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E J Hensen

Publications and source records attributed to E J Hensen.

At least 19 recordsLinked to original sources
PubMed2002

Immunoprecipitation and isoelectric focusing of sheep MHC class I antigens reveal higher complexity than serology.

Biochemical and serological methods were used to characterize sheep MHC class I polymorphism at the product level. The cells of 65 selected animals were subjected to immunoprecipitation and one-dimensional isoelectric focusing (IEF) with two cross-reacting monoclonal antibodies, B1.1G6 and HC-10. We were able to define up to 18 distinct haplotypes in the Latxa breed sample. Most of the locally defined serological specificities were confirmed or subdivided by biochemical typing. As IEF detected antigens not yet defined by serology, it provided us with a guide to producing more specific antisera by appropriate immunizations. Lastly, evidence of expression of two class I loci products was found.

Animals↗
PubMed2000

Selection of T-cell epitopes from foot-and-mouth disease virus reflects the binding affinity to different cattle MHC class II molecules.

The major histocompatibility complex (MHC)-restricted selection of T-cell epitopes of foot-and-mouth disease virus (FMDV) by individual cattle MHC class II DR (BoLA-DR) molecules was studied in a direct MHC-peptide binding assay. By in vitro priming of T lymphocytes derived from animals homozygous for both MHC class I and II, five T-cell epitopes were analyzed in the context of three MHC class II haplotypes. We found that the presentation of these T-cell epitopes was mediated by DR molecules, since blocking this pathway of antigen presentation using monoclonal antibody TH14B completely abolished the proliferative responses against the peptides. To study the DR-restricted presentation of these T-cell epitopes, a direct MHC-peptide binding assay on isolated cattle DR molecules was developed. Purified cattle MHC class II DR molecules of the BoLA-DRB3*0201, BoLA-DRB3*1101, and BoLA-DRB3*1201 alleles were isolated from peripheral blood mononuclear cells. For each allele, one of the identified T-cell epitopes was biotinylated, and used as a marker peptide for the development of a competitive MHC-peptide binding assay. Subsequently, the T-cell epitopes of FMDV with functionally defined MHC class II specificity were analyzed in this binding assay. The affinity of the epitopes to bind to certain DR molecules was significantly correlated to the capacity to induce T-cell proliferation. This demonstrated at the molecular level that the selection of individual T-cell epitopes found at the functional level was indeed the result of MHC restriction.

Alleles↗
PubMed1998

DNA typing for BoLA class I using sequence-specific primers (PCR-SSP).

The analysis of cattle MHC (BoLA) class I gene expression is an essential component of studies on immune responses and susceptibility to disease. International BoLA workshops have generated data and reagents that allow discrimination of class I molecules at the haplotype level, but progress has been limited by difficulties encountered in defining single alleles. Our aim in this study was to develop a DNA-based system for improved identification of expressed class I alleles, utilizing available cDNA sequences derived from cattle carrying a series of serologically defined class I specificities. This method has allowed more accurate typing of animals for expression of the class I genes present within a small number of haplotypes. The method has also reliably differentiated between allelic variants (identified by prior sequence analysis) and has split existing serological specificities. The data show that MHC class I genes in cattle are more polymorphic than demonstrated by serology and biochemical analysis.

Alleles↗
PubMed1997

Antibody responses against epitopes on the F protein of bovine respiratory syncytial virus differ in infected or vaccinated cattle.

The fusion protein F of bovine respiratory syncytial virus (BRSV) is an important target for humoral and cellular immune responses, and antibodies against the F protein have been associated with protection. However, the F protein can induce antibodies with different biological activity, possibly related to distinct antigenic regions on the protein. Therefore, epitopes were mapped on the F protein using monoclonal antibodies. Two epitopes (A and B) were identified that induced neutralizing antibodies, and one epitope (C) that did not elicit neutralizing antibodies. Subsequently, antibody responses were analysed against these epitopes in cattle sera after natural infection, experimental infection or vaccination. After natural infection or reinfection, the antibody titres against epitope A were significantly higher than those against epitope B or C. After experimental infection and after vaccination with an inactivated vaccine, antibody titres against epitope B and C were significantly higher than after natural infection. Conversely, virus neutralizing antibody titres were significantly lower in these animals with higher antibody titres against epitopes B and C than in naturally infected cattle. Because after natural infection the epitope-specific-antibody titres against epitope A, B or C differed markedly between the cattle, the magnitude of the antibody titres against epitope A, B or C in relation to the major histocompatibility complex (MHC) genes of cattle (BoLA) was studied. The magnitude of the antibody responses against epitope A of the F protein, but not against the G protein, appeared to be associated with the bovine lymphocyte antigen (BoLA) haplotype.

