Aad Hoek - Academia.edu (original) (raw)

Papers by Aad Hoek

Research paper thumbnail of Promiscuous epitopes of Mycobacterium paratuberculosis 70kDa heatshock protein activating bovine T cells

Veterinary Immunology and Immunopathology, 2009

. Mycobacterial 70 kDa heat-shock protein is an effective subunit vaccine against bovine paratube... more . Mycobacterial 70 kDa heat-shock protein is an effective subunit vaccine against bovine paratuberculosis.

Research paper thumbnail of Identification of monoclonal antibodies with specificity to α- or β-chains of β2-integrins using peripheral blood leucocytes of normal and Bovine Leucocyte Adhesion Deficient (BLAD) cattle

Veterinary Immunology and Immunopathology, 1996

The reactivity of monoclonal antibodies entered in the panels of the Third Workshop on Ruminant L... more The reactivity of monoclonal antibodies entered in the panels of the Third Workshop on Ruminant Leucocyte Antigens with lymphocytes and monocytes of normal and beta 2-integrin deficient (Bovine Leucocyte Adhesion Deficiency: BLAD) animals was determined by flow cytometry to investigate potential specificities to alpha- or beta-chains of beta 2-integrins. From the 13 monoclonal antibodies that were entered as having specificity for CD11/CD18 antigens, ten were confirmed correct, but three had reactivity with cells from BLAD animals. We conclude that our approach provides an easy way to reliably identify the majority of beta 2-integrin specific monoclonal antibodies.

Research paper thumbnail of Cell mediated responses to mycobacterial PPD and heat shock proteins in various stages of bovine paratuberculosis

Immunology Letters, 1997

Introduction: Infection with Myccbacterium paratu&emuhxis during catfhood causes chronic inflamma... more Introduction: Infection with Myccbacterium paratu&emuhxis during catfhood causes chronic inflammation of the ileum and cecum in ruminants. Following an incubation period of 4-5 years a fatal protein loosing enteropathy will develop in 10% of the infected animals. Vaccination at early age prevents the occurance of clinical disease. Histologically the terminal lesions resemble those observed in lepromatous leprosy. In mycobacteriai diseases like tuberculosis and leprosy heat shock protein (HSP) specific T-cells have been shown to be involved in the immunopathogenesis, but little is known about bovine paratuberculosis in this respect.

Research paper thumbnail of Functional characterization of T-cells involved in skin allograft rejection in case of inherited β2-integrin deficiency of cattle

Immunology Letters, 1997

Poster presentations APO and the induction of T-and B-cell responses 265 of the toxin to introduc... more Poster presentations APO and the induction of T-and B-cell responses 265 of the toxin to introduce soluble proteins into the MHC class I presentation pathway was unaltered. Thus we were able to generate CD8+ T ceils against the membrane protein ActA and propagate them k, vi& Wti these ActA specific cytotoxic T cells we could show that in L&&r& infected calls this antigen is not available for MHC class I presentation as long as the pathogen is viable. Furthermore, no protection was observed when ActA-specific CD8+ T cells were adoptively transfered into syngeneic mice.

Research paper thumbnail of High volume continuous venovenous haemofiltration (HV-CVVH) in an equine endotoxaemic shock model

Equine Veterinary Journal, 2010

Equine acute abdominal disease is often associated with shock. Important aspects in the onset of ... more Equine acute abdominal disease is often associated with shock. Important aspects in the onset of this complication include hypovolaemia, the translocation of endotoxins from the gut and the subsequent activation of the cytokine network. The clinical efficacy of high volume continuous venovenous haemofiltration (HV-CVVH) and the clearance of cytokines were therefore investigated in an equine endotoxaemic model. Ten male Shetland ponies received a slow infusion of LPS (2 microg/kg bwt) under general anaesthesia. The treatment group (n = 5) received HV-CVVH (2 ml/kg bwt/min) using a 75 kD polymethylmethacrylate (PMMA filter). Haemodynamic, blood variable and cytokine (TNF, IL-1 and IL-6) measurements were performed every 30 min for a 6 h period. The ponies showed a typical reaction in mean pulmonary arterial pressure, blood chemical and haematological markers after LPS challenge. No significant differences were found between the treatment group and the control group. Only a slight increase in cardiac index and no marked decrease in mean arterial pressure were seen. A clear cytokine response was found in all ponies, though substantially different in magnitude between individuals. The clearance of cytokines from the blood increased in time, but did not lead to significant decrease in serum levels. In this study, HV-CVVH with a PMMA filter did not prove to have a significant beneficial effect on the course of experimental endotoxaemia in horses. However, in a more severe model, better efficacy might be obtained. Testing additional filters might lead to a more suitable therapy for horses.

Research paper thumbnail of Generation of the First TCR Transgenic Mouse with CD4+ T Cells Recognizing an Anti-inflammatory Regulatory T Cell-Inducing Hsp70 Peptide

Frontiers in Immunology, 2016

Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study sinc... more Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such -antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen--specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4(+) T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4(+) T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4(+)CD25(+) Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis.

