Analysis of cell hyperplasia and parietal cell dysfunction induced by Ostertagia ostertagi infection

Infections in cattle with the gastric nematode Ostertagia ostertagi are associated with decreased acid secretion and profound physio-morphological changes of the gastric mucosa. The purpose of the current study was to investigate the mechanisms triggering these pathophysiological changes. O. ostertagi infection resulted in a marked cellular hyperplasia, which can be explained by increased transcriptional levels of signaling molecules related to the homeostasis of gastric epithelial cells such as HES1, WNT5A, FGF10, HB-EGF, AREG, ADAM10 and ADAM17. Intriguingly, histological analysis indicated that the rapid rise in the gastric pH, observed following the emergence of adult worms, cannot be explained by a loss of parietal cells, as a decrease in the number of parietal cells was only observed following a long term infection of several weeks, but is likely to be caused by an inhibition of parietal cell activity. To investigate whether this inhibition is caused by a direct effect of the parasites, parietal cells were co-cultured with parasite Excretory/Secretory products (ESP) and subsequently analyzed for acid production. The results indicate that adult ESP inhibited acid secretion, whereas ESP from the L4 larval stages did not alter parietal cell function. In addition, our data show that the inhibition of parietal cell activity could be mediated by a marked upregulation of inflammatory factors, which are partly induced by adult ESP in abomasal epithelial cells. In conclusion, this study shows that the emergence of adult O. ostertagi worms is associated with marked cellular changes that can be partly triggered by the worm's Excretory/secretory antigens

Comparative immune responses against Psoroptes ovis in two cattle breeds with different susceptibility to mange

The sheep scab mite, Psoroptes ovis, is a major problem in the beef cattle industry, especially in Belgian Blue (BB) cattle. This breed is naturally more predisposed to psoroptic mange but reasons for this high susceptibility remain unknown. Different immune responses could be a potential cause; thus in this study, the cutaneous immune response and in vitro cellular immune response after antigen re-stimulation were examined in naturally infested BB. Cytokine production in the skin and in circulating re-stimulated peripheral blood mononuclear cells (PBMC) demonstrated a mixed pro-inflammatory Th2/Th17 profile, with transcription of IL-4, IL-13, IL-6 and IL-17. Strong IL-17 up-regulation in the skin of BB was associated with an influx of eosinophils and other immune cells, potentially leading towards more severe symptoms. Virtually no changes in cutaneous IFN-gamma transcription were detected, while there was substantial IFN-gamma up-regulation in re-stimulated PBMC from infested and uninfested animals, potentially indicating a role of this pro-inflammatory cytokine in the innate immune response. In Holstein-Friesian (HF) cattle, generally more resistant to P.ovis infection, a largely similar immunologic response was observed. Differences between HF and BB were the lack of cutaneous IL-17 response in infested HF and low transcription levels of IFN-gamma and high IL-10 transcription in re-stimulated PBMC from both infested and uninfested animals. Further research is needed to identify potential cell sources and biological functions for these cytokines and to fully unravel the basis of this different breed susceptibility to P. ovis

Host protective ASP-based vaccine against the parasitic nematode Ostertagia ostertagi triggers NK cell activation and mixed IgG1-IgG2 response

The mucus-dwelling parasite Ostertagia ostertagi is one of the most important gastrointestinal nematodes in cattle. Our group has previously demonstrated the protective capacity of a vaccine against this parasite based on a native activation-associated secreted protein ASP1 (nASP) in combination with the saponin adjuvant QuilA. The aim of the current study was to analyse the effect of both antigen and adjuvant on the cellular and humoral vaccine-induced immune responses by comparing the native ASP to a recombinant version expressed in Pichia pastoris (pASP) and replacing QuilA by Al(OH)(3). Immunization of cattle with the protective nASP+QuilA vaccine was associated with antigen-induced proliferation of natural killer (NK) cells combined with IFN-gamma. secretion and the induction of a mixed IgG1/IgG2 antibody response. ASP-specific activation and proliferation of NK cells was also observed in mice following the same vaccination regime. Replacing QuilA by Al(OH)(3) or nASP by pASP significantly decreased the capacity of the vaccines to trigger both NK cell activation and antibody responses and failed to induce protection against a challenge infection. Reduction of the structurally anchoring disulphide bonds of the nASP completely abolished its ability to induce NK cell activation and antibody responses, highlighting the importance of protein conformation for the immunostimulatory activity

Study of human T cell differentiation using the OP9-DL1 co-culture system as a model

