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Biomedical subjects

J Engberg

Publications and source records attributed to J Engberg.

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

Equipotent in vitro actions of alpha- and beta-CGRP on guinea pig basilar artery are likely to be mediated via CRLR derived CGRP receptors.

The aim of the present study was to investigate and compare specific in vitro pharmacological actions of human alpha- and beta-CGRP applied as single concentrations to prostaglandin F2alpha precontracted segments of guinea pig basilar artery. To support the suggestion of a possible link between the pharmacological actions of alpha- and beta-CGRP and a specific receptor, we wished to determine whether mRNAs required for the expression of calcitonin receptor-like receptor (CRLR) derived CGRP receptors were present in the guinea pig basilar artery. In the pharmacological experiments we demonstrated an increase in the cAMP content by 2.5-fold and a concomitant significant vasorelaxation of the precontracted basilar artery segments following 1 min of stimulation by 10(-7) M alpha- or beta-CGRP. In another set of experiments, the time course of alpha- and beta-CGRP induced vasodilatation was investigated and concentration dependent responses of the two peptides were demonstrated. No significant differences between the actions of alpha- and beta-CGRP regarding induction of cAMP formation, amount of vasodilatation, time course of vasodilatation and mode of inhibition by the CGRP receptor antagonist, human alpha-CGRP(8-37), could be detected. The presence of mRNA encoding the guinea pig CRLR and the guinea pig CGRP receptor component protein (RCP) in the guinea pig basilar artery was demonstrated by RT-PCR methods. Furthermore, a partial sequence of mRNA encoding the guinea pig CRLR was determined. The expression in this tissue of a CRLR derived CGRP receptor and a functional RCP is therefore likely, and the equipotent pharmacological actions of alpha- and beta-CGRP might be mediated via CRLR derived CGRP receptors.

Animals↗
PubMed1999

An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype typhimurium DT104.

BACKGROUND: Food-borne salmonella infections have become a major problem in industrialized countries. The strain of Salmonella enterica serotype typhimurium known as definitive phage type 104 (DT104) is usually resistant to five drugs: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. An increasing proportion of DT104 isolates also have reduced susceptibility to fluoroquinolones. METHODS: The Danish salmonella surveillance program determines the phage types of all typhimurium strains from the food chain, and in the case of suspected outbreaks, five-drug-resistant strains are characterized by molecular methods. All patients infected with five-drug-resistant typhimurium are interviewed to obtain clinical and epidemiologic data. In 1998, an outbreak of salmonella occurred, in which the strain of typhimurium DT104 was new to Denmark. We investigated this outbreak and report here our findings. RESULTS: Until 1997, DT104 infections made up less than 1 percent of all human salmonella infections. The strain isolated from patients in the first community outbreak of DT104 in Denmark, in 1998 was resistant to nalidixic acid and had reduced susceptibility to fluoroquinolones. The outbreak included 25 culture-confirmed cases. Eleven patients were hospitalized, and two died. The molecular epidemiology and data from patients indicated that the primary source was a Danish swine herd. Furthermore, the investigation suggested reduced clinical effectiveness of treatment with fluoroquinolones. CONCLUSIONS: Our investigation of an outbreak of DT104 documented the spread of quinolone-resistant bacteria from food animals to humans; this spread was associated with infections that were difficult to treat. Because of the increase in quinolone resistance in salmonella, the use of fluoroquinolones in food animals should be restricted.

Abattoirs↗
PubMed1999

Mice lacking all conventional MHC class II genes.

MHC class II (MHC-II) molecules play a central role in the selection of the T cell repertoire, in the establishment and regulation of the adaptive immune response, and in autoimmune deviation. We have generated knockout mice lacking all four of the classical murine MHC-II genes (MHCII(Delta/Delta) mice), via a large (80-kilobase) deletion of the entire class II region that was engineered by homologous recombination and Cre recombinase-mediated excision. These mice feature immune system perturbations like those of Aalpha and Abeta knockout animals, notably a dearth of CD4(+) lymphocytes in the thymus and spleen. No new anatomical or physiological abnormalities were observed in MHCII(Delta/Delta) mice. Because these animals are devoid of all classical MHC-II chains, even unpaired chains, they make excellent recipients for MHC-II transgenes from other species, avoiding the problem of interspecies cross-pairing of MHC-II chains. Therefore, they should be invaluable for engineering "humanized" mouse models of human MHC-II-associated autoimmune disorders.

