96)-Y (here named 1) which is immunogenic in H-2k mice and known to bind to the murine major histocompatibility complex (MHC) class II molecule Ek, was synthesized in five different glycosylated forms. The N-terminal serine of HEL(81-96)-Y was derivatized with D-glucose (2), maltotriose (3), and a branched D-glucose pentasaccharide (4). Furthermore, 1 was prepared with a central serine or asparagine derivatized with the branched D-glucose pentasaccharide (5) and GlcNAc (6), respectively. The ability of the five glycopeptides and the non-glycosylated peptide, labeled with 125I, to bind to the two MHC class II molecules, Ak and Ek, was studied using a gel filtration assay. None of them could bind to Ak. Neither 5 nor 6 were able to bind to Ek. Surprisingly 2, 3 and 4 bound better to Ek than did the non-glycosylated peptide 1. The increased binding varied depending on the type of oligosaccharide attached to the N terminus of the peptide. The better binding to Ek of glycopeptide 4 was found to be due to an increased association rate. The binding of 1 as well as 4 was optimal at pH 5.0. Functional studies showed that 4 was able to elicit a heteroclitic proliferative response from T cells of mice immunized with the native non-glycosylated peptide. Circular dichroism studies of 1 and 4 indicated a more unordered structure of 4 and a predominant alpha-helical conformation of 1, suggesting that the MHC class II molecule may bind to peptides which are in a non-alpha-helical conformation. These results demonstrate that glycosylation has considerable influence on peptide immunogenicity for T lymphocytes.

Amino Acid Sequence↗
PubMed1994

MHC class II-bound self-peptides can be effectively separated by isoelectric focusing and bind optimally to their MHC class II restriction elements around pH 5.0.

More than 90% of the major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APC) have in their binding site a peptide derived from an extracellular protein ingested by the APC or from a protein of the APC itself. These self-peptides can be eluted from affinity-purified MHC class II molecules by acid elution, and have been studied with a variety of techniques. We show here that the self-peptides eluted from the mouse MHC class II molecules Ad, Ed and Ek bind specifically to MHC class II molecules of the allelic type from which they were derived. The pH optimum for binding is around 5.0, i.e. the same optimum at which synthetic peptides representing sequences of foreign antigens bind to MHC class II molecules. This suggests that the physiological compartment where MHC class II molecules bind self-peptides may be very late in the endocytic pathway. The chemical properties of the eluted and labelled MHC class II peptides were studied by isoelectric focusing. This method was able to separate the peptides very efficiently, and enabled a rapid comparison of peptides eluted from different MHC molecules. The 125I-labelled peptides displayed a broad range of isoelectric points with values predominantly below neutral. This suggests that such peptides bind to MHC in a predominantly non-charged state.

Amino Acid Sequence↗
PubMed1993

Expression of beta 2-microglobulin by premalignant epithelium.

Many human tumors express low amounts of HLA class I molecules relative to the normal cells from which they are derived. From experimental work it is clear that the malignant behavior of a tumor cell may depend on its MHC class I expression. Therefore, it is of obvious interest to study the HLA class I expression of human tumors in their various stages. We have studied the HLA class I expression by the cells in premalignant epithelial lesions and invasive carcinoma of the bladder and uterine cervix using immunoperoxidase staining for beta 2-microglobulin of paraffin-embedded tissue. We here assume that beta 2-microglobulin expression by malignant and premalignant cells equals HLA class I expression. Thirty-two of the 36 invasive tumors expressed less overall beta 2-microglobulin than cells from the normal epithelium. In contrast, approximately two-thirds of 34 premalignant bladder epithelia and 47 premalignant cervix epithelia displayed higher overall beta 2-microglobulin expression than the normal epithelium. Thus, a systematic large-scale elimination of HLA class I high-expressing tumor cell variants may take place only after the tumor penetrates the basement membrane.

Carcinoma in Situ↗