Research paper thumbnail of The immune response of cattle, persistenly infected with non-cytopathic BVDV, after superinfection with antigenically homologous cytopathic BVDV

Research paper thumbnail of An arthritis suppressive and regulatory T cell-inducing CD4 + T cell epitope is functional in the context of HLA-restricted T cell responses

Arthritis & Rheumatology, 2015

Previously, we have shown that mycobacterial heat shock protein 70 (HSP70)-derived peptide B29 in... more Previously, we have shown that mycobacterial heat shock protein 70 (HSP70)-derived peptide B29 induces B29-specific regulatory T cells, which suppressed experimental arthritis in mice by cross-recognition of their mammalian HSP70 homologs (1). The aim of this study is to characterize the B29 binding and specific CD4(+) T cell responses in the context of human MHC molecules (HLA). Binding of B29 peptide and its mammalian homologs to HLA molecules was examined with competitive binding assays. The effect of B29 immunization was assessed in proteoglycan-induced arthritis in HLA-DQ8 transgenic mice, followed by ex vivo restimulation with B29 to examine the T cell response. Human PBMC were used to investigate the presence of B29-specific T cells with immunoregulatory potential. We found a high to moderate binding affinity for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was functional, as B29 immunization resulted in suppression of arthritis and T cell responses in HLA-DQ8 transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4(+) T cells, which were cross-reactive with the mammalian homologs. With HLA-DR4(+) tetramers specific for B29 or mB29b we showed expansion of cross-reactive T cells, especially the human CD4(+) CD25(+) FoxP3(+) T cell population after in vitro stimulation with B29. These results demonstrated a conserved fine-specificity and functionality of the B29-induced regulatory T cell responses in the context of the human MHC. Based on these findings, a translational path of the B29 experimental findings into a clinical immunomodulatory therapeutic approach comes within reach. This article is protected by copyright. All rights reserved.

Research paper thumbnail of Evidence for distinct host response patterns in cows experimentally infected with M. avium subspecies paratuberculosis

Research paper thumbnail of Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis

Proceedings of the National Academy of Sciences

Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T ... more Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T cells (Tregs). Exploiting these antigen-specific regulators is hampered by the obscure nature of disease-relevant autoantigens. We have uncovered potent disease-suppressive Tregs recognizing Heat Shock Protein (Hsp) 70 self-antigens, enabling selective activity in inflamed tissues. Hsp70 is a major contributor to the MHC class II ligandome. Here we show that a conserved Hsp70 epitope (B29) is present in murine MHC class II and that upon transfer, B29-induced CD4(+)CD25(+)Foxp3(+) T cells suppress established proteoglycan-induced arthritis in mice. These self-antigen-specific Tregs were activated in vivo, and when using Lymphocyte Activation Gene-3 as a selection marker, as few as 4,000 cells sufficed. Furthermore, depletion of transferred Tregs abrogated disease suppression. Transferred cells exhibited a stable phenotype and were found in joints and draining lymph nodes up to 2 mo after ...

Research paper thumbnail of Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis

Veterinary immunology and immunopathology, 1999

Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused... more Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses ...

Research paper thumbnail of Effect of a Dietary n-6 Polyunsaturated Fatty Acid Supplement on Distinct Immune Functions of Goats

Journal of Veterinary Medicine Series A, 2004

The aim of this study was to investigate whether dietary n-6 polyunsaturated fatty acids (PUFA) a... more The aim of this study was to investigate whether dietary n-6 polyunsaturated fatty acids (PUFA) alter immune functions as lymphocyte blastogenesis, expression of lymphocyte and monocyte markers, and cell adhesion molecules of the integrin family in goats. Dutch White castrated male goats received a diet either rich in olive oil (control group, n = 4) or rich in corn oil (test group, n = 4) over a period of 3 weeks. The animals in the test group had significantly higher linoleic acid (LA, 18:2n-6) levels in plasma on days 7, 14 and 21 of the diet than control animals. This was also true for washed erythrocyte membranes at day 21. A significant reduction in the percentage of alpha-4 integrin (CD49d) expressing lymphocytes was observed in goats fed LA rich corn oil at day 21. However, no changes were observed in either lymphocyte proliferative responses to ConA and PHA or the expression of other lymphocyte/monocyte markers. In conclusion, feeding corn oil rich in LA caused significant increases in the proportion of LA in the fatty acid composition of plasma and erythrocyte membranes and was accompanied by a significant decrease of the adhesion molecule alpha-4 integrin (CD49d) on lymphocytes but not on monocytes.

Research paper thumbnail of Antigen-specific immune responses in cattle with inherited β2-integrin deficiency

Veterinary Immunology and Immunopathology, 1997

The significance of beta2-integrins for the generation of antigen-specific immune responses in vi... more The significance of beta2-integrins for the generation of antigen-specific immune responses in vivo was studied employing the bovine model of beta2-integrin deficiency. To that end four cattle with bovine leukocyte adhesion deficiency (BLAD) and healthy age-matched controls were immunized with tetanus toxoid (TT) and rabies virus (RV) vaccines three times in monthly intervals. In addition, two animals with BLAD and three controls received a fourth vaccination 8 months after the start of the study. Proliferative responses of peripheral blood mononuclear cells (PBMC) to the antigens TT and RV as well as specific serum immunoglobulin G (IgG) titers were determined in intervals for up to 10 months after primary vaccination. Proliferative responses of PBMC to TT and RV were substantially lower in cattle with BLAD than in controls, although PBMC from cattle with BLAD were shown to have the capacity to proliferate in the response to the mitogen concanavalin A. Occurrence of antigen-specific IgG titers was delayed and they were considerably lower in cattle with BLAD compared to controls. Finally, treatment of TT- and RV-stimulated PBMC from an immunized control with different concentrations of the anti-CD18 monoclonal antibody R15.7 resulted in a dose-dependent inhibition of lymphocyte proliferation to almost 100%. The results of the present study show that beta2-integrin deficiency leads to delayedand severely impaired immune responsiveness in vivo. The observations that antibody production, although considerably delayed and impaired, does occur and that apparently class-switching takes place in BLAD indicate T-cell reactivity in vivo.