Animals↗
PubMed1999

Cloning and characterization of the gene encoding the highly expressed ribosomal protein l3 of the ciliated protozoan Tetrahymena thermophila. Evidence for differential codon usage in highly expressed genes.

We have cloned and characterized the cDNA and the macronuclear genomic copy of the highly conserved ribosomal protein (r-protein) L3 of Tetrahymena thermophila. The r-protein L3 is encoded by a single copy gene interrupted by one intron. The organization of the promoter region exhibits features characteristic of ribosomal protein genes in Tetrahymena. The codon usage of the L3 gene is highly biased. A thorough analysis of codon usage in Tetrahymena genes revealed that genes could be categorized into two classes according to codon usage bias. Class A comprises r-protein genes and a number of other highly expressed genes. Class B comprises weakly expressed genes such as the conjugation induced CnjB and CnjC genes, but surprisingly, this class also contains abundantly expressed genes such as the genes encoding the surface antigens SerH3 and SerH1. Codon usage is slightly more restricted in class A than in class B, but both classes exhibit distinct and different codon usage biases. Class A genes preferentially use C and U in the silent third codon positions, whereas class B genes preferentially use A and U in the silent third codon positions. The analysis suggests that two different strategies have been employed for optimization of codon usage in the A+T-rich genome of Tetrahymena.

Animals↗
PubMed1999

Recombinant antibodies with the antigen-specific, MHC restricted specificity of T cells: novel reagents for basic and clinical investigations and immunotherapy.

MHC class I and II molecules play a central role in the immune response against a variety of invading microorganisms and cells that have undergone malignant transformation by shaping the T cell repertoire in the thymus and by presenting peptide antigens from endogenous and exogenous antigens in the periphery to CD8+ cytotoxic T cells and CD4+ helper T cells. In certain situations MHC-peptide complexes may, however, also initiate and perpetuate an autoimmune attack mediated by autoaggressive T cells leading to diseases such as insulin dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA) and multiple sclerosis (MS). Such MHC-peptide complexes are a desirable target for novel approaches in immunotherapy. Targeted delivery of toxins or other cytotoxic drugs to cells which express specific MHC-peptide complexes that are involved in the immune response against cancer or viral infections and specific masking of MHC-peptide complexes that are involved in autoimmune reactions would allow for a specific immunotherapeutic treatment of these diseases. We have recently demonstrated that antibodies with the antigen-specific, MHC restricted specificity of T cells can be readily generated by taking advantage of the selection power of phage display technology.

Antibodies, Monoclonal↗
PubMed1999

An scFv phage clone that binds to human beta2-microglobulin.

Using phage display technology, a novel mouse scFv phage clone D1 was generated from mice immunized with single-chain HLA-A2, the human MHC class I molecules. D1 recognized a determinant in beta2-microglobulin. It reacted with beta2-microglobulin associated with the heavy chain of HLA-A2, recombinant beta2-microglobulin made in Escherichia coli or beta2-microglobulin prepared from human urine. The heavy chain variable region of D1 belongs to gene family VII and the light chain variable region belongs to the kappa chain gene family XI according to the Kabat database.

Amino Acid Sequence↗
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

Identification of patient-specific peptides for detection of M-proteins and myeloma cells.

We have taken advantage of the selection power of phage display technology to define specific peptide mimotopes that recognize individual M-proteins, isolated from patients with multiple myeloma. Preferred amino acid motifs of phages binding to M-proteins were identified in 6/9 patients investigated. Chemically synthesized peptides, corresponding to the phage-displayed peptide inserts, were used to verify the specificity of binding in competition assays. The peptides were able to bind to the M-proteins, as well as the myeloma cells, with high sensitivity and specificity. Employing simple immunological techniques, < 0.01 g/l of M-protein could be quantified, suggesting a novel way for monitoring minimal residual disease in the production of guidelines for adjusting or reintroducing conventional chemotherapy. The peptide mimotopes defined by this technology may be useful as tumour-specific targeting agents and as a tool for purging cells in autologous bone marrow transplantation.

Adult↗
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

Validity of SmaI-defined genotypes of Campylobacter jejuni examined by SalI, KpnI, and BamHI polymorphisms: evidence of identical clones infecting humans, poultry, and cattle.