While most hematopoietic lineages develop in the bone marrow, waves of hematopoietic precursor cells (HPC) need to travel from the bone marrow to the thymus, to allow T cell development. The unique thymic microenvironment provides all elements necessary for both the initiation of T cell differentiation and terminal differentiation. While several models have been developed to study human T cell differentiation in vitro, the only model that allows for the in vitro generation of mature T cells in the absence of a thymic microenvironment, is the OP9-Delta ligand (DL) 1 co-culture system. OP9-DL1 co-culture generated T cells could be of great therapeutic value for both the treatment of malignant disease in an immunotherapeutic setting and to restore T cell based immunity in immunodeficient patients. However, the clinical use of OP9-DL1 o-culture generated T cells has been impeded, partly because of the lack of proof that mature T cells thus generated are functional major histocompatibility complex (MHC) class I and class II restricted T cell receptor (TCR) alpha beta+ T cells or functional TCRgammadelta+ T cells. In this work we present evidence that both unconventional and conventional T cells are generated on OP9-DL1. A prominent unconventional CD8alpha alpha single positive (SP) TCRalphabeta+ population was observed that was interleukin (IL)-15 responsive and we believe this population is the in vitro equivalent of the gut intraepithelial lymphocyte (IEL) population. CD8alphabeta SP and CD4 SP TCRalphabeta+ cells did not only present a mature phenotype, but also displayed conventional CD8alphabeta SP and CD4 SP TCRalphabeta+ function. Although this suggests that generation of these cells is dependent on MHC class I and class II induced positive selection mechanisms, similar to those operative in the thymus, we show that functional maturation of these cells is independent of the presence of MHC class I and class II. MHC class I and class II independent selection may impede the use of in vitro generated T cells, because of the risk of the generation of auto-reactive T cells. Therefore, further investigation will be needed in order to better understand selection mechanisms in OP9-DL1 co-cultures. We also present a detailed precursor-progeny relationship between the different TCRgammadelta+ populations that are present in human thymus. We show that, in contrast to the murine situation, human mature TCRgammadelta+ cells are generated along 2 different pathways: a Notch independent double negative (DN) pathway generating functional mature DN and CDB alpha alpha SP TCRgammadelta+ cells and a Notch dependent double positive (DP) pathway generating functional mature CD8alphabetaSP TCRgammadelta+ cells. Maturation of TCRgammadelta6 expressing DN and DP T cell precursors is enhanced by TCR ligation. We furthermore show that DP TCRgammadelta6+ cells are not lineage committed, since they not only give rise to mature CD8alphabeta SP TCRgamma delta6+, but also to TCRalphabeta + cells. These data open new challenges in the search for functional differences between ontogenetically different TCRgammadelta+ populations. In conclusion, our data contribute to the understanding of both in vitro and in vivo T cell differentiation. However, prior to the use of in vitro generated T cells in a clinical setting, selection mechanisms in the absence of a thymic microenvironment will have to be further clarified in order to allow for the efficient production of a self-tolerant T cell repertoire

Granulysin produced by globule leukocytes as a potential key element in the development of vaccine-induced immunity against Ostertgia ostertagi

Ostertagia ostertagi is considered the most economically important bovine parasite. An experimental host-protective vaccine against Ostertagia ostertagi was developed based on ASP-proteins derived from excretory-secretory material of the helminth. Further optimization and commercialization of this vaccine, however, requires a thorough understanding of the vaccine-induced immune response. Previous studies in which immune cell counts and cytokine transcription levels were investigated, did not detect differences between vaccinated and susceptible animals. Therefore, a broader whole-transcriptomic approach using a micro-array was applied. Interesting targets discovered by the micro-array were further analysed on a protein level. This approach revealed a significant upregulation in expression of the granule-proteins granulysin (GNLY) and granzyme B (GZMB) and the high affinity IgE-receptor 1 (FCER1A) in infected vaccinated animals compared to infected non-vaccinated animals. Moreover, these genes significantly correlated with faecal egg count reduction and worm counts. We have further shown that granulysin is produced by globule leukocytes in the abomasum and is secreted into the mucus, presumably through antibody-dependent triggering of the IgE receptor 1. Correlations between globule leukocytes and protection, and the suggestion of a protective agent being present in the mucus, have been described previously. This is however the first time to our knowledge that such an agent is actually identified. Our results, along with the knowledge that granulysin has recently been associated with immunity against a variety of helminths, render granulysin a very interesting topic for further research