Research paper thumbnail of Impaired specific immunoreactivity in cows with hepatic lipidosis

Veterinary Immunology and Immunopathology, 1997

In this study, hepatic lipidosis in cows was experimentally induced by offering an energy surplus... more In this study, hepatic lipidosis in cows was experimentally induced by offering an energy surplus during the dry period. Liver triacylglycerol (TAG) was 16% in the experimental group. In the control group fed the same diet in restricted quantities, liver TAG was about 7%.The animals of both groups were vaccinated with tetanus vaccine at Day 3 after parturition. It was

Research paper thumbnail of The effects of a single injection of dexamethasone-21-isonicotinate on the lymphocyte functions of dairy cows at two weeks post partum

Veterinary Research, 2004

Dexamethasone is a potent therapeutic for treatment of the fatty liver syndrome or primary ketosi... more Dexamethasone is a potent therapeutic for treatment of the fatty liver syndrome or primary ketosis in post partum dairy cows. Reservations exist, however, among practitioners with respect to the risk of immunosuppression induced by corticosteroids. The aim of this study was to investigate the effect of a single injection of dexamethasone-21-isonicotinate on distinct immune functions of postpartum dairy cows because only scarce information is available on the effects of corticosteroid preparations when administered at a dosage and frequency for treatment of the fatty liver syndrome or primary ketosis. Sixteen Swedish red-pied dairy cows, between days 9 and 15 post partum, were allotted to either a control group (n = 8) or a treatment group (n = 8). The cows in the treatment group received a single intramuscular injection of a dexamethasone-21-isonicotinate suspension at a dosage of 0.02 mg/kg i.m. at the start of the experiment. White blood cell counts and selected lymphocyte functions (lymphocyte proliferation, expression of lymphocyte markers and the β2 and α4 chain of adhesion molecules belonging to the integrin family) and some parameters of the energy metabolism (glucose, insulin) were determined before the administration of corticosteroids (day 0) and subsequently at days 2, 4, 7 and 9 of the experiment. Changes in glucose and insulin were within the target range for treatment of the fatty liver syndrome or primary ketosis. Significant (P < 0.05) increases in the number of circulating white blood cells were observed in treated cows on the second day following treatment which was exclusively caused by an increase in the number of circulating neutrophils. Lymphocyte blastogenesis in response to ConA and the percentages of lymphocytes positive for CD2, CD4, CD8, CD49d and CD18 as well as the intensity of CD49d expression did not differ between the treatment and control groups. There was, however, a significant reduction (P < 0.01) in the intensity of CD18 expression on lymphocytes in the treated animals on the fourth day after treatment. In conclusion, a single administration of dexamethasone-21-isonicotinate in a dosage of 0.02 mg/kg i.m. at two weeks post partum in healthy cows had a significant but highly transient effect on CD18 expression on lymphocytes and the number of peripheral blood neutrophils, but did not affect lymphocyte blastogenesis or lymphocyte subpopulation patterns in peripheral blood. glucocorticosteroid / ketosis / lymphocyte proliferation / adhesion molecules / dairy cows * Corresponding author: j.thanasak@vet.uu.nl 104 J. Thanasak et al.

Research paper thumbnail of Subpopulations of bovine WC1 + γδ T cells rather than CD4 + CD25 high Foxp3 + T cells act as immune regulatory cells ex vivo

Veterinary Research, 2009

Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis... more Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis. Especially CD4 + CD25 high T cells are considered to be important regulators of immune reactivity. In humans and rodents these natural Treg are characterized by their anergic nature, defined as a non-proliferative state, suppressive function and expression of Foxp3. In this study the potential functional role of flowcytometry-sorted bovine white blood cell populations, including CD4 + CD25 high T cells and T cell subpopulations, as distinct ex vivo regulatory cells was assessed in co-culture suppression assays. Our findings revealed that despite the existence of a distinct bovine CD4 + CD25 high T cell population, which showed Foxp3 transcription/expression, natural regulatory activity did not reside in this cell population. In bovine co-culture suppression assays these cells were neither anergic nor suppressive. Subsequently, the following cell populations were tested functionally for regulatory activity: CD4 + CD25 low T cells, WC1 + , WC1.1 + and WC1.2 + T cells, NK cells, CD8 + T cells and CD14 + monocytes. Only the WC1.1 + and WC1.2 + T cells and CD14 + monocytes proved to act as regulatory cells in cattle, which was supported by the fact that these regulatory cells showed IL-10 transcription/expression. In conclusion, our data provide first evidence that cattle CD4 + CD25 high Foxp3 + and CD4 + CD25 low T cells do not function as Treg ex vivo. The bovine Treg function appears to reside in the T cell population, more precisely in the WC1.1 + and the WC1.2 + subpopulation, major populations present in blood of cattle in contrast to non-ruminant species.

