| 1. |
- Ahlberg, Viktor, et al.
(författare)
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Innate immune responses induced by the saponin adjuvant Matrix-M in specific pathogen free pigs
- 2017
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Ingår i: Veterinary Research. - : Springer Science and Business Media LLC. - 0928-4249 .- 1297-9716. ; 48
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Tidskriftsartikel (refereegranskat)abstract
- Saponin-based adjuvants have been widely used to enhance humoral and cellular immune responses in many species, but their mode of action is not fully understood. A characterization of the porcine transcriptional response to Matrix-M was performed in vitro using lymphocytes, monocytes or monocyte-derived dendritic cells (MoDCs) and in vivo. The effect of Matrix-M was also evaluated in specific pathogen free (SPF) pigs exposed to conventionally reared pigs. The pro-inflammatory cytokine genes IL1B and CXCL8 were up-regulated in monocytes and lymphocytes after Matrix-M exposure. Matrix-M also induced IL12B, IL17A and IFNG in lymphocytes and IFN-a gene expression in MoDCs. Several genes were indicated as up-regulated by Matrix-M in blood 18 h after injection, of which the genes for IFN-a and TLR2 could be statistically confirmed. Respiratory disease developed in all SPF pigs mixed with conventional pigs within 1-3 days. Two out of four SPF pigs injected with saline prior to contact exposure displayed systemic symptoms that was not recorded for the four pigs administered Matrix-M. Granulocyte counts, serum amyloid A levels and transcription of IL18 and TLR2 coincided with disease progression in the pigs. These results support further evaluation of Matrix-M as a possible enhancer of innate immune responses during critical moments in pig management.
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| 2. |
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| 3. |
- Fossum, Caroline, et al.
(författare)
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Early inflammatory response to the saponin adjuvant Matrix-M in the pig
- 2014
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Ingår i: Veterinary Immunology and Immunopathology. - : Elsevier BV. - 0165-2427 .- 1873-2534. ; 158, s. 53-61
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Tidskriftsartikel (refereegranskat)abstract
- The early inflammatory response to Matrix-M was evaluated in pigs. Adverse reactions measured as body temperature, appetite, activity level and reaction at the site of injection were not observed after s.c. injection with three doses of the adjuvant (75, 100 or 150 mu g) into one week old piglets. Analyses of the immediate cytokine response of PBMC after in vitro exposure to Matrix-M (AbISCO-100 (R)) revealed only a low expression of mRNA for tumour necrosis factor-alpha (p < 0.05) after 6 h incubation. Histological examination revealed an infiltration of leukocytes, haemorrhage and necrosis in muscle 24 h after i.m. injection of 150 mu g Matrix-M in pigs aged eleven weeks. At this time, different grades of reactive lymphoid hyperplasia were recorded in the draining lymph node that was enlarged in three of these six pigs injected with Matrix-M. The global transcriptional response at the site of injection and in the draining lymph node was analyzed using Affymetrix GeneChip Porcine Genome Array. A significant enrichment of gene signatures for the cell types described as "myeloid cells" and "plasmacytoid dendritic cells" was observed at the site of injection in Matrix-M injected pigs compared with pigs injected with saline. A number of genes encoding cytokines/chemokines or their receptors were upregulated at the injection site as well as in the draining lymph node. In the draining lymph node, a majority of the upregulated genes were interferon-regulated genes (IRGs). The expression of IFN-beta, but not IFN-alpha, was increased in the draining lymph nodes of a majority of the pigs exposed to Matrix-M. These IFN-beta expressing pigs also expressed increased levels of osteopontin (OPN) or stimulator of interferon genes (STING), two factors known to facilitate the expression of type I IFNs in response to viral infection. Thus, Matrix-M does not appear to induce any harmful inflammatory response in piglets whilst contributing to the innate immunity by activating the type I IFN system, possibly through several alternative signalling pathways. (C) 2013 Elsevier B.V. All rights reserved.
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| 4. |
- Fossum, Caroline, et al.