Animals↗
PubMed1997

The specificity of anti-HLA class II monoclonal antibodies in cattle.

At the Eleventh International HLA Histocompatibility Workshop, numerous anti-HLA class II monoclonal antibodies (mAb) were tested. For several of the polymorphic mAb, one epitope for binding has been mapped within the antigen-binding site of the class II molecules. Screening of the available bovine DRB3 and DQB exon 2 sequences revealed that some of the key amino acid (AA) motifs of these epitopes were present in cattle as well, and the question was raised whether this sharing of key AA motifs might cause interspecies cross-reactivity. Eight polymorphic anti-HLA class II mAb (seven anti-HLA DRB1 and one anti-HLA DQB) were selected for analysis of their reactivity towards bovine lymphocytes. In addition, the monomorphic anti-HLA class II mAb, 7.5.10.1, was selected for analysis, as this mAb was described to detect class II polymorphism in cattle. Flow cytometry and lymphocyte microcytotoxicity testing revealed that five of the polymorphic anti-HLA mAb were reactive with bovine lymphocytes. Furthermore, the anti-bovine reactivity of 7.5.10.1 was confirmed. These findings were supported by biochemical analysis. The anti-bovine reaction of the anti-HLA mAb did not correspond with the expected reaction, which was based on the presence of the AA, postulated to be responsible for recognition. Therefore, we suggest that the patterns of reactivity of the anti-HLA mAb are not always determined by one epitope.

Amino Acid Sequence↗
PubMed1995

Sequences derived from the highly antigenic VP1 region 140 to 160 of foot-and-mouth disease virus do not prime for a bovine T-cell response against intact virus.

Although VP1 region 140 to 160 of foot-and-mouth disease virus (FMDV) is able to elicit neutralizing antibody in cattle, the protection against virus challenge that is conferred by peptide immunization is often poor. Here, we show that bovine T cells primed with peptides derived from this region generally show no reactivity to intact FMDV. In contrast, T-cell epitope VP4[20-34] is able to prime for a virus-specific response.

Amino Acid Sequence↗
PubMed1995

The influence of MHC polymorphism on the selection of T-cell determinants of FMDV in cattle.

There is a quest for the development of a new generation of vaccines consisting of well-defined subunit antigens. For a number of practical reasons it is attractive to develop vaccines on the basis of synthetic peptides. However, their efficacy may be limited by genetic restrictions imposed on T-cell recognition via major histocompatibility complex (MHC) polymorphism, as shown by many studies using inbred animal species. To study the effect of MHC polymorphism in an outbred species, we selected four cattle homozygous for different A-DR-DQ haplotypes, and another four cattle which shared one haplotype in combination with a haplotype of one of the MHC homozygous animals. We analysed responses to synthetic peptides comprising defined T-cell epitopes of foot-and-mouth disease virus (FMDV) in this selected group of FMDV-vaccinated cattle. This analysis shows that even in outbred animals. MHC polymorphism influences the responses to synthetic peptides. Interestingly, one of the peptides, VP4[20-34], was recognized in association with at least four different MHC haplotypes. Fine specificity analysis of this peptide revealed subtle shifts in the core epitope recognized. All peptides that induced lymphocyte proliferation in vitro were found to induce a T-helper type-1 (Th1) type of response, irrespective of the MHC haplotype involved. Together, these data support the notion that individuals carrying distinct MHC types can be vaccinated successfully by vaccines that include only a limited number of peptides. In the design of a peptide vaccine against FMDV we suggest inclusion of the highly conserved VP4 sequence 20-34.

Amino Acid Sequence↗
PubMed1994

Effect of climatic stress on the immunological reactivity of weaned pigs.