Research paper thumbnail of Bovine leukocyte adhesion deficiency ‐ clinical course and laboratory findings in eight affected animals

Veterinary Quarterly, 1994

The clinical course of Bovine Leukocyte Adhesion Deficiency (BLAD) in eight Holstein Friesian cat... more The clinical course of Bovine Leukocyte Adhesion Deficiency (BLAD) in eight Holstein Friesian cattle is described. Affected animals were presented with a history of poor thriving and recurrent bacterial infections. Five of these animals had to be killed because of severe respiratory disease shortly after admittance. Three affected animals survived calfhood only as a result of frequent antibacterial treatments. At one year of age, failure to thrive and stunted growth were still evident, but infections requiring antibiotic treatments occurred only sporadically. Clinical manifestations of BLAD were found in the digestive system (gingivitis, periodontitis, alveolar periostitis, diarrhoea), the respiratory system and the skin (impaired wound healing, chronic dermatitis). A leukocytosis based on a mature neutrophilia, which persisted during infection-free periods, was observed in all animals. Granulocytes were substantially deficient of beta 2-integrin expression on their membranes. Anaemia, which was noted in four animals, may be related to the Anaemia of Inflammatory Disease Complex (AID). The serum total protein content increased with time and was associated with elevated gamma-globulin levels. We suggest that, at a certain age, animals affected with BLAD are able to cope with environmental agents due to compensatory mechanisms of the immune system.

Research paper thumbnail of Characterization of bovine regulatory cells

Veterinary Immunology and Immunopathology, 2009

E-mail address: a.hoek@vet.uu.nl (A. Hoek).

Research paper thumbnail of Allograft rejection in cattle with bovine leukocyte adhesion deficiency

Veterinary Immunology and Immunopathology, 1995

In the present investigation cell-mediated immunity in animals with bovine leukocyte adhesion def... more In the present investigation cell-mediated immunity in animals with bovine leukocyte adhesion deficiency (BLAD) was studied by means of skin transplantation experiments.Autograft and allograft behaviour in animals with BLAD was compared with the behaviour of simultaneously transplanted autografts and allografts in healthy controls. Allograft survival time was prolonged in three BLAD cattle (28, 30, and 72 days) compared to six

Research paper thumbnail of The immune response of cattle, persistently infected with noncytopathic BVDV, after superinfection with antigenically semi-homologous cytopathic BVDV

Veterinary Immunology and Immunopathology, 1998

Cattle persistently infected (PI) with noncytopathic (ncp) bovine virus diarrhea virus (BVDV) are... more Cattle persistently infected (PI) with noncytopathic (ncp) bovine virus diarrhea virus (BVDV) are at risk for developing fatal mucosal disease (MD), which is considered to occur after superinfection with antigenically homologous cytopathic (cp) BVDV. In this study, we intranasally inoculated four PI-animals, that were PI with 2 ncp BVDV strains with 10(5) TCID50 antigenically closely related cp BVDV. Two PI-animals were inoculated with 10(5) TCID50 ncp BVDV and one PI-animal, with virus free cell culture medium. Two out of four PI-animals that were inoculated with cp BVDV, developed MD and were euthanized at day 17 and at day 24 after infection. Postmortem, both animals showed typical lesions of MD and cp BVDV was isolated. The other two PI-animals that were inoculated with cp BVDV did not develop MD and were euthanized at day 51. They showed ulcerations in the gastrointestinal tract, cp BVDV was isolated and neutralizing antibodies were detected. From the three PI-animals, that were inoculated with ncp BVDV or cell culture medium, cp BVDV was also isolated. Cross neutralization tests were performed and no antigenic differences could be detected between the cp strains isolated from the PI-animals. Lymphocyte subsets of these PI-animals were determined by flow cytometric analysis. Before superinfection, the percentages of gamma delta subsets were much higher in the PI-animals that did not develop MD than in nonviremic control animals and in the PI-animals that died of MD. From this study we conclude that the presence of antigenically closely related cp BVDV in PI-animals does not necessarily lead to the development of MD and that besides the antigenic relatedness between the persisting ncp BVDV and cp BVDV other factors, for instance the number of circulating gamma delta cells, might determine whether or not PI-animals develop MD.

Research paper thumbnail of Promiscuous epitopes of Mycobacterium paratuberculosis 70kDa heatshock protein activating bovine T cells

Veterinary Immunology and Immunopathology, 2009

. Mycobacterial 70 kDa heat-shock protein is an effective subunit vaccine against bovine paratube... more . Mycobacterial 70 kDa heat-shock protein is an effective subunit vaccine against bovine paratuberculosis.