(författare)
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Expression of tlr4, md2 and cd14 in equine blood leukocytes during endotoxin infusion and in intestinal tissues from healthy horses
- 2012
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Ingår i: Veterinary Immunology and Immunopathology. - : Elsevier BV. - 0165-2427 .- 1873-2534. ; 150:3-4, s. 141-148
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Tidskriftsartikel (refereegranskat)abstract
- The expression of tlr4, md2 and cd14 was studied in equine blood leukocytes and in intestinal samples using real time PCR. The stability of three commonly used reference genes, glyceraldehyde-3P-dehydrogenase (GAPDH), hypoxantine ribosyltransferase (HPRT) and succinate dehydrogenase complex subunit A (SDHA), was evaluated using qbase(PLUS). The equine peripheral blood mononuclear cells (eqPBMC) examined were either stimulated in vitro with Phorbol 12-myristate 13-acetate (PMA) and ionomycin or with the CpG oligodeoxynuclotide 2216 (CpG-ODN 2216) or obtained from horses before, during and after infusion of endotoxin. Intestinal tissue from healthy horses was sampled at ileum, right dorsal colon and rectum. Ranking of the three reference genes used for normalisation identified the combination HPRT/SDHA as most suitable both when determined ex vivo in leukocytes obtained from experimentally induced endotoxaemia and in eqPBMC activated in vitro while HPRT/GAPDH were most appropriate for the intestinal samples. The relative amounts of mRNA for TLR4 and MD-2 increased threefold during in vitro activation of the cells with CpG-ODN 2216 but was decreased in cultures stimulated with PMA/ionomycin. A transient elevation in the transcription of tlr4 and md2 was also evident for equine blood leukocytes following endotoxaemia. The levels of mRNA for CD14 on the other hard remained unaffected both during the induction of endotoxaemia and in the in vitro stimulated PBMCs. A low steady expression of TLR4, MD-2 and CD14 mRNA was demonstrated for the intestinal samples with no variation between the intestinal segments analysed. Thus, the foundation for real time PCR based levels of analysis of mRNA for all three components in the equine LPS receptor complex in different intestinal segments was set, making it possible to carry out future expression studies on clinical material.
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| 5. |
- Fossum, Caroline, et al.
(författare)
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PCV2 on the spot-A new method for the detection of single porcine circovirus type 2 secreting cells
- 2014
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 196, s. 185-192
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Tidskriftsartikel (refereegranskat)abstract
- A porcine circovirus type 2 SPOT (PCV2-SPOT) assay was established to enumerate virus-secreting lymphocytes obtained from naturally infected pigs. The assay is based on the same principle as general ELISPOT assays but instead of detecting cytokine or immunoglobulin secretion, PCV2 particles are immobilized and detected as filter spots. The method was used to evaluate the influence of various cell activators on the PCV2 secretion in vitro and was also applied to study the PCV2 secretion by lymphocytes obtained from pigs in healthy herds and in a herd afflicted by postweaning multisystemic wasting disease (PMWS). Peripheral blood mononuclear cells (PBMCs) obtained from a pig with severe PMWS produced PCV2-SPOT5 spontaneously whereas PBMCs obtained from pigs infected subclinically only generated PCV2-SPOT5 upon in vitro stimulation. The PCV2 secretion potential was related to the PCV2 DNA content in the PBMCs as determined by two PCV2 real-time PCR assays, developed to differentiate between Swedish PCV2 genogroups 1 (PCV2a) and 3 (PCV2b). Besides the current application these qPCRs could simplify future epidemiological studies and allow genogroup detection/quantitation in dual infection experiments and similar studies. The developed PCV2-SPOT assay offers a semi-quantitative approach to evaluate the potential of PCV2-infected porcine cells to release PCV2 viral particles as well as a system to evaluate the ability of different cell types or compounds to affect PCV2 replication and secretion. (C) 2013 Elsevier B.V. All rights reserved.
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| 6. |
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| 7. |
- Hellman, Stina, et al.
(författare)
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Cytokine responses to various larval stages of equine strongyles and modulatory effects of the adjuvant G3 in vitro
- 2020
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Ingår i: Parasite Immunology. - : Wiley. - 0141-9838 .- 1365-3024. ; 43
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Tidskriftsartikel (refereegranskat)abstract
- Aims To generate different larval stages ofStrongylus vulgarisand to study cytokine responses in cultures of eqPBMC exposed to defined larval stages ofS. vulgarisand cyathostomins with the aim to understand the early immune reaction to these parasites. Methods and results EqPBMC were exposed toS. vulgarislarvae (L3, exsheated L3 and L4) and cyathostomin L3 and analysed for cytokine gene expression. Procedures for decontamination, culturing and attenuation of larvae were established. Transcription of IL-4, IL-5 and IL-13 was induced by bothS. vulgarisand cyathostomin L3. Moulting ofS. vulgarisfrom L3 to L4 stage was accompanied by a shift to high expression of IL-5 and IL-9 (exsheated L3 and L4) and IFN-gamma (L4 only). In parallel, the adjuvant G3 modified the cytokine profile induced by both parasites by reducing the expression of IL-4, IL-5 and IL-10 while concomitantly enhancing the expression of IFN-gamma. Conclusion The L4 stage ofS. vulgarisgenerated a cytokine profile different from that induced by the earlier L3 stage ofS. vulgarisand cyathostomins. This diversity depending on the life cycle stage will have implications for the choice of antigen and adjuvant in future vaccine design.