Weaned pigs exposed daily to either unpredictable draught (experiment 1) or intermittent unpredictable draught (experiment 2) showed different lymphocyte blastogenic responses after mitogenic stimulation with phytohaemagglutinin (PHA) and concanavalin A (ConA). In both experiments PHA skin test responses were lower for draught exposed pigs than for control pigs and leucocyte numbers or profiles were altered compared to those of control pigs. Superoxide production and chemiluminescence of porcine granulocytes were similar for draught exposed and control animals. Furthermore, serum globulin content did not differ significantly between pigs in the experimental and control room. The strong increase in serum gamma-globulin after the Aujeszky Disease Virus (ADV)-challenge was the same for draught exposed and control pigs. The same held for the lymphocyte blastogenic response with ADV protein as antigenic stimulus. The present study shows the effects of climatic stress on immunological reactivity, which may reflect a homeostatic disturbance of the pig's immune system elicited by exposure to unpredictable draught.

Animals↗
PubMed1994

Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules.

New strategies applied in the treatment of experimental autoimmune disease models involve blocking or modulation of MHC-peptide-TCR interactions either at the level of peptide-MHC interaction or, alternatively, at the level of T cell recognition. In order to identify useful competitor peptides one must be able to assess peptide-MHC interactions. Several well described autoimmune disease models exist in the Lewis rat and thus this particular rat strain provides a good model system to study the effect of competitor peptides. So far no information has been available on the peptide binding characteristics of the Lewis rat MHC class II RT1.B1 molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptides can be assessed while employing detection of biotinylated marker peptides by chemiluminescence. The assay is sensitive and specific. We have used this assay to determine the binding characteristics of several disease associated T cell determinants and their sequence analogues in the Lewis rat. Notably, most of the autoimmune disease associated peptide sequences tested were found to be intermediate to poor binders. Single amino acid substitutions at defined positions were sufficient to turn certain peptides into good binders. These results are relevant to the design of competitor peptides in the treatment of experimental autoimmune diseases.

Amino Acid Sequence↗
PubMed1994

T cell-stimulatory fragments of foot-and-mouth disease virus released by mild treatment with cathepsin D.

Cathepsin D and cathepsin B are endosomal/lysosomal proteases that are thought to play a role during in vivo antigen processing, releasing fragments for binding to major histocompatibility complex class II products and subsequent presentation to T cells. Here we treated purified foot-and-mouth disease virus (FMDV) strain A10Holland with both enzymes. Cathepsin D, but not cathepsin B, was shown to release fragments from reduced or non-reduced FMDV under mild conditions in vitro. Twenty-eight predominant cathepsin D-released fragments were purified by HPLC and identified by amino acid composition analysis and sequencing. The unseparated set of fragments produced (the digest) was able to stimulate T cells from eight vaccinated cattle. With respect to the response to intact virus the extent of the response to the digest differed between animals: four animals could be classified as good responders, three as intermediate responders and one as a low responder. Subsequently, we investigated the proliferative T cell response to a large set of synthetic peptides in detail for two animals, one belonging to the group of good responders, the other being the low responder. The peptides covered all 28 cathepsin D-released fragments analysed and also several sequences not recovered from the digest. In this way seven T cell sites could be identified, five of which coincided with cathepsin D-released fragments. The other two T cell sites were VP2[54-72], being a homologue of a T cell site identified for FMDV strain O1K and the N terminus of VP4. Whether the most dominantly recognized T cell site was recovered from the digest or not was shown to be related to the good or low response to the digest. These findings suggest a role for cathepsin D in the release of some but not all T cell-stimulatory fragments from FMDV.

Amino Acid Sequence↗
PubMed1994

Biochemical characterization of bovine MHC DQ allelic variants by one-dimensional isoelectric focusing.

Previous studies on expressed bovine MHC class II polymorphism using one-dimensional isoelectric focusing (1D-IEF) allowed the identification of at least 12 allelic variants of the DRB3 gene. So far, only limited data have been available on the expression of other class II genes. The present study involved biochemical analysis of bovine MHC class II molecules using a set of monoclonal antibodies presupposed to be bovine DR and DQ reactive. After essential modification of the standard electrophoresis conditions used for 1D-IEF typing of bovine DR products, biochemical polymorphism was observed for non-DR molecules, revealing polymorphic sets of basic and acidic focusing bands. Because of the extensive DNA polymorphism described for bovine DQA and DQB genes, and the apparent similarity with the focusing pattern of human DQ products, these molecules were considered to be the bovine DQ homologues. The definition of the DQ-associated banding patterns was made possible by using two half-sib sire families. Four different DQA-like patterns and nine DQB-like patterns were detected. Segregation of the DQ types was supported by serological class I and class II typing. These results show that it is now possible to discriminate between expressed bovine DR and DQ polymorphism.