Research paper thumbnail of Identification of monoclonal antibodies with specificity to α- or β-chains of β2-integrins using peripheral blood leucocytes of normal and Bovine Leucocyte Adhesion Deficient (BLAD) cattle

Veterinary Immunology and Immunopathology, 1996

The reactivity of monoclonal antibodies entered in the panels of the Third Workshop on Ruminant L... more The reactivity of monoclonal antibodies entered in the panels of the Third Workshop on Ruminant Leucocyte Antigens with lymphocytes and monocytes of normal and beta 2-integrin deficient (Bovine Leucocyte Adhesion Deficiency: BLAD) animals was determined by flow cytometry to investigate potential specificities to alpha- or beta-chains of beta 2-integrins. From the 13 monoclonal antibodies that were entered as having specificity for CD11/CD18 antigens, ten were confirmed correct, but three had reactivity with cells from BLAD animals. We conclude that our approach provides an easy way to reliably identify the majority of beta 2-integrin specific monoclonal antibodies.

Research paper thumbnail of Cell mediated responses to mycobacterial PPD and heat shock proteins in various stages of bovine paratuberculosis

Immunology Letters, 1997

Introduction: Infection with Myccbacterium paratu&emuhxis during catfhood causes chronic inflamma... more Introduction: Infection with Myccbacterium paratu&emuhxis during catfhood causes chronic inflammation of the ileum and cecum in ruminants. Following an incubation period of 4-5 years a fatal protein loosing enteropathy will develop in 10% of the infected animals. Vaccination at early age prevents the occurance of clinical disease. Histologically the terminal lesions resemble those observed in lepromatous leprosy. In mycobacteriai diseases like tuberculosis and leprosy heat shock protein (HSP) specific T-cells have been shown to be involved in the immunopathogenesis, but little is known about bovine paratuberculosis in this respect.

Research paper thumbnail of Functional characterization of T-cells involved in skin allograft rejection in case of inherited β2-integrin deficiency of cattle

Immunology Letters, 1997

Poster presentations APO and the induction of T-and B-cell responses 265 of the toxin to introduc... more Poster presentations APO and the induction of T-and B-cell responses 265 of the toxin to introduce soluble proteins into the MHC class I presentation pathway was unaltered. Thus we were able to generate CD8+ T ceils against the membrane protein ActA and propagate them k, vi& Wti these ActA specific cytotoxic T cells we could show that in L&&r& infected calls this antigen is not available for MHC class I presentation as long as the pathogen is viable. Furthermore, no protection was observed when ActA-specific CD8+ T cells were adoptively transfered into syngeneic mice.

Research paper thumbnail of High volume continuous venovenous haemofiltration (HV-CVVH) in an equine endotoxaemic shock model

Equine Veterinary Journal, 2010

Equine acute abdominal disease is often associated with shock. Important aspects in the onset of ... more Equine acute abdominal disease is often associated with shock. Important aspects in the onset of this complication include hypovolaemia, the translocation of endotoxins from the gut and the subsequent activation of the cytokine network. The clinical efficacy of high volume continuous venovenous haemofiltration (HV-CVVH) and the clearance of cytokines were therefore investigated in an equine endotoxaemic model. Ten male Shetland ponies received a slow infusion of LPS (2 microg/kg bwt) under general anaesthesia. The treatment group (n = 5) received HV-CVVH (2 ml/kg bwt/min) using a 75 kD polymethylmethacrylate (PMMA filter). Haemodynamic, blood variable and cytokine (TNF, IL-1 and IL-6) measurements were performed every 30 min for a 6 h period. The ponies showed a typical reaction in mean pulmonary arterial pressure, blood chemical and haematological markers after LPS challenge. No significant differences were found between the treatment group and the control group. Only a slight increase in cardiac index and no marked decrease in mean arterial pressure were seen. A clear cytokine response was found in all ponies, though substantially different in magnitude between individuals. The clearance of cytokines from the blood increased in time, but did not lead to significant decrease in serum levels. In this study, HV-CVVH with a PMMA filter did not prove to have a significant beneficial effect on the course of experimental endotoxaemia in horses. However, in a more severe model, better efficacy might be obtained. Testing additional filters might lead to a more suitable therapy for horses.

Research paper thumbnail of Generation of the First TCR Transgenic Mouse with CD4+ T Cells Recognizing an Anti-inflammatory Regulatory T Cell-Inducing Hsp70 Peptide

Frontiers in Immunology, 2016

Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study sinc... more Antigen-specific regulatory T cells (Tregs) directed at self-antigens are difficult to study since suitable specific tools to isolate and characterize these cells are lacking. A T cell receptor (TCR)-transgenic mouse would generate possibilities to study such -antigen-specific T cells. As was shown previously, immunization with the mycobacterial heat shock protein (Hsp) 70-derived peptide B29 and its mouse homologs mB29a and mB29b induced anti-inflammatory responses. Furthermore, B29 induced antigen--specific Tregs in vivo. To study mB29b-specific Tregs, we isolated the TCR from T cell hybridomas generated against mB29b and produced a TCR transgenic mouse that expresses a MHC-class II restricted mB29b-specific TCR. These TCR transgenic CD4(+) T cells were found to cross-react with the B29 epitope as identified with peptide-induced proliferation and IL-2 production. Thus, we have successfully generated a novel mouse model with antigen-specific CD4(+) T cells that recognize self and bacterial Hsp 70-derived peptides. With this novel mouse model, it will be possible to study primary antigen-specific T cells with specificity for a regulatory Hsp70 T cell epitope. This will enable the isolation and characterization CD4(+)CD25(+) Tregs with a proven specificity. This will provide useful knowledge of the induction, activation, and mode of action of Hsp70-specific Tregs, for instance, during experimental arthritis.