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| 8. |
- Hellman, Stina, et al.
(författare)
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Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection
- 2024
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Ingår i: Veterinary research (Print). - : Springer Nature. - 0928-4249 .- 1297-9716. ; 55:1
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Tidskriftsartikel (refereegranskat)abstract
- Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.
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| 9. |
- Hellman, Stina, et al.
(författare)
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The adjuvant G3 promotes a Th1 polarizing innate immune response in equine PBMC
- 2018
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Ingår i: Veterinary Research. - : Springer Science and Business Media LLC. - 0928-4249 .- 1297-9716. ; 49
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Tidskriftsartikel (refereegranskat)abstract
- The immunomodulatory effect of a new particulate adjuvant, G3, alone or in combination with agonists to TLR2/1 or TLR5 was evaluated in cultures of equine PBMC. Exposure to the G3 adjuvant up-regulated genes encoding IFN-, IL-1, IL-6, IL-8, IL-12p40 and IL-23p19 in the majority of the horses tested, indicating that the G3 adjuvant induced a pro-inflammatory and Th1 dominated profile. In accordance, genes encoding IL-13, IL-4, IL-10 and TGF- remained unaffected and genes encoding IFN-, IL-17A and TNF- were only occasionally and weakly induced. The two TLR agonists Pam3CSK4 (TLR2/1) and FliC (TLR5) induced cytokine profiles characterized by a clear induction of IL-10 as well as up-regulation of the genes encoding IL-1, IL-6 and IL-8. The presence of G3 modified this response, in particular by reducing the FliC and Pam3CSK4 induced production of IL-10. Furthermore, G3 acted in synergy with Pam3CSK4 in enhancing the production of IFN- whereas G3 combined with FliC increased the gene expression of IL-8. Thus, the G3 adjuvant seems to have the capacity to promote a Th1 polarizing innate immune response in eqPBMC, both by favouring IFN- production and by reducing production of IL-10 induced by co-delivered molecules. These features make G3 an interesting candidate to further evaluate for its potential as an adjuvant in equine vaccines.
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| 10. |
- Hjertner, Bernt, et al.
(författare)
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A novel adjuvant G3 induces both Th1 and Th2 related immune responses in mice after immunization with a trivalent inactivated split-virion influenza vaccine
- 2018
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Ingår i: Vaccine. - : Elsevier BV. - 0264-410X .- 1358-8745. ; 36, s. 3340-3344
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Tidskriftsartikel (refereegranskat)abstract
- A preferred adjuvant should promote both Th1 and Th2 responses. However, most adjuvants in common use are biased towards a Th2-driven response. Therefore, the ability of a novel saponin-based adjuvant G3 to inducing balanced Thl and Th2 responses in BALB/c mice immunized with a split trivalent seasonal influenza vaccine was evaluated in comparison to that of the adjuvant Al(OH) 3 . Clear differences in the IgG profiles induced by G3, Al(OH)(3) or non-adjuvanted vaccine were recorded. Both adjuvants enhanced high and similar levels of the Th2 associated IgG1 subtype compared to mice given vaccine alone. Only G3 enhanced the IgG2a subclass reflecting a Th1 response, whereas Al(OH)(3) even abrogated the IgG2a production. Accordingly, G3 enhanced the production of IL-2 and IFN-gamma and also of IL-2/IFN-gamma double secreting cells, emphasizing the strong Th1 driving effect of G3. Only Al(OH)(3) increased splenocyte production of IL-17. Taken together, the results indicate a strong propensity for G3 to induce both Th1 and Th2 driven immune responses. (C) 2018 The Authors. Published by Elsevier Ltd.
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