We describe here an examination of the validity of molecular types of Campylobacter jejuni as defined by separation of SmaI-digested DNA using pulsed-field gel electrophoresis (PFGE), recently suggested as part of a molecular subtyping scheme. Thirty-four Danish strains from humans, water, poultry and cattle were assigned to one of six SmaI 'profile groups' (PGs), with two additional strains included as genotypically distinct controls. The interstrain relationships were reexamined by PFGE of SalI, KpnI and BamHI-digested DNA, and also by serotyping with heat-stable antigens. All outbreak-related strains were indistinguishable by all criteria, as were two sets of two randomly-isolated human strains. Two groups of indistinguishable isolates contained randomly isolated strains from more than one source (poultry, humans and/or cattle), a finding with significant epidemiological connotations. All 'genetically identical' strains belonged to the same serotype, whereas genetic differences were detected between strains assigned to the same SmaI PG but differing in serotype. We conclude that PFGE-based genetic fingerprinting can yield invaluable data for epidemiological studies of sporadic C. jejuni infection, but that results based on one restriction site polymorphism must be checked with another enzyme.

Animals↗
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

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

Distribution of serotypes of Campylobacter jejuni and C. coli from Danish patients, poultry, cattle and swine.

The number of human cases of enteritis caused by Campylobacter jejuni and C. coli is increasing in Denmark and other European countries. No systemic typing has earlier been performed on Campylobacter isolates of Danish origin. The primary purpose of this study was to provide a serotype distribution of Campylobacter isolates from Danish patients and the major food production animals. In addition, the occurrence of intestinal carriers of thermophilic campylobacters among these food production animals was examined. In a nationwide survey, the individual isolation rate was 36% for broiler chickens, 47% for cattle and 46% for swine when sampled at the slaughterhouse. C. jejuni accounted for 83-91% of the thermophilic Campylobacter spp. in broiler chickens and cattle, whereas 95% of the isolates from swine was C. coli. In human patients with Campylobacter enteritis, 94% of the isolates were C. jejuni and 6% were C. coli. Heat-stable serotyping (the 'Penner scheme') was performed on a total of 398 isolates from the four sources: human patients (n = 145), broiler chickens (n = 94), swine (n = 111) and cattle (n = 48). Among human isolates, serotype O:1,44, O:2 and the O:4-complex accounted for 62% of the C. jejuni isolates. These serotypes were also common in samples from broilers and cattle. In swine, C. coli O:30 and O:46 were most common. The serotype distribution of human clinical isolates showed large overlap with the serotype distribution of campylobacters in cattle and chickens, and on this basis both could be major sources of human campylobacteriosis.

Abattoirs↗
PubMed1997

Antimicrobial susceptibility patterns of thermophilic Campylobacter spp. from humans, pigs, cattle, and broilers in Denmark.

The MICs of 16 antimicrobial agents were determined for 202 Campylobacter jejuni isolates, 123 Campylobacter coli isolates, and 6 Campylobacter lari isolates from humans and food animals in Denmark. The C. jejuni isolates originated from humans (75), broilers (95), cattle (29), and pigs (3); the C. coli isolates originated from humans (7), broilers (17), and pigs (99); and the C. lari isolates originated from broilers (5) and cattle (1). All isolates were susceptible to apramycin, neomycin, and gentamicin. Only a few C. jejuni isolates were resistant to one or more antimicrobial agents. Resistance to tetracycline was more common among C. jejuni isolates from humans (11%) than among C. jejuni isolates from animals (0 to 2%). More resistance to streptomycin was found among C. jejuni isolates from cattle (10%) than among those from humans (4%) or broilers (1%). A greater proportion of C. coli than of C. jejuni isolates were resistant to the other antimicrobial agents tested. Isolates were in most cases either coresistant to tylosin, spiramycin, and erythromycin or susceptible to all three antibiotics. More macrolide-resistant isolates were observed among C. coli isolates from swine (79%) than among C. coli isolates from broilers (18%) and humans (14%). Twenty-four percent of C. coli isolates from pigs were resistant to enrofloxacin, whereas 29% of C. coli isolates from humans and none from broilers were resistant. More resistance to streptomycin was observed among C. coli isolates from swine (48%) than among C. coli isolates from broilers (6%) or humans (0%). The six C. lari isolates were susceptible to all antimicrobial agents except ampicillin and nalidixic acid. This study showed that antimicrobial resistance was found only at relatively low frequencies among C. jejuni and C. lari isolates. Among C. coli isolates, especially from swine, there was a high level of resistance to macrolides and streptomycin. Furthermore, this study showed differences in the resistance to antimicrobial agents among Campylobacter isolates of different origins.

Animals↗
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↗
PubMed1996

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

Amino Acid Sequence↗