Research paper thumbnail of The immune response of cattle, persistenly infected with non-cytopathic BVDV, after superinfection with antigenically homologous cytopathic BVDV

Research paper thumbnail of An arthritis suppressive and regulatory T cell-inducing CD4 + T cell epitope is functional in the context of HLA-restricted T cell responses

Arthritis & Rheumatology, 2015

Previously, we have shown that mycobacterial heat shock protein 70 (HSP70)-derived peptide B29 in... more Previously, we have shown that mycobacterial heat shock protein 70 (HSP70)-derived peptide B29 induces B29-specific regulatory T cells, which suppressed experimental arthritis in mice by cross-recognition of their mammalian HSP70 homologs (1). The aim of this study is to characterize the B29 binding and specific CD4(+) T cell responses in the context of human MHC molecules (HLA). Binding of B29 peptide and its mammalian homologs to HLA molecules was examined with competitive binding assays. The effect of B29 immunization was assessed in proteoglycan-induced arthritis in HLA-DQ8 transgenic mice, followed by ex vivo restimulation with B29 to examine the T cell response. Human PBMC were used to investigate the presence of B29-specific T cells with immunoregulatory potential. We found a high to moderate binding affinity for multiple HLA-DR and HLA-DQ molecules, including those highly associated with rheumatoid arthritis. This binding was functional, as B29 immunization resulted in suppression of arthritis and T cell responses in HLA-DQ8 transgenic mice. In humans, we demonstrated the presence and expansion of B29-specific CD4(+) T cells, which were cross-reactive with the mammalian homologs. With HLA-DR4(+) tetramers specific for B29 or mB29b we showed expansion of cross-reactive T cells, especially the human CD4(+) CD25(+) FoxP3(+) T cell population after in vitro stimulation with B29. These results demonstrated a conserved fine-specificity and functionality of the B29-induced regulatory T cell responses in the context of the human MHC. Based on these findings, a translational path of the B29 experimental findings into a clinical immunomodulatory therapeutic approach comes within reach. This article is protected by copyright. All rights reserved.

Research paper thumbnail of Evidence for distinct host response patterns in cows experimentally infected with M. avium subspecies paratuberculosis

Research paper thumbnail of Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis

Proceedings of the National Academy of Sciences

Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T ... more Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T cells (Tregs). Exploiting these antigen-specific regulators is hampered by the obscure nature of disease-relevant autoantigens. We have uncovered potent disease-suppressive Tregs recognizing Heat Shock Protein (Hsp) 70 self-antigens, enabling selective activity in inflamed tissues. Hsp70 is a major contributor to the MHC class II ligandome. Here we show that a conserved Hsp70 epitope (B29) is present in murine MHC class II and that upon transfer, B29-induced CD4(+)CD25(+)Foxp3(+) T cells suppress established proteoglycan-induced arthritis in mice. These self-antigen-specific Tregs were activated in vivo, and when using Lymphocyte Activation Gene-3 as a selection marker, as few as 4,000 cells sufficed. Furthermore, depletion of transferred Tregs abrogated disease suppression. Transferred cells exhibited a stable phenotype and were found in joints and draining lymph nodes up to 2 mo after ...

Research paper thumbnail of Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis

Veterinary immunology and immunopathology, 1999

Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused... more Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses ...

Research paper thumbnail of Effect of a Dietary n-6 Polyunsaturated Fatty Acid Supplement on Distinct Immune Functions of Goats

Journal of Veterinary Medicine Series A, 2004

The aim of this study was to investigate whether dietary n-6 polyunsaturated fatty acids (PUFA) a... more The aim of this study was to investigate whether dietary n-6 polyunsaturated fatty acids (PUFA) alter immune functions as lymphocyte blastogenesis, expression of lymphocyte and monocyte markers, and cell adhesion molecules of the integrin family in goats. Dutch White castrated male goats received a diet either rich in olive oil (control group, n = 4) or rich in corn oil (test group, n = 4) over a period of 3 weeks. The animals in the test group had significantly higher linoleic acid (LA, 18:2n-6) levels in plasma on days 7, 14 and 21 of the diet than control animals. This was also true for washed erythrocyte membranes at day 21. A significant reduction in the percentage of alpha-4 integrin (CD49d) expressing lymphocytes was observed in goats fed LA rich corn oil at day 21. However, no changes were observed in either lymphocyte proliferative responses to ConA and PHA or the expression of other lymphocyte/monocyte markers. In conclusion, feeding corn oil rich in LA caused significant increases in the proportion of LA in the fatty acid composition of plasma and erythrocyte membranes and was accompanied by a significant decrease of the adhesion molecule alpha-4 integrin (CD49d) on lymphocytes but not on monocytes.

Research paper thumbnail of Antigen-specific immune responses in cattle with inherited β2-integrin deficiency

Veterinary Immunology and Immunopathology, 1997

The significance of beta2-integrins for the generation of antigen-specific immune responses in vi... more The significance of beta2-integrins for the generation of antigen-specific immune responses in vivo was studied employing the bovine model of beta2-integrin deficiency. To that end four cattle with bovine leukocyte adhesion deficiency (BLAD) and healthy age-matched controls were immunized with tetanus toxoid (TT) and rabies virus (RV) vaccines three times in monthly intervals. In addition, two animals with BLAD and three controls received a fourth vaccination 8 months after the start of the study. Proliferative responses of peripheral blood mononuclear cells (PBMC) to the antigens TT and RV as well as specific serum immunoglobulin G (IgG) titers were determined in intervals for up to 10 months after primary vaccination. Proliferative responses of PBMC to TT and RV were substantially lower in cattle with BLAD than in controls, although PBMC from cattle with BLAD were shown to have the capacity to proliferate in the response to the mitogen concanavalin A. Occurrence of antigen-specific IgG titers was delayed and they were considerably lower in cattle with BLAD compared to controls. Finally, treatment of TT- and RV-stimulated PBMC from an immunized control with different concentrations of the anti-CD18 monoclonal antibody R15.7 resulted in a dose-dependent inhibition of lymphocyte proliferation to almost 100%. The results of the present study show that beta2-integrin deficiency leads to delayedand severely impaired immune responsiveness in vivo. The observations that antibody production, although considerably delayed and impaired, does occur and that apparently class-switching takes place in BLAD indicate T-cell reactivity in vivo.

Research paper thumbnail of Impaired specific immunoreactivity in cows with hepatic lipidosis

Veterinary Immunology and Immunopathology, 1997

In this study, hepatic lipidosis in cows was experimentally induced by offering an energy surplus... more In this study, hepatic lipidosis in cows was experimentally induced by offering an energy surplus during the dry period. Liver triacylglycerol (TAG) was 16% in the experimental group. In the control group fed the same diet in restricted quantities, liver TAG was about 7%.The animals of both groups were vaccinated with tetanus vaccine at Day 3 after parturition. It was

Research paper thumbnail of The effects of a single injection of dexamethasone-21-isonicotinate on the lymphocyte functions of dairy cows at two weeks post partum

Veterinary Research, 2004

Dexamethasone is a potent therapeutic for treatment of the fatty liver syndrome or primary ketosi... more Dexamethasone is a potent therapeutic for treatment of the fatty liver syndrome or primary ketosis in post partum dairy cows. Reservations exist, however, among practitioners with respect to the risk of immunosuppression induced by corticosteroids. The aim of this study was to investigate the effect of a single injection of dexamethasone-21-isonicotinate on distinct immune functions of postpartum dairy cows because only scarce information is available on the effects of corticosteroid preparations when administered at a dosage and frequency for treatment of the fatty liver syndrome or primary ketosis. Sixteen Swedish red-pied dairy cows, between days 9 and 15 post partum, were allotted to either a control group (n = 8) or a treatment group (n = 8). The cows in the treatment group received a single intramuscular injection of a dexamethasone-21-isonicotinate suspension at a dosage of 0.02 mg/kg i.m. at the start of the experiment. White blood cell counts and selected lymphocyte functions (lymphocyte proliferation, expression of lymphocyte markers and the β2 and α4 chain of adhesion molecules belonging to the integrin family) and some parameters of the energy metabolism (glucose, insulin) were determined before the administration of corticosteroids (day 0) and subsequently at days 2, 4, 7 and 9 of the experiment. Changes in glucose and insulin were within the target range for treatment of the fatty liver syndrome or primary ketosis. Significant (P < 0.05) increases in the number of circulating white blood cells were observed in treated cows on the second day following treatment which was exclusively caused by an increase in the number of circulating neutrophils. Lymphocyte blastogenesis in response to ConA and the percentages of lymphocytes positive for CD2, CD4, CD8, CD49d and CD18 as well as the intensity of CD49d expression did not differ between the treatment and control groups. There was, however, a significant reduction (P < 0.01) in the intensity of CD18 expression on lymphocytes in the treated animals on the fourth day after treatment. In conclusion, a single administration of dexamethasone-21-isonicotinate in a dosage of 0.02 mg/kg i.m. at two weeks post partum in healthy cows had a significant but highly transient effect on CD18 expression on lymphocytes and the number of peripheral blood neutrophils, but did not affect lymphocyte blastogenesis or lymphocyte subpopulation patterns in peripheral blood. glucocorticosteroid / ketosis / lymphocyte proliferation / adhesion molecules / dairy cows * Corresponding author: j.thanasak@vet.uu.nl 104 J. Thanasak et al.