Alleles↗
PubMed1994

Polymorphism of bovine MHC class I genes. Joint report of the Fifth International Bovine Lymphocyte Antigen (BoLA) Workshop, Interlaken, Switzerland, 1 August 1992.

The objectives of the Fifth International BoLA Workshop were to: standardize nomenclature, compare typing methods, and characterize BoLA haplotypes. The workshop was based on the distribution of blood samples (cells) from 60 selected cattle to 14 laboratories. Results for the class I (BoLA-A) region are presented in this paper while results for the class II regions are presented in a separate report. Thirty-six of the 50 previously established serological class I specificities were represented in the cell panel. However, only 30 specificities could be confirmed. Two specificities, A16 and A32, were upgraded from provisional, workshop (w) specificities to BoLA-A locus specificities and three new specificities, w51(w28), w52 and w53(w28), were defined. The 39 specificities distinguished 30 class I haplotypes in the 60 animals. Class I isoelectric focusing proved to be a useful adjunct to the serology. Isoelectric focusing confirmed several serologically defined splits and detected splits of A15(A8), A18(A6) and A22(w49) that had not been detected by serology. Subsequently, serological support for splits of A15(A8) and A22(w49) was found.

Animals↗
PubMed1993

Mapping of susceptibility to Marek's disease within the major histocompatibility (B) complex by refined typing of White Leghorn chickens.

The major histocompatibility (B) complex of a distinct commercial pure White Leghorn chicken line was characterized using serological, biochemical and restriction fragment length polymorphism (RFLP) typing. Line B chickens displayed a high recombination frequency within the B complex. Three recombinant haplotypes were identified. The influence of these haplotypes was determined in relation to the haplotypes B19 and B21 on their resistance to Marek's disease (MD) in an experimental infection with the virus. Offspring of sires with a recombinant haplotype in combination with B19 or B21, and dams, which were homozygous B19/B19 or B21/B21 were infected. The B type of the offspring had a significant effect upon survival. Animals with B complex types B21/B21, B134/B21 and B234/B21 were relatively resistant to MD (24-32% mortality), whereas B19/B19 birds were highly susceptible (68% mortality). Animals with a recombinant haplotype B19r21 (B-G21, B-F19) were equally susceptible to MD as birds with the complete B19 haplotype. In contrast to earlier publications, resistance was not inherited as a dominant trait. Apparently, B19 was associated with a dominant susceptibility. The gene(s) associated with the B complex and involved in resistance to MD were localized within the B-F/B-L region. However, the association with a presumably non-coding subregion of B-G could not be excluded.

Animals↗
PubMed1993

A high recombination frequency within the chicken major histocompatibility (B) complex.

Chickens of a commercial pure White Leghorn line were typed for B-F and B-G by serological, biochemical and molecular biological methods. Amongst 287 typed animals of one particular line, three animals with recombinant haplotypes were identified. Compared to earlier reports this revealed a statistically significant (P < 0.05), tenfold higher recombination frequency in this chicken line.

Animals↗
PubMed1993

Use of isoelectric focusing to define major histocompatibility complex class I polymorphism in goats.

We have used biochemical methods to extend and improve serological class I typing using a panel of 77 Swiss goats of the Saanen breed, comprising dam-offspring combinations from six half-sib sire families and several unrelated animals. Of these animals class I molecules were precipitated from cell lysates with the mAb B1.1G6 and HC10. Immunoprecipitates were analysed by SDS-PAGE and 1D-IEF. There was a good agreement between class I serological types and IEF banding patterns. We have identified three new class I specificities and subdivided the Be17 specificity. IEF has enabled us to make planned immunizations to produce antisera to the new specificities. New evidence for the expression of a second class I locus product in the Be7 haplotype has been found.

Animals↗