Research paper thumbnail of Subpopulations of bovine WC1 + γδ T cells rather than CD4 + CD25 high Foxp3 + T cells act as immune regulatory cells ex vivo

Veterinary Research, 2009

Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis... more Regulatory T cells (Treg) are regarded essential components for maintenance of immune homeostasis. Especially CD4 + CD25 high T cells are considered to be important regulators of immune reactivity. In humans and rodents these natural Treg are characterized by their anergic nature, defined as a non-proliferative state, suppressive function and expression of Foxp3. In this study the potential functional role of flowcytometry-sorted bovine white blood cell populations, including CD4 + CD25 high T cells and T cell subpopulations, as distinct ex vivo regulatory cells was assessed in co-culture suppression assays. Our findings revealed that despite the existence of a distinct bovine CD4 + CD25 high T cell population, which showed Foxp3 transcription/expression, natural regulatory activity did not reside in this cell population. In bovine co-culture suppression assays these cells were neither anergic nor suppressive. Subsequently, the following cell populations were tested functionally for regulatory activity: CD4 + CD25 low T cells, WC1 + , WC1.1 + and WC1.2 + T cells, NK cells, CD8 + T cells and CD14 + monocytes. Only the WC1.1 + and WC1.2 + T cells and CD14 + monocytes proved to act as regulatory cells in cattle, which was supported by the fact that these regulatory cells showed IL-10 transcription/expression. In conclusion, our data provide first evidence that cattle CD4 + CD25 high Foxp3 + and CD4 + CD25 low T cells do not function as Treg ex vivo. The bovine Treg function appears to reside in the T cell population, more precisely in the WC1.1 + and the WC1.2 + subpopulation, major populations present in blood of cattle in contrast to non-ruminant species.

Research paper thumbnail of Bovine leukocyte adhesion deficiency ‐ clinical course and laboratory findings in eight affected animals

Veterinary Quarterly, 1994

The clinical course of Bovine Leukocyte Adhesion Deficiency (BLAD) in eight Holstein Friesian cat... more The clinical course of Bovine Leukocyte Adhesion Deficiency (BLAD) in eight Holstein Friesian cattle is described. Affected animals were presented with a history of poor thriving and recurrent bacterial infections. Five of these animals had to be killed because of severe respiratory disease shortly after admittance. Three affected animals survived calfhood only as a result of frequent antibacterial treatments. At one year of age, failure to thrive and stunted growth were still evident, but infections requiring antibiotic treatments occurred only sporadically. Clinical manifestations of BLAD were found in the digestive system (gingivitis, periodontitis, alveolar periostitis, diarrhoea), the respiratory system and the skin (impaired wound healing, chronic dermatitis). A leukocytosis based on a mature neutrophilia, which persisted during infection-free periods, was observed in all animals. Granulocytes were substantially deficient of beta 2-integrin expression on their membranes. Anaemia, which was noted in four animals, may be related to the Anaemia of Inflammatory Disease Complex (AID). The serum total protein content increased with time and was associated with elevated gamma-globulin levels. We suggest that, at a certain age, animals affected with BLAD are able to cope with environmental agents due to compensatory mechanisms of the immune system.

Research paper thumbnail of Characterization of bovine regulatory cells

Veterinary Immunology and Immunopathology, 2009

E-mail address: a.hoek@vet.uu.nl (A. Hoek).

Research paper thumbnail of Allograft rejection in cattle with bovine leukocyte adhesion deficiency

Veterinary Immunology and Immunopathology, 1995

In the present investigation cell-mediated immunity in animals with bovine leukocyte adhesion def... more In the present investigation cell-mediated immunity in animals with bovine leukocyte adhesion deficiency (BLAD) was studied by means of skin transplantation experiments.Autograft and allograft behaviour in animals with BLAD was compared with the behaviour of simultaneously transplanted autografts and allografts in healthy controls. Allograft survival time was prolonged in three BLAD cattle (28, 30, and 72 days) compared to six

Research paper thumbnail of The immune response of cattle, persistently infected with noncytopathic BVDV, after superinfection with antigenically semi-homologous cytopathic BVDV

Veterinary Immunology and Immunopathology, 1998

Cattle persistently infected (PI) with noncytopathic (ncp) bovine virus diarrhea virus (BVDV) are... more Cattle persistently infected (PI) with noncytopathic (ncp) bovine virus diarrhea virus (BVDV) are at risk for developing fatal mucosal disease (MD), which is considered to occur after superinfection with antigenically homologous cytopathic (cp) BVDV. In this study, we intranasally inoculated four PI-animals, that were PI with 2 ncp BVDV strains with 10(5) TCID50 antigenically closely related cp BVDV. Two PI-animals were inoculated with 10(5) TCID50 ncp BVDV and one PI-animal, with virus free cell culture medium. Two out of four PI-animals that were inoculated with cp BVDV, developed MD and were euthanized at day 17 and at day 24 after infection. Postmortem, both animals showed typical lesions of MD and cp BVDV was isolated. The other two PI-animals that were inoculated with cp BVDV did not develop MD and were euthanized at day 51. They showed ulcerations in the gastrointestinal tract, cp BVDV was isolated and neutralizing antibodies were detected. From the three PI-animals, that were inoculated with ncp BVDV or cell culture medium, cp BVDV was also isolated. Cross neutralization tests were performed and no antigenic differences could be detected between the cp strains isolated from the PI-animals. Lymphocyte subsets of these PI-animals were determined by flow cytometric analysis. Before superinfection, the percentages of gamma delta subsets were much higher in the PI-animals that did not develop MD than in nonviremic control animals and in the PI-animals that died of MD. From this study we conclude that the presence of antigenically closely related cp BVDV in PI-animals does not necessarily lead to the development of MD and that besides the antigenic relatedness between the persisting ncp BVDV and cp BVDV other factors, for instance the number of circulating gamma delta cells, might determine whether or not PI-animals